CA1340231C - Polynucleotede dertermination with selectable cleavage sites - Google Patents

Polynucleotede dertermination with selectable cleavage sites

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Publication number
CA1340231C
CA1340231C CA000579309A CA579309A CA1340231C CA 1340231 C CA1340231 C CA 1340231C CA 000579309 A CA000579309 A CA 000579309A CA 579309 A CA579309 A CA 579309A CA 1340231 C CA1340231 C CA 1340231C
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Prior art keywords
polynucleotide
support
label
nucleic acid
reagent
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired - Fee Related
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CA000579309A
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French (fr)
Inventor
Michael S. Urdea
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Bayer Corp
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Chiron Corp
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Publication date
Priority to US07/251,152 priority Critical patent/US5118605A/en
Priority to DE3854969T priority patent/DE3854969T2/en
Priority to EP88309203A priority patent/EP0360940B1/en
Priority to ES88309203T priority patent/ES2083955T3/en
Application filed by Chiron Corp filed Critical Chiron Corp
Priority to CA000579309A priority patent/CA1340231C/en
Priority to JP63250726A priority patent/JP2676535B2/en
Priority to US07/806,642 priority patent/US5380833A/en
Application granted granted Critical
Publication of CA1340231C publication Critical patent/CA1340231C/en
Anticipated expiration legal-status Critical
Expired - Fee Related legal-status Critical Current

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    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813Hybridisation assays
    • C12Q1/6827Hybridisation assays for detection of mutation or polymorphism
    • C12Q1/683Hybridisation assays for detection of mutation or polymorphism involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07HSUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813Hybridisation assays
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    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813Hybridisation assays
    • C12Q1/6816Hybridisation assays characterised by the detection means
    • C12Q1/6823Release of bound markers
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    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813Hybridisation assays
    • C12Q1/6827Hybridisation assays for detection of mutation or polymorphism
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10STECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00Chemistry: molecular biology and microbiology
    • Y10S435/803Physical recovery methods, e.g. chromatography, grinding
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10STECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00Chemistry: molecular biology and microbiology
    • Y10S435/81Packaged device or kit

Abstract

Novel methods for assaying a nucleic acid analyte are provided, which employ polynucleotides having oligonucleotide sequences substantially homologous to a sequence of interest in the analyte, where the presence or absence of hybridization at a predetermined stringency provides for the release of a label from a support.
Particularly, various techniques are employed for binding a label to a support, whereupon cleavage of either a single or double strand, a label may be released from a support, where the release of the label can be detected as indicative of the presence of a particular oligonucleotide sequence in a sample. The method finds use in diagnosis of disease, genetic monitoring, and analysis of nucleic acid mixtures.

Description

1~40231 POLYNUCLEOTIDE DETERMINATION WITH
SELECTABLE CLEAVAGE SITES

Background of the Invention 1. Field of the Invention The ability to synthesize oligonucleotide sequences at will and to clone polynucleotide sequences prepared by synthetic procedures or obtained from naturally occurring sources has greatly expanded the opportunities for detecting the presence of specific sequences in an extended oligonucleotide sequence, e.g., chromosome(s), mixture of sequences, mRNAs, or the like.
Interest in specific sequences may involve the diagnosis of the presence of pathogens, the determination of the presence of alleles, the presence of lesions in a host genome, the detection of a particular mRNA or the monitoring of a modification of a cellular host, to mention only a few illustrative opportunities. While the use of antibodies to perform assays diagnostic of the presence of various antigens in samples has seen an explosive expansion in techniques and protocols since the advent of radioimmunoassay, there has been until recently 1~0231 no parallel activity in the area of the DNA probes.
Therefore, for the most part, detection of sequences has involved various hybridization techniques requiring the binding of a polynucleotide sequence to a support and employing a radiolabeled probe.
In view of the increasing capability to produce oligonucleotide sequences in large amounts in an economical way, the attention of investigators will be directed to providing for simple, accurate and efficient techniques for detecting specific oligonucleotides sequences. Desirably, these techniques will be rapid, minimize the opportunity for technician error, be capable of automation, and allow for simple and accurate methods of detection. Toward this end, there have already been efforts to provide for means to label oligonucleotide probes with labels other than radioisotopes and for improving the accuracy of transfer of DNA sequences to a support from a gel, as well as improved methods for derivatizing oligonucleotides to allow for binding to a label. There continues to be a need for providing new protocols which allow for flexibility in detecting DNA
sequences of interest in a variety of situations where the DNA may come from diverse sources.
2. Description of the Prior Art Meinkoth and Wahl, Anal. Biochemistry (1984) 138:267-284, provide an excellent review of hybridization techniques. Leary, et al., Proc. Natl. Acad. Sci. USA
(1983) 80:4045-4049, describe the use of biotinylated DNA
in conjunction with an avidin-enzyme conjugate for detection of specific oligonucleotide sequences. Ranki et al., Gene (1983) 21:77-85 describe what they refer to as a sandwich" hybridization for detection of oligonucleotide sequences. Pfeuffer and Helmrich, J. of Biol. Chem.
(1975) 250:867-876 describe the coupling of guanosine-5'-... . . , . . , " ~, .. . .

0-(3-thiotriphosphate) to Sepharose~ 4B. Bauman, et al., J.
of Histochem. and Cytochem. (1981) 29:227-237, describe the 3'-labeling of RNA with fluorescers. PCT application W0/8302277 published 07 July 1983 describes the addition to DNA fragments of modified ribonucleotides for labeling and methods for analyzing such DNA fragments. Renz and Kurz, Nucl. Acids Res. (1984) 12:3435-3444, describe the covalent linking of enzymes to oligonucleotides. Wallace, DNA
Recombinant Technology (Woo, S., Ed.) CRC Press, Boca Raton, Florida, provides a general background of the use of probes in diagnosis. Chou and Merigan, N. Eng. J. of Med. (1983) 308:921-925, describe the use of a radioisotope labeled probe for the detection of CMV. Inman, Methods in Enzymol. 34B, 24 (1974) 30-59, describes procedures for linking to polyacrylamides, while Parikh, et al., Methods in Enzymol.
34B, 24 (1974) 77-102, describe coupling reactions with agarose. Alwine, et al., Proc. Natl. Acad. Sci. USA (1977) 74:5350-5354, describe a method of transferring oligonucleotides from gels to a solid support for hybridization. Chu, et al., Nucl. Acids Res. (1983) _ :6513-6529, describe a technique for derivatizing terminal nucleotides. Ho. et al., Biochemistry (1981) 20:64-67, describe derivatizing terminal nucleotides through phosphate to form esters. Ashley and MacDonald, Anal. Biochem. (1984) 140:95-103, report a method for preparing probes from a surface bound template.

Summary of the Invention Methods are provided for the detection of specific nucleotide sequences employing a solid support, at least one label, and hybridization involving a sample and a labeled probe, where the presence or absence of (*) Trademark B
.1 , .

~~ ~4~ 13~231 duplex formation results in the ability to modify the spatial relationship between the support and label(s).
Exemplary of the technique is to provide a cleavage site between the label and support through duplexing of a labeled probe and sample DNA, where the duplex is bound to a support. The cleavage site may then be cleaved resulting in separation of the support and the label(s).
Detection of the presence or absence of the label may then proceed in accordance with conventional techniques.
A primary advantage of the invention over the art is that the present method enables one to distinguish between specific and nonspecific binding of the label.
That is, in the prior art, label is typically detected on a solid support, i.e., the sample is affixed to the support and contacted with a complementary, labeled probe;
duplex formation is then assayed on the support. The problem with this method is that label can and does bind to the support in the absence of analyte. This direct binding of the label to the support is referred to herein as ~nonspecific' binding. If any significant amount of nonspecific binding occurs, label will be detected on the support regardless of the presence of analyte, giving false positive results.
By contrast, in the present method, label is detected only when the analyte of interest is present, i.e., only "specific" binding is detected. In a preferred embodiment, this is done by introducing a cleavage site between a support and the selected label, through a duplex between the sample and one or more probes. The cleavage site may be a restriction endonuclease cleavable site, as described in the parent case hereto, U.S.4,775,619 issued 04 October 1988, or it may be one of a number of types of chemically cleavable sites, e.g., a disulfide linkage, periodate-cleavable 1,2-diols, or the like. In an alternative embodiment, specifically bound label is 1~40231 released by a strand replacement procedure, wherein after binding of the label to the support through an analyte/probe complex, a DNA strand is introduced that iscomplementary to a segment of the analyte/probe complex and is selected so as to replace and release the labeled portion thereof.
s According to a first aspect of the invention, there is provided a method for detecting the presence of an oligonucleotide sequence of interest in a nucleic acid analyte present in a nucleic acid sample, said method comprising:
combining under hybridizing conditions in an aqueous medium, said nucleic acid sample with a polynucleotide reagent, where one of said sample or acomponent of said reagent is bound to a support and hybridization of said analyte and said polynucleotide reagent results in a label being bound to said support through a selectable cleavage site;
separating said support having bound polynucleotide reagent and nucleic acid analyte from said aqueous medium;
washing said support with a medium of different hybridizing stringency from said aqueous medium to remove label bound to said support other than through said selectable cleavage site;
cleaving said cleavage site; and detecting label free of said support.
Preferably, the polynucleotide reagent comprises a first polynucleotide capture probe bound to a support and a second polynucleotide label probe, wherein said first and second probes have oligonucleotide sequencescomplementary to sequences present in said analyte to form duplexes therewith under said hybridizing conditions, at least one of said oligonucleotide sequences being a sequence of interest, wherein said capture probe contains said selectable cleavage site.

.., ~
8~ .

-5a--~ 13~0231 According to a second aspect of the invention, there is provided a method for detecting the presence of an oligonucleotide sequence of interest in a nucleic acid analyte present in a nucleic acid sample, said method comprising:
combining under hybridizing conditions said nucleic acid with a first 5 polynucleotide capture probe and a second polynucleotide label probe, wherein said first and second probes have oligonucleotide sequences complementary to sequences present in said analyte so as to form first and second duplexes therewith under said hybridizing conditions, wherein said capture probe is boundto a solid support;
introducing a replacement polynucleotide strand selected so as to form a duplex with said capture probe that is more stable than said first duplex, thereby substantially freeing said support of label bound to said support; and detecting label free of support.
According to a third aspect of the invention, there is provided an 15 article useful for detecting the presence of an oligonucleotide sequence of interest in a nucleic acid analyte present in a nucleic acid sample, comprising a single stranded polynucleotide bound proximal to one end to a support and at its opposite end having a sequence complementary to said sequence of interest, said polynucleotide containing in addition a selectable cleavage site, wherein said 20 cleavage site (a) is chemically cleavable; (b) is other than a restriction enzyme cleavable site; (c) is other than a phosphodiester linkage; and (d) provides for a complete break between adjacent nucleotides in the polynucleotide reagent upon cleavage.
According to a fourth aspect of the invention, there is provided a 25 polynucleotide reagent having the structure o o 5 ' -HO [ DNAl ] 3 -o-P-o-X-o-P-o-5 [ DNA2 ] 3 -OH
O O

., ;~,~
. ~

13~0231 where DNA, is a first strand of DNA, DNA2 is a second strand of DNA, and X
comprises a selectable cleavage site, wherein said cleavage site X (a) is chemically cleavable; (b) is other than a restriction enzyme cleavable site; (c) is other than a phosphodiester linkage; and (d) provides for a complete break 5 between adjacent nucleotides in the polynucleotide reagent upon cleavage.
X may comprise a linkage cleavable by OH~, hydroxylamine, SH-, or periodate. or a linkage selected from the group consisting of -C-O-CH2-CH2-O-C-, -C-NH-, -~-, -S-S-, and -CH--CH--Preferably, X contains the moiety OH OH

--CH--CH.

Preferably, X is ,CH3 H3C\

-CH2CH2N~ ~ NCH2C 2 COCH--CHCO

OH OH

According to a fifth aspect of the invention, there is provided a 20 reagent useful in polynucleotide synthesis, given by the structure CH3 CH3\
Rl-O-CH2 CH2-N N-CH2 CH2-0-R2 CH - CIH O
O O
C=O C=O
wherein R' is an acid-sensitive, base-stable protecting group, and R2 is selected from the group consisting of H, phosphoramidite, phosphotriester, phosphodiester, phosphite, H-phosphonate and phosphonothioate.

, -5c-Preferably, R' is DMT and R2 is N(iPr)2 5 where DMT is mimethoxy and iPr is isopropyl.

Brief DescriPtion of the Drawin~s Figure 1 illustrates the difference between specific and nonspecific binding of a label to a solid support.
Figures 2A through 2D schematically illustrate the preferred method of the invention, wherein a selectively cleavable site is introduced between a support and a label through an analyte/probe complex.
Figure 3 schematically illustrates an alternative method of the invention, wherein specifically bound label is released through a strand 15 replacement technique.

Description of the SPecific Embodiments Detection of specific sequences is achieved using hybridization, whereby duplexing of the sample DNA and a probe affects the ability to modify the 20 spatial relationship between a label and a support. In this manner, the presence or absence of a particular sequence in a sample can be readily determined by theamount of label which is freed into the medium.
The subject method allows for varying protocols and reagents where the sample nucleic acid may be bound to a support or free in solution. In a 25 preferred embodiment, the method involves forming a nucleic acid duplex where a label is separated from a support by a selectively cleavable bond, so that the amount of label released under conditions providing selective cleavage is a measure of C
,.
s~, .

.. . ..

-6- 13~0231 the presence and amount of a sequence of interest in a nucleic acid sample. The selectable cleavage site may be as a result of formation of a restriction enzyme recognition site through homoduplexing, or the presence of such selectable cleavage site in the single-stranded polynucleotide chain may be a result of the prior introduction of such site into the single-stranded chain.
A reagent will be employed which will include a polynucleotide sequence having an oligonucleotide sequence of interest that hybridizes to the nucleic acid analyte.
This reagent will sometimes be referred to herein as a ~capture probe", which in the present method, is bound to the selected solid support. A labeling probe will also be employed, which may or may not include the sequence of interest.
In the first, preferred embodiment, the subject method involves the forming of a polynucleotide duplex in a hybridization medium resulting in a label bound to a support through a selectable cleavage site. Various protocols may be employed where the sample DNA is bound to a support or dispersed in a solution.
In order to distinguish the various nucleotide sequences involved, the following terms will be used:
nucleic acid sample - sample suspected of containing a nucleic acid sequence having an oligonucleotide sequence of interest;
nucleic acid analyte - DNA or RNA in said nucleic said sample having an oligonucleotide sequence of interest;
oligonucleotide sequence of interest - a DNA or RNA sequence which may be all or part of a nucleotide chain, usually at least six bases, more usually at least about 10 bases, preferably at least about 16 bases, which may be 5kb or more, usually not more than .2kb, which is diagnostic of a property to be detected, where the . . , , ., ~ . ., ~7~ 13~231 property may be a gene or sequence diagnostic of a hereditary trait, pathogen, etc.;
polynucleotide sequence - DNA or RNA sequences employed as reagents for detection of the oligonucleotide sequence of interest, which polynucleotide sequence may be labeled or unlabeled, bound or unbound to a support, and may or may not include a sequence complementary to the oligonucleotide sequence of interest. There will be one to two polynucleotide sequences, which individually or in conjunction with the nucleic acid analyte will act as a bridge between a label and a support, with a selectably cleavable site intermediate the label and support; and selectably cleavable site - a functionality or plurality of functionalities which can be selectively cleaved and may include restriction sites, phosphate esters, purines, peptide bonds, etc.
For convenience of description, the preferred embodiment of the subject invention wherein a selectable cleavage site is created will be divided into four primary sub-embodiments. In the first of these (see Fig. 2A) the reagent employed is a single component, which is a polynucleotide joined proximal to one end to a support and joined proximal to the opposite end to one or more detectable labels. The polynucleotide will include a region of at least four successive nucleotides homoduplexing with a sequence of interest, where such sequence includes a restriction site, which is intermediate the support and label.
In the second case (See Fig. 2B), the reagent employed will have two components which will vary with whether the nucleic acid sample is bound or unbound to a support and the nature of the selectable cleavage site.
Where the nucleic acid sample is bound to the support, the two components will be (1) a bridging polynucleotide sequence and (2) a polynucleotide sequence complementary 13~0231 and hybridizing to a portion of the bridging polynucleotide sequence. Either the bridging or complementary polynucleotide sequence may be labeled. The presence of the label bound to the bridging sequence will be limited to when the duplex of the bridging and analyte polynucleotide sequences define a restriction site as the selectable cleavage site. Otherwise, only the complementary sequence will be labeled. Besides having a sequence duplexing with the complementary sequence, the bridging polynucleotide sequence will have a region duplexing with the oligonucleotide sequence of interest.
Where the sample nucleic acid is in solution, the two components will be (1) a first polynucleotide sequence bound to a support, which has a region complementary to a sequence present in the nucleic acid analyte, which sequence may or may not define a restriction site and may or may not define the oligonucleotide sequence of interest; and (2) a labeled second polynucleotide sequence which as a region complementary to a sequence present in the nucleic acid analyte, which region is subject to the same limitations as the region of the first polynucleotide sequence. At least one of the duplexed regions will define a sequence of interest. In the absence of one of the regions defining a restriction site or in addition to the presence of a restriction site, there will be a selectable cleavage site present with the first or second polynucleotide sequence.
In a third case (see Fig. 2C), the analyte is bound to a support and the reagent employed is a single component which is a labeled polynucleotide sequence having a region complementary to the oligonucleotide sequence of interest which may define a restriction site.
The restriction site and/or a functionality present on the labeled polynucleotide sequence may serve as a selectable cleavage site.
In a fourth case (see Fig. 2D), a capture probe is provided which is a polynucleotide chain bound to a solid support via a linkage "Y", and at its opposing end is complementary to a first sequence present in the nucleic acid analyte. A labeling probe comprising a labeled second polynucleotide chain has a region complementary to a second sequence in the analyte that is distinct from and does not overlap with the first sequence. The linkage designated "Y" in Fig. 2D
represents any conventional means of binding a probe to a support. The linkage ~X" is a selectable cleavage site, i.e., a chemically cleavable linkage such as a disulfide lS bond, periodate-cleavable 1,2-diols, or the like.
The nucleic acid containing sample will be combined with the appropriate reagent under conditions where duplex formation occurs between complementary sequences. The mixture is allowed to hybridize under conditions of predetermined stringency to allow for at least heteroduplex formation or homoduplex formation over an oligonucleotide sequence of interest. After a sufficient time for hybridization to occur, the support may be separate from the supernatant and washed free of at 2S least substantially all of the non-specifically bound label. The oligonucleotides bound to the support are then treated with one or more reagents, which results in cleavage of at least one strand and release of label bound to support.
Depending upon the presence of a particular sequence in the sample resulting in duplex formation, release of the label(s) bound to the support will be observed. Various protocols may be employed, where normally the supernatant medium may be assayed for the presence of the label, although in some instances the -10- 13~0231 support may be measured. Protocols and reagents may be employed, where a physical separation of the support from the supernatant may or may not be required.
The subject method can be used for the detection of oligonucleotide sequences, either DNA or RNA, in a wide variety of situations. One important area of interest is the detection of pathogens, viruses, bacteria, fungi, protozoa, or the like, which can infect a particular host.
See for example, U.S. Patent No. 4,358,535. Another area of interest is the detection of alleles, mutations or lesions present in the genome of a host, such as involved in amniocentesis, genetic counseling, host sensitivity or susceptibility determinations, and monitoring of cell populations. A third area of interest is the determination of the presence of RNA for such diverse reasons as monitoring transcription, detecting RNA
viruses, differentiating organisms through unexpressed RNA, and the like. Other areas of interest, which are intended to be illustrative, but not totally inclusive, include monitoring modified organisms for the presence of extrachromosomal DNA or integrated DNA, amplifications of DNA sequences, the maintenance of such sequences.
The physiological samples may be obtained from a wide variety of sources as is evident from the varied purposes for which the subject method may be used.
Sources may include various physiological fluids, such as excreta, e.g., stool, sputum, urine, saliva, etc.; plasma, blood, serum, ocular lens fluids, spinal fluid, lymph, and the like. The sample may be used without modification or may be modified by expanding the sample, cloning, or the like, to provide an isolate, so that there is an overall enhancement of the DNA or RNA and reduction of extraneous RNA or DNA. Viruses may be plated on a lawn of compatible cells, so as to enhance the amount of viral DNA; clinical isolates may be obtained by the sample being streaked or , . . ~ .

spotted on a nutrient agar medium and individual colonies assayed; or the entire sample introduced into a li~uid broth and the cells selectively or non-selectively expanded. The particular manner in which the sample is treated will be dependent upon the nature of the sample, the nature of the DNA or RNA source, the amount of oligonucleotide sequence of interest which is anticipated as being present as compared to the total amount of nucleic acid present, as well as the sensitivity of the protocol and label being employed.
Either the sample nucleic acid or the reagent polynucleotide may be bound, either covalently or non-covalently, but in any event non-diffusively, to the support. (In the case of the embodiment represented by lS Fig. 2D, the capture probe alone is bound to the solid support.) Where a sample nucleic acid is bound to the support, various supports have found particular use and to the extent, those supports will be preferred. These supports include nitrocellulose filters, diazotized paper, ecteola paper, or other support which provides such desired properties as low or no non-specific binding, retention of the nucleic acid sample, ease of manipulation, and allowing for various treatments, such as growth or organisms, washing, heating, transfer, and label 2S detection, as appropriate.
To the extent that a component of the polynucleotide reagent is bound to the support, the type of support may be greatly varied over the type of support involved with the sample oligonucleotide. The support may include particles, paper, plastic sheets, container holder walls, dividers, Millipore*filters, etc., where the materials may include organic polymers, both naturally occurring and synthetic, such as polysaccharides, polystyrene, polyacrylic acid and derivatives thereof, e.g., polyacrylamide, glass, ceramic, metal, carbon, (*) Trademark E~ ~
~' -12- 13~231 polyvinyl chloride, protein, and the like. The various materials may be functionalized or non-functionalized, depending upon whether covalent or non-covalent bonding is desired.
Where the sample nucleic acid is bound to the support, depending upon the particular support, heating may be sufficient for satisfactory binding of the nucleic acid. In other situations, diazo groups may be employed for linking to the nucleic acid. Where, however, the polynucleotide reagent component is bound to the support, a wide variety of different techniques may be employed for ensuring the maintenance of the polynucleotide reagent bound to the support. For example, supports can be functionalized, to have active amino groups for binding, resulting from the binding of alkylamines, hydrazides, or thiosemicarbazides to the support. One can then add, by means of a terminal transferase, a ribonucleotide to a DNA
polynucleotide reagent. Upon glycol cleavage with an appropriate oxidant, e.g., periodate, osmium tetroxide plus hydrogen peroxide, lead tetraacetate, or the like, a dialdehyde is formed, which will then bind to the amino group on the surface to provide a monosubstituted amino or disubstituted amino group. Alternatively, one can provide for a maleimide group which with thiophosphate will form the alkylthioester. Various techniques described by Parikh, et al., supra and by Inman, supra for agarose and polyacrylamide may be employed, which techniques may have application with other materials.
The total number of polynucleotide reagent components on the support available in the assay medium will vary, for the most part being determined empirically.
Desirably, a relatively high concentration per unit surface area of polynucleotide to available functional groups on the support should be employed, so long as the , . ~ . . , ... ~ .

-13- 1~40231 polynucleotide density does not interfere with hybridization.
The size of the polynucleotide will vary widely, usually being not less than about 15 bases and may be 50 bases or more, usually not exceeding about 500 bases, more usually not exceeding 250 bases. There will usually be a region in the polynucleotide reagent component homologous with a sequence in the nucleic acid sample, usually the sequence of interest, of at least six bases, usually at least 12 bases. The region for hybridization may be 16 bases or more, usually not exceeding about lkbp, where perfect homology is not required, it being sufficient that there be homology to at least about 50%, more preferably homology to at least 80~. (By percent homology is intended complementary, ignoring non-complementary insertions which may loop out, insertions being greater than five bases.) Particularly, where one is interested in a group of allelic genes, a number of different strains, or related species, where the messenger RNA or genomic portion is highly conserved but nevertheless is subject to polymorphisms, it will frequently be desirable to prepare a probe which reflects the differences and optimizes the homology for all the sequences of interest, as against any particular sequence.
The label of the labeled polynucleotide reagent component may be joined to the polynucleotide sequence through the selectively cleavable site or through a link which is retained during the assay. A wide variety of labels may be employed, where the label may provide for a detectable signal or means for obtaining a detectable signal.
Labels therefore include such diverse substituents as ligands, radioisotopes, enzymes, fluorescers, chemiluminescers, enzyme suicide inhibitors, -14- 13~023i enzyme cofactors, enzyme substrates, or other substituent which can provide, either directly or indirectly, a detectable signal.
Where ligands are involved, there will normally be employed a receptor which specifically binds to the ligand, e.g., biotin and avidin, 2,4'-dinitrobenzene and anti(2,4-dinitrobenzene)IgG, etc., where the receptor will be substituted with appropriate labels, as described above. In this manner, one can augment the number of labels providing for a detectable signal per polynucleotide sequence.
For the most part, the labels employed for use in immunoassays can be employed in the subject assays.
These labels are illustrated in U.S. Patent Nos. 3,850,752 (enzyme); 3,853,914 (spin label); 4,160,016 (fluorescer);
4,174,384 (fluorescer and quencher); 4,160,645 (catalyst);
4,277,437 (chemiluminescer); 4,318,707 (quenching particle); and 4,318,890 (enzyme substrate).
Illustrative fluorescent and chemiluminescent labels include fluorescein, rhodamine, dansyl, umbelliferone, biliproteins, luminol, etc.
Illustrative enzymes of interest include horse radish peroxidase, glucose-6-phosphate dehydrogenase, acetylcholinesterase, ~-galactosidase, ~-amylase, uricase, malate dehydrogenase, etc. That is, the enzymes of interest will primarily be hydrolases and oxidoreductases.
The manner in which the label becomes bound to the polynucleotide sequence will vary widely, depending upon the nature of the label. As already indicated, a ribonucleotide may be added to the oligonucleotide sequence, cleaved, and the resulting dialdehyde conjugated to an amino or hydrazine group. The permanence of the binding may be further enhanced by employing reducing conditions, which results in the formation of an alkyl 3S amine. Alternatively, the label may be substituted with -15- 1340~31 an active halogen, such as alpha-bromo or -chloroacetyl.
This may be linked to a thiophosphate group or thiopurine to form a thioether. Alternatively, the label may have maleimide functionality, where a mercapto group present on the polynucleotide will form a thioether. The terminal phosphate of the polynucleotide may be activated with carbodiimide, where the resulting phosphorimidazolide will react with amino groups or alcohols to result in phosphoramidates or phosphate esters. Polypeptide bonds may be formed to amino modified purines. Thus, one has a wide latitude in the choice of label, the manner of linking, and the choice of linking group.
By combining the polynucleotide reagent with the sample, any nucleic acid analyte present will become bound to the support. The amount of label released from the support upon cleavage of the selectable cleavage site will be related to the presence of analyte, where the amount of analyte may also be determined quantitatively.
The modification of the spatial relationship between the label and the support can be achieved in a number of ways. As indicated, there can be at least one recognition site common to the probe and the same polynucleotide, which can be digested with a restriction enzyme, thus releasing the probe from the support. A wide variety of restriction enzymes are available which can detect four base, six base, or eight base recognition sites, where cleavage can be blunt-ended or staggered, may occur at the recognition site or distant from the recognition site. In this manner, the duplex formation of the recognition site(s) provides for the opportunity to cleave the double strand with release of the label.
The nature of the selective cleavage site may or may not depend upon the linking group. Where a restriction site is involved, the bonds involved with the reagent components need only be stable under the assay -16- 1~40231 conditions. Where a restriction site is not involved, then the site will involve a bond(s) which allows for separation of the label from the support.
A phosphodiesterase may be employed where random hydrolysis will separate the label from the support. The polynucleotide may be tailed with modified nucleotides which are or may be subsequently labeled.
A wide variety of linking groups can be employed, where the nucleotides may be modified or unmodified for linkage of the label. W083/02277 reports the use of 8-aminoalkyladenosine, where a label can be bound to the amino group. The DNA polynucleotide reagent may then be tailed with the ribonucleotides so that a plurality of labels will be present at the terminus of each labeled polynucleotide. The tailed ribonucleotides may be selectively cleaved employing an RNase. This will be particularly advantageous when employing labels which interact to modify the signal. For example, fluorescers in close proximity tend to self-quench. The observed fluorescent signal can be greatly enhanced by hydrolyzing the phosphate bonds, so that the individual fluorescer molecules are randomly present in the solution. Of course, fluorescers need not be the only labels demonstrating this phenomenon, but other of the labels may also display similar effects. Where enzyme substrates or cofactors are employed, their presence on a polymer bound to a support will result in substantial steric interference with enzyme approach. Thus the depolymerization of the label and release from the support will substantially enhance the enzyme rate.
Another technique is to add a ribonucleotide to a DNA polynucleotide reagent and then cleave the ribosyl moiety to produce a dialdehyde. (See, for example, Lee, et al., Biochemistry (1970) 9:113-118.) The dialdehyde may be linked to an amino group joined to a label through -17- 13~231 a selectively cleavable site. For example, a disulfide link may be present between the Schiff's base and the label which can be cleaved by reduction, with Ellman's reagent, or the like, to release the label. Where a S restriction endonuclease will be used to release of the label, then the dialdehyde can be combined with the amino functionality under reductive amination conditions.
Various amino sources, such as proteins, e.g., enzymes, phycobiliprotein fluorescers, receptors, such as immunoglobulins or avidin, or non-proteinaceous labels may be employed.
Another linking method involves activating a terminal phosphate with carbodiimide to form a phosphorimidazolide. (Chu, et al., Nucleic Acids Res.
(1983) 11:6513-6628.) The phosphorimidazolide may be reacted with amines to form phosphoramidates. As before, the amino linking group will include the selectable cleavage site, as appropriate, which could be a pyrophosphate diester, cleavable by a pyrophosphatase, a short polypeptide which could be cleaved by a peptidase, a light-sensitive functionality such as azo, peroxy, or the like.
Another method for attaching the label involves chemical synthesis of polynucleotides with a modifiable nucleoside derivative such as a cytosine or uracil containing a 12-atom amine linker arm, followed by incorporation of a reporter group such as fluorescein or dinitrobenzene (Ruth, DNA (1984) 3:123).
Ligand substituted nucleotides can be employed where the ligand does not give a detectable signal directly, but bonds to a receptor to which is conjugated one or more labels. Illustrative examples include biotinylated nucleotides which will bind to avidin, haptens which will bind to immunoglobulins, and various naturally occurring compounds which bind to proteinaceous -18- 1 3 ~n 23 receptors, such as sugars with lectins, hormones and growth factors with cell surface membrane proteins, and the like.
In the embodiment represented by Fig. 2D, the selectable cleavage site may be introduced in one of two ways.
First, a crosslinking compound may be incorporated into the capture probe 1 itself, i.e., at position "X" as indicated in the figure. Any number of crosslinking agents may be used for this purpose, the only limitation being that the cleavage site introduced into the capture probe must be cleavable with reagents that are compatible with the various probes, labels, etc., used in the remainder of the method. Examples of suitable crosslinkers include the following:
N-hydroxy succinimide (NHS), which introduces an amide bond into the probe; ethylene glycolbis (succinimidylsuccinate) (EGS), which creates a hydroxylamine-sensitive linkage; bis[2-succinimido-oxycarbonyloxy)ethyl]sulfone (BSOCOES), which gives abase-sensitive sulfone linkage; disuccinimidyl tartarate (DST), which introduces 1,2-diols cleavable by periodate;
and dithiobis(succinimidylpropionate)(DSP), which results in thiol-cleavable disulfide bonds. The crosslinker is preferably introduced into the capture probe by (1) preparation of an alkylamine probe as described by Urdea et al. in Nucleic Acids Research 16 (11):4937-4956 (1988);
(2) reaction of the free amine functionalities on the probe with the selected crosslinking agent to give probe-bound crosslinking agent; (3) purification of the probe-bound crosslinking agent using chromatographic or other means; and (4) reaction of the probe-bound crosslinking agent with a solid support having free reactive moieties, e.g., free amine groups, to provide a support-bound probe having the desired cleavage site.

The cleavage site may therefore include, for example, the following types of linkages:
O O
-C-O-CH2-CH2-O-C- (hydroxylamine-sensitive);
o -C-NH- ( base-sensitive);

-~O- tbase-sensitive);

-S-S- (thiol-sensitive); and OH OH
-CH- CH- (periodate-sensitive).

The selectable cleavage site "X" in Fig. 2D may also be introduced by appropriate modification of the capture probe prior to attachment to the solid support.
This method involves preparation of a polynucleotide having the structure O O
5'-Ho5 [DNAl]3 -o-1-o-x-o-P-o-5 [DNA2]3 -OH
O O

where X is or contains the selectable cleavage site as described above. In a particularly preferred embodiment, the polynucleotide has the structure O ~H3 H3C O
5 ' -Ho-5 [ DNAl ] 3 -O- P-CH2CH2N ~NCH2CH2o-P-o-3 [ DNA ] -OH
O COCH -CHCO O
OH OH

- ~- 1~40231 This compound may then be attached to a solid support, using conventional means well known in the art, to give the capture probe illustrated in Fig. 2D. This latter compound is prepared using a reagent derived from tartaric acid, where the 1,2-diol system is protected as the dibenzoyl compound during DNA synthesis and which further contains a dimethoxytrityl (DMT)-protected hydroxyl group and a phosphoramidite-derived hydroxyl group (wherein ~iPr" represents isopropyl):
/CH3 CH3~ N(iPr)2 O"C\ ~ C"O OCH3 CH -CH
O O
. CO CO
)~ ~
allowing for incorporation into a DNA fragment using standard phosphoramidite chemistry protocols. After synthesis and complete deprotection the DNA/DNA hybrid molecule, as noted above, contains a 1,2-diol, i.e., a linkage that can be cleaved specifically with NaIO4. As will be readily appreciated by those skilled in the art, the DMT protecting group can be replaced with any suitable moiety R1 that is acid-sensitive and base-stable, e.g., unsubstituted or substituted aryl or arylkyl groups, where the alkyl is, e.g., phenyl, naphthyl, furanyl, biphenyl, or the like, and where the substituents are from 0 to 3, usually 0 to 2, and include any non-interfering stable groups, neutral or polar, electron-donating or withdrawing. Similarly the phosphoramidite moiety may be replaced with other species R2 including phosphorus derivatives (e.g., a phosphotriester, a phosphodiester, a -21- 13~0231 phosphite, an H-phosphonate, a phosphorothioate, etc.) suitable for polynucleotide synthesis, or with hydrogen.
See, for example, EP Publication No. 0225807 (Urdea et al., ~Solution Phase Nucleic Acid Sandwich Assay and Polynucleotide Probes Useful Therein").
As in the embodiment represented by Figs. 2A-2C, the embodiment of Fig. 2D enables detection of specifically bound label in solution (and thus accurate measurement of analyte 2) while nonspecifically bound label 6 remains bound to the solid suport 5.
In an alternative embodiment of the invention illustrated by Figs. 3A and 3B, a complex is formed beween a capture probe 1 (bound to solid support 5 through linkage Y), the nucleic acid analyte 2, and labeling probe 3, as in the embodiment of Fig. 2D. The procedure followed to obtain this hybridization complex is more fully described in EP Publication No. 0225807, cited supra. In order to release the specifically bound label into solution, a "replacement" polynucleotide strand 4 is introduced, selected so as to form a more stable hybrid with capture probe 1 than the analyte forms with the capture probe. Although G/C content is also a factor, this procedure typically requires that the length "B" of the replacement strand be somewhat longer than the length "A" of the duplex formed between the capture probe and the analyte.
A wide variety of supports and techniques for non-diffusive binding of oligonucleotide chains have been reported in the literature. For a review, see Meinkoth and Wahl, Anal. Biochem. (1984) 138:267-284. Supports include nitrocellulose filters, where temperatures of 80~C
for 2 hr suffices, diazotized papers, where bonding occurs without further activation, ecteola paper, etc. Agarose beads can be activated with cyanogen bromide for direct reaction with DNA. (Bauman, et al., J. Histochem.

Cytochem. (1981) 29:227-237); or reacted with cyanogen bromide and a diamine followed by reaction with an ~-haloacetyl, e.g., bromoacetyl or with an active carboxylic substituted olefin, e.g., maleic anhydride, to provide beads capable of reacting with a thiol functionality present on a polynucleotide chain. For example, DNA can be modified to form a ~-thiophosphate for coupling.
tPfeuffer and Hilmreich, J. Biol. Chem. (1975) 250:867-876.) It is also possible to synthesize by chemical means an oligonucleotide bound to a Teflon*support and then fully deblock the material without removing it (Lohrmann, et al., DNA (1984) 3:122).
In view of the wide diversity of labels and reagents, the common aspects of the method will be described, followed by a few exemplary protocols. Common to the procedures will be hybridization. The hybridization can be performed at varying degrees of stringency, so that greater or lesser homology is required for duplexing. For the most part, aqueous media will be employed, which may have a mixture of various other components. Particularly, organic polar solvents may be employed to enhance stringency. Illustrative solvents include dimethylformamide, dimethylacetamide, dimethylsulfoxide, that is, organic solvents which at the amounts employed, are miscible with water. Stringency can also be enhanced by increasing salt concentration, so that one obtains an enhanced ionic strength. Also, increasing temperature can be used to stringency. In each case, the reverse direction results in reduced stringency. Other additives may also be used to modify the stringency, such as detergents.
The period of time for hybridization will vary with the concentration of the sequence of interest, the stringency, the length of the complementary sequences, and the like. Usually, hybridization will require at least - (*) Trademark ~_ -23- 1340231 about 15 min, and generally not more than about 72 hr, more usually not more than about 24 hr. Furthermore, one can provide for hybridization at one stringency and then wash at a higher stringency, so that heteroduplexes lacking sufficient homology are removed.
The nucleic acid sample will be treated in a variety of ways, where one may employ the intact genome, mechanically sheared or restriction enzyme digested fragments of the genome, varying from about .Skb to 30kb, or fragments which have been segregated according to size, for example, by electrophoresis. In some instances, the sequences of interest will be cloned sequences, which have been cloned in an appropriate vector, for example, a single-stranded DNA or RNA virus, e.g., M13.
Included in the assay medium may be other additives including buffers, detergents, e.g., SDS*
Ficoll* polyvinyl pyrrolidone and foreign DNA, to minimize non-specific binding. All of these additives find illustration in the literature, and do not need to be described in detail here.
In accordance with a particular protocol, the sample nucleic acid and polynucleotide reagent(s) are brought together in the hybridization medium at the predetermined stringency. After a sufficient time for hybridization, the support will be washed at least once with a medium of greater or lesser stringency than the hybridization medium. The support with the bound polynucleotide and analyte will then be contacted with the necessary reactants (includes physical treatment, e.g., light) for cleaving the selectable cleavage site, providing for single- or double-stranded cleavage. For the most part hydrolase enzymes will be used, such as restriction endonucleases, phosphodiesterases, pyrophosphatase, peptidases, esterases, etc., although other reagents, such as reductants, Ellman's reagent, or (*) Trademark , .. .. .

~'~40231 light may find use. After cleavage, the support and the supernatant may or may not be separated, depending upon the label and the manner of measurement, and the amount of label released from the support determined.
S To further illustrate the subject invention, a few exemplary protocols will be described. In the first exemplary protocol, a microtiter plate is employed, where fluorescent labeled polynucleotides are bound to the bottom of each well. DNA from a pathogen which has been cloned, is restricted with one or more restriction enzymes to provide fragments of from about 0.5 2kb. The fragments are isolated under mild basic conditions for denaturing and dispersed in the hybridization medium, which is then added sequentially to the various wells, each of the wells having different sequences which are specifically homologous with sequences of different strains of a particular pathogen species.
The wells are maintained at an elevated temperature, e.g., 60~C, for sufficient time for hybridization to occur, whereupon the supernatant is removed and wells are thoroughly washed repeatedly with a buffered medium of lower stringency than the hybridization medium. Duplex formation results in a recognition site for a restriction enzyme common to all of the strains. To each well is then added a restriction enzyme medium for digestion of double-stranded DNAs which are digested result in the release of the fluorescent label into the supernatant. The supernatant is aspirated from each of the wells and irradiated. The amount of fluorescence is then determined as indicative of the presence of the sequence of interest. In this manner, one can rapidly screen for which of the strains is present, by observing the presence of fluorescence in the liquid phase.
In the second exemplary protocol, one employs a column containing glass beads to which are bound unlabeled polynucleotide. To the column is then added the sample nucleic acid containing DNA fragments obtained from mammalian cells. The fragments range from about 0.5 to lOkb. The sample DNA is dispersed in an appropriate hybridization medium and added to the column and retained in the column for sufficient time for hybridization to occur. After the hybridization of the sample, the hybridization medium is released from the column and polynucleotide reagent labeled with horse radish peroxidase (HRP) through a disulfide linkage is added in a second hybridization medium under more stringent conditions than the first medium and the second medium released in the column for sufficient time for hybridization to occur. The labeled polynucleotide has a sequence complementary to the sequence of interest. The hybridization medium is evacuated from the column.
The column may then be washed one or more times with a medium of higher stringency to remove any polynucleotide sequences which have insufficient homology with the labeled polynucleotide. Ellman's reagent is then added to the column resulting in cleavage of the disulfide linkage and release of the HRP. The HRP containing medium is evacuated from the column and collected, as well as a subsequent wash to ensure that freed enzyme is not held up in the column. The resulting medium which contains the HRP label may now be assayed for the HRP label. Instead of HRP a wide variety of other enzymes can be used which produce products which can be detected spectrophotometrically or fluorometrically.
In a third protocol, the nucleic acid sample is non-diffusively bound to one end of a nitrocellulose filter by absorbing the sample with the filter and heating at 80~C for 2 hr. The filter is washed and then added under hybridization conditions to a hybridization solution of a polynucleotide labeled with umbelliferone through an .. . .

ester linkage to an alkylcarboxy substituted adenine. The labeled polynucleotide has a sequence complementary to the sequence of interest. After sufficient time for hybridization the filter is removed from the hybridization medium, washed to remove non-specifically bound nucleotides, and then submerged in a measured solution of an esterase. The rate of increase of fluorescence is monitored as a measure of the amount of analyte in the nucleic acid sample.
In another protocol, dipstick can be used of a plastic material where a holder is employed which holds a strip having a labeled polynucleotide sequenced complementary to the analyte sequence with a polyfluoresceinylated terminus. The nucleic acid sample is prepared in the appropriate hybridization medium and the dipstick introduced and hybridization allowed to proceed. After sufficient time for the hybridization to have occurred, the dipstick is removed and washed to remove any non-specific binding polynucleotide. The presence of a polynucleotide sequence of interest results in the formation of a restriction enzyme recognition site and the dipstick is then introduced into the restriction enzyme reaction mixture and digestion allowed to proceed.
After sufficient time for digestion to have proceeded, the dipstick is removed, thoroughly washed, and the fluorescence in the solution read, while fluorescence above a baseline value indicates the presence of the analyte.
In another protocol, the polynucleotide reagent components are a first polynucleotide which has a sequence complementary to one region of the nucleic acid analyte and is bound to the walls of wells of a microtiter plate and a labeled second polynucleotide which has a sequence complementary to another region of the nucleic acid analyte. The label is the result of tailing the ~ -27- 1~40231 polynucleotide with N8-aminohexyl deoxyadenosine triphosphate umbelliferyl carboxamide. The nucleic acid sample is introduced into the wells with an excess of the labeled polynucleotide under hybridizing conditions.
After sufficient time for hybridization, the hybridization solution is aspirated out of the wells, the wells washed and the residual DNA in the wells depurinated by adding a solution of 90% formic acid and heating at 60~C for 1 hr or adding piperidine and heating at 90~C for 30 min.
Alternatively, the label can be a result of ligating the polynucleotide to be labeled with an excess of an oligomer obtained by treating poly-dA with chloroacetaldehyde according to Silver and Feisht, Biochemistry (1982) 21:6066 to produce the fluorescent N6-ethenoadenosine. Release of the label is achieved with micrococcal nuclease in a solution of lOOugM CaC12 for 1 hr at 37~C.
In both instances the fluorescence of the polymer is substantially diminished due to self-quenching.
Upon dissolution, a substantial enhancement in fluorescence is observed. Thus, non-specifically bound labeled polynucleotide resistant to the depolymerization would not interfere with the observed signal.
Furthermore, one could measure the rate of increase of fluorescence as a quantitative measure of nucleic acid analyte, since the background fluorescent level would be low. Instead of self quenching, systems can be employed where fluorescers and quenchers alternate or in two component reagent systems, non-quenching fluorescers are present on one component and quenchers are present on the other component.
The following examples are offered by way of illustration and not by way of limitation.

EXPERIMENTAL
I. Attachment of ribonucleotides to the 3'-end of DNA.
a. Tailing with terminal deoxynucleotide transferase (TdT).
S The following is a modification of the method described by R. Roychoudry, Method in Enzymology (1980) 65:43. A synthetic oligonucleotide of the composition (5 to 3') ATTCGACGCTTATGG (fragment 1) was tailed with rATP.
To a solution of 4005 pmoles of fragment 1 (based on 20 OD260 units per mg) in lS~l of 0.83 mM ATP, 2.5~41 of lOx TdT buffer (1.4 M potassium cacodylate, 0.6 M Tris pH 7.6, 10 MM CoC12, 1 mM dithiothreitol (DTT)), 2~1 of TdT (calf thymus, P-L Biochemicals, Inc.; 13.5 units) is added. The 24.5~1 sample was left for 1 hr at 37~C, the evaporated lS to dryness. The pellet was dissolved in 10 ~ of 90%
- formamide, 0.05% bromophenol blue, 1% Ficoll*, heated to 90~C for 5 min and loaded on a 20% denaturing polyacrylamide gel run at 40ma. A band corresponding to fragment 1 extended by one riboadenosine unit was visualized by U.V. shadowing, cut from the gel and eluted overnight in 0.1 M Tris pH 8.0, SmM EDTA, O.Sm NaCl (Maxam and Gilbert, Methods in Enzymology (1980) 65:499-560).
Desalting was achieved with a C-18 SEP-PAX*(Waters Associates) as follows: The cartridge wa~ first washed with 5 ml of reagent grade methanol then 10 ml of distilled water. The filtered sample was then applied by syringe to the SEP-PAX. After washing with 20 ml of water, the DNA was eluted with 3 ml of 1:1 (V:V), triethylamine acetate, pH 7.3 : methanol. The solution was then evaporated to dryness (yield ~ 80%).
b. Ligation with T4 ligase.
The following process was used to produce a 137 base fragment that contained a 3'-terminal riboadenosine.
Fragment 1 from above is used as a "universal~ adaptor in (*) Trademark order to produce by ligation a ribonucleotide tailed DNA
sequence.

Fragment 2 AGTTGGCAGTACAGCCTAGCAGCCATGGAAACGATGTATATTTCCGCGAGAGGACGACAG

Fragment 3 GGTCGTCCGCGGGATTCAGCGCCGACGGGACGTAAACAAAGGACGTCCCGCGAAGGATCC

Fragment 4 TTCCATGGCTGCTAGGCTGTACTGCCAACTGGATCCTTCGCGGGACGTCCTTTGTTTACG

Fragment 5 AATTCTGTCGTCCTCTCGCG
Fragment 6 CCATAAGCGTCG

All sequences unless otherwise indicated were 5', 3' hydroxyl. The sequences can be ligated as follows:

5' 3 2 1 rA 3' 3' _ 5' 1) ligation 2) gel isolation Fragment 2 was 5' phosphorylated with T4 polynucleotide kinase. To 900 pmoles of the fragment that had been evaporated to dryness were added 2~1 of 10 x KB-TS (500 mM Tris, 100 mM MgC12, lO~ug/ml spermidine), 2 ~1 10 mM ATP, 2~1 10 mM DTT, 1 ~ (8 units) T4 kinase (New England Nuclear) and 13~1 of water. After 30 min at 37~C, another 8U of T4 kinase were added. After an .. .. ....... .

additional 30 min at 37~C, 45f~1 (990 pmoles) of fragment 1 that had previously been 5~-phosphorylated in a similar manner were added. After adding 22 ~ of 2 M sodium acetate and 8~1 of 250mM EDTA, the solution was heated for 5 min at 65 C to inactivate the T4 kinase. Fragments 3 through 6 were then added (2.6,~1, 902 pmoles; 8 ~ , 1008 pmoles; 32~1, 1003 pmoles, 45 ~ , 936 pmoles, respectively). The solution was vortexed, 680 ~ of cold ethanol were added and the solution was placed at -80~C
for 20 min. The pellet was then centrifuged at 12,800 RPM
for 10 min, decanted, washed with cold ethanol twice and dried.
To anneal the pieces, 18~1 of water were added to dissolve the pellet, the mixture heated in added to dissolve the pellet, the mixture heat in boiling water and cooled to room temperature slowly (~10 min). At this point were added 3 ~1 of 10xKB-TS, 3~1 of 10mM ATP and 3 ~1 of T4 DNA ligase (New England Biolabs; 40,000 units per ml). After 30 min at 14~C, the solution was dried and purified on a 10% denaturing polyacylamide gel as described above for fragment 1. (Yield ~75 pmoles.) c. Synthesis of DNA on a 2'-nitrobenzyluridine control pore glass support.
The 5'-dimethoxytrityl 2'-nitrobenzyluridine derivative of control pore glass (long chain alkylamino;
Pierce Chemical Company) was prepared by Cruachem, Bend Oregon, according to Gough, et al., Tetrahedron Lett.
(1981) 22:4177. Oligonucleotide synthesis was carried out on an automatic synthesizer (Warner et al., DNA 3, in press).
The 2'-nitrobenzyl functionality was removed by U.V. irradiation as described by Ohtsuka, et al., Nucleic Acids Res. (1974) 1:1351, except that a 2100 watt mercurv bulb was employed. Five min irradiation in a Pyrex*
(*) Trademark ~ -31- 1340231 cuvette was used for all samples (~14.5~moles of 2'-nitrobenzyluridine).
A sequence corresponding to 5' TTCCATGGCTGCTAGGC
TGTACTGCCAACTGGATCCTTCGCGGGACGTCcTTTGTTTAcGru 3' (fragment 7) was produced in this manner and used for the coupling described below.

II. Attachment of DNA by the 3' end to solid supports.
a. Synthesis of thiosemicarbazido control pore glass (TSC-CPG).
Isothiocyanate control pore glass (Pierce Chemical) was modified with hydrazine to yield the thiosemicarbazido derivative as follows. 400 mg of isothiocyanate CPG was placed in a 50 ml round bottom flask. 25 ml of dimethylsulfoxide, 200f~1 of distilled pyridine and 500~1 of a 0.6% hydrazine in dimethylsulfoxide solution were added (see, for example, J. 8auman, et al., J. of Histochem. and Cytochem. (1981) 29:227). After 18 hr with occasional mixing in the dark, the support was washed with 50 ml each of dimethylsulfoxide, pyridine, methanol and 2 L of 0.01 M
Tris, pH 7.5.
b. Attachment of fragment 7 to the solid support.
Approximately 2000 pmoles of fragment 7 was dried from water by evaporation. To this was added lO0 i of ~3 P-ATP (New England Nuclear), 2 ~ of lOxRB (0.5 M Tris HCl pH 7.8, lO0 mM MgC12, lO0 mM DDT), l ~ (8U) T4 kinase (New England Nuclear). After 30' at 37~C, the solution was diluted to l ml with gel elution buffer and SEP-PAK*de-salted as described above. Fragment 7 (20/~1, 982 pmoles) was treated with 20 ~ of l mM sodium periodate (Sigma) in 0.01 M Tris-HCl, pH 8.0 for 1 hr at 0~C in the dark. To this was added lO mg of TSC-CPG in 100fL1 of 0.1 M sodium acetate, pH 5.6 and the mixture (*) Trademark -32- 134023i allowed to set for 1 hr at 0~C in the dark, and then at 4~C overnight.
In order to block the remaining thiosemicarbazido functionalities, periodate oxidized ATP
was used. A 20 ~ sample of 100 mM ATP was treated with 20 mg of sodium periodate in 100~1 of 0.01 M Tris-HCl, pH
8Ø After 1 hr in the dark, 45~1 of the solution was added to the 10 mg of fragment 7-TSC-CPG in 150~1 of 0.lM
sodium acetate. After 6 hr at 4~C, the support was washed extensively with 4x standard sodium citrate (SSC).
Based on the incorporated counts, 13~ of fragment 7 (128 pmoles) were attached to the glass support.

III. Attachment of 5' ends of DNA to solid supports.
a. Preparation of bromoacetyl control pore glass (BA-CPG).
Synthesis of O-bromoacetyl N-hydroxysuccinimide was carried out approximately as described by Cuatreacasas, J. Biol. Chem. (1974) 245:3059.
A mixture of 347 mg of bromoacetic acid and 345 mg of N-hydroxysuccinimide (Sigma) was made up in 20 ml of distilled dioxane. To this mixture was added 532 mg of dicyclohexylcarbodiimide. After 70 min of shaking at room temperature, the cloudy solution was filtered through glass-wool.
To 500 mg of long chain alkylamino control pore glass (Pierce Chemical; 0.1 meq/g) was added 10 ml of 0.10 M sodium phosphate, pH 7.6. The slurry was placed on ice and the O-bromoacetyl N-hydroxysuccinimide solution was slowly added. After 30' with occasional stirring, the BA-CPG was washed with 5L of 0.1 M NaCl.
The number of equivalents of bromoacetate on the support was determined with a 5',5'-dithiobis(2-nitrobenzoic acid) acid (DTNB) test (Butterworth, et al., ..

~ ~33~ 1340231 Arch. Biol. Biophysl. (1967) 118:716). Stock solutionscontaining 200 mg of DTNB in 50 ml of water and 114 mg of 2-mercaptoethylamine in 100 ml of water were prepared.
BA-CPG (10 mg) was reacted with lO~ul of the 2-mercaptoethylamine solution plus 500~u1 of 0.OSM sodiumphosphate at pH 8.0 for 10 min at room temperature. The solution was then tested with 100~1 of DTNB and the visible spectrum was recorded (E=1.36x104 mol lcm 1 at pH
8). A control was run with 2-mercaptoethylamine without BA-CPG. From the amount of 2-mercaptoethylamine lost upon treatment with BA-CPG, it was determined that BA-CPG
contained 10 mmoles bromoacetate per mg.
b. 5' attachment of DNA to BA-CPG.
To 10~1 (333 pmoles) of fragment 3 (see above) was added 10 ~1 of 3- S-ATP (adenosine 5'-0-(3-thiotriphosphate; 0.25mCi, New England Nuclear)), 2-5~1 of lOxKB and 1~1 (8U) of T4 polynucleotide kinase. After 30 min at 37 C, 1~1 of 50mM 3-S-ATP (lithium salt; P.-L.
Biochemicals) and 1 ~1 (8U) T4 kinase were added. After an additional 30min at 37~C, the fragment was gel isolated as described above (yield 266 pmoles). Samples were counted on a Beckman*LS7000 liquid scintillation counter in Atomlite (New England Nuclear).
A 5 mg sample of BA-CPG was washed by centrifugation 3 times with water and 2 times with O.lOM
sodium phosphate, pH 7.6. The 5'thiophosphate fragment 2 was dissolved in 100 ~-1 of the phosphate buffer and added to the washed BA-CPG. The slurry was mixed by rotation on a rotary e~aporator for 2 hr at room temperature. The solution was decanted and discarded. In order to block the remaining bromoacetate functionalities, the support was treated with 200~1 of 50mM sodium phosphate, pH 8.0 and 50~1 of 2-mercaptoethanol for an additional 2 hr.
Subsequently the solution was decanted and the support was (*) Trademark ~34~ 134023~

extensively washed with 4XSSC (yield ~10 pmoles per mg of CPG).

IV. Synthesis of Horseradish Peroxidase-DNA Conjugates.
a. Purification by Elutip*.
Horseradish peroxidase (HRP) (2 mg; Type VI, Sigma; lO,OOOU/38 mg) was dissolved in 0.5 ml of O.lM
sodium phosphate buffer, pH 7.5. O-bromoacetyl N-hydroxysuccinimide (15~1) was added to the above solution and reaction allowed to proceed for 30 min at room temperature. The solution was passed over a PD-10 Sephadex*G-25M column (Pharmacia) that had previously been equilibrated with 30 ml of 0.1 M sodium phosphate, pH 7.5.
The brown fraction (1-1.2 ml) was collected. Fragment 8 (5~ to 3', GGTATTGTTGAACAATGTTGTACTTCTATTTG) that had previously been 5'-thiophosphorylated with 3'-35S-ATP as described above and dried (30 pmoles) was taken up in 50 ~1 of the phosphate buffer. To this thiophosphorylated fragment 8 solution was added the functionalized HRP and the mixture allowed to set for 30 min at room temperature.
The mixture was passed over an Elutip-d (Schleicher and Schuell) column. The peroxidase-DNA conjugate is eluted in the void volume (26% of the counts were recovered). A
control experiment conducted as described above but using 5'-32P-phosphate labeled fragment 8 showed less than 0.5%
of the counts were e~uted under these conditions.
b. Separation by gel.
A peroxidase conjugate of fragment 9 (5' to 3', TTGAAGAACTACG(~lllGllGTCll~lLlCAGAAAGGACTTGCACAAGACCCAAACC) was produced as above except that 360 pmoles of bromoacetyl horseradish peroxidase was combined with 156 pmoles of fragment 9 in 120 ~ of 0.025M sodium phosphate, pH 7.5. Instead of passing over an Elutip-d*column, the mixture was evaporated to dryness, suspended in 1~1 of 75~ glycerol, lO~ul of H20 and 1~1 of 1% bromophenol ~(*) Trademark blue. This material was then run on a 10% native protein gel. (Lindle, et al., Methods in Enzymol. (1983) 92:309).
A control experiment with 5'- P-phosphate fragment 9 was also run. The enzyme-DNA conjugate was well resolved from the unconjugated peroxidase as a faster running 35S-labeled species. The gel was stained with 100 ml of 10 mM
Tris-HCl, pH 7.5, 50 mM NaCl, 60~1 of 30% H202 to which was added 60 mg of 4-chloro-1-naphthol dissolved in 20 ml of cold methanol. Since this stain is based on the horseradish peroxidase activity, it was possible to show that the peroxidase-DNA conjugate was itself active. No new active species was produced with the 32P-fragment 9 control.
c. Hybridization of DNA-peroxidase to complementary DNA.
5'-Thiophosphorylated fragment 11 (S' to 3', CCAAGAGCTGGTCAAATCTTGAAGCAAACCTACGACAAGTTCGACACCAACATG
AGATCTGACGACGCTTTG) was 32P-labeled as above. A 10%
excess of fragment 12 (with reference to fragment 11) was added to the reaction mixture of S'-thiophosphate fragment 11 plus bromoacetyl peroxidase. The solution was heated to 60~C for 3 min and cooled to room temperature. A
control was performed with fragment 12 plus bromoacetyl enzyme without S'-thiophosphate fragment 11. A gel was run as before and the reaction mixture of fragment 11 plus bromoacetyl enzyme was run as a standard. The lane of fragment 12 plus enzyme and fragment 11 revealed a new slower running band (relative to the fragment 11-peroxidase conjugate) that contained 32p label. This band was also positive for peroxidase activity. No enzyme positive-32P labeled band was found for the fragment 12-peroxidase control.

v. Assay.
The fragments in this assay represent a model system and comprise a portion of the Hepatitis B virus genome extending about 60 bases in the 5'- (fragment 3) and 3'-direction (fragment 2) from the BamHI site at base No. 1403 of HBV DNA. The analyte, fragment 4, is com-plementary to the 3' end of fragment 3 and the 5' end of fragment 2. The solid-supported fragment 3 was produced as described in Section IIIb. Fragment 2 was 5' labeled with ~32P-ATP according to the initial part of Section IIb ( as applied to fragment 7).
a. Hybridization, Probe Capture.
Approximately 3 pmoles of a suspension of solid-supported fragment 3 (0.3 mg) and S pmoles of 32p_ fragment 2 (in lO~Kl H20) were used in each experiment.
Appropriate quantities of reagents (see Table 1 below) were added to give a final volume S0-100/~1, heated to 90~C and cooled to room temperature over a 1 hr period.
After washing with 4 x SSC at room temperature, samples of the solid support were Chernenkov*counted on a Beckman LS7000 liquid scintillation counter.

Table 1 pmole of ~l of Fragment 4,~1 20 x SCC ~1 H20CPM Bound A 0,0 8 20 31,260 B 1,10 8 10 132,293 C O.S,S 8 lS 113,039 b. Restriction Cleavage Release.
Typically, the solid support after probe-capture as described above is washed with BamHI buffer (20 mM Tris-HCl), pH 8.0, 7 mM MgC12, 100 mM NaCl, 2 mM 2-mercaptoethanol) twice and resuspended in 20 ~1 of the (*) Trademark , _ ~37~ 1340231 same buffer. To this 1~1 of BamHI (BRL; 5000 Units/715 ~1) is added and mixed. After 30 min incubation at 37~Cthe supernatant and one water wash are removed from the tube containing the settled solid support and counted. An additional 20~1 of buffer and 2~1 of enzyme are then added and left overnight at 37~C. The supernatant and one water wash are counted as before. Controls without enzyme are also run.

Table 2 Initial CPM CPM released CPM released on Support after 30 min after 18 h Sample 1 w/enzyme 69660 2064 10513 Sample 1 w/o enzyme 67353 536 2524 (control) Sample 2 w/enzyme 34982 1848 6336 Sample 2 w/o enzyme 44113 504 2191 (control) 2 VI. Preparation of PCL (Periodate-Cleavable Linker) O,O-Dibenzoyl tartaric acid monohydrate (18.8 g, 50 mmole) was dissolved in 250 ml CH3CN and the solvent was removed in vacuo. This process was repeated. The resulting oil was dissolved in 250 ml THF and dicyclohexylcarbodiimide (DCC) (10.6 g, 50 mmole) dissolved in 50 ml THF was added. A precipitate started forming in a few minutes. After stirring for 18 hr at 20~C the reaction mixture was filtered, and the precipitate washed with a little THF. The precipitate was then dried in high vacuum to give 10.8 g (100~, 50 mmole) dicyclohexylurea (DCHU). To the combined filtrate was added 2-(N-methyl)aminoethanol (4.0 ml, 50 mmole) and the reaction mixture was stirred for 1 hr at 20~C. Then, DCC
(10.6 g, 50 mmole) in 50 ml THF was added; a small .......

-38- 13~231 precipitate was formed. After about 1 hr, 2-(N-methyl)aminoethanol (4.0 ml, 50 mmole) was added and the reaction mixture stirred for 18 hours at 20~C.
The formed precipitate was filtered often and washed with a little THF. The dried precipitate of DCHU
weighed 10.8 g (100%). The combined filtrate was evaporated to an oil; chromatography on silica afforded 8 g (17 mmole) O,O-dibenzoyl tartaric di(N-methyl-2-hydroxyethyl)amide (1). The product was eluted with 6%
10 MeOH/CH2C12.
To (1) (8.6 mmole) in S0 ml CH2C12 containing dimethylamino pyridine (DMAP) (0.11 g) and triethylamine (TEA) (2.4 ml) was added dropwise, DMT-Cl (8.6 mmole) dissolved in 50 ml CH2C12. After addition of DMT-Cl, the reaction mixture was stirred for 1 hr at 20~C, and the solvent was removed by evaporation. The residue was dissolved in 600 ml ethyl acetate and the organic phase washed with 400 ml 5% NaHCO3 and 400 ml 80% saturated aqueous NaCl. The organic phase was dried over solid Na2SO4. After 30 min, the Na2SO4 was filtered off, and the supernatant was concentrated to an oil and then coevaporated with toluene and CH3CN.
The crude material was subjected to silica gel chromatography using n-butanol/CH2C12 for elution. The pure mono-DMT product eluted with 2-3% n-butanol/CH2C12 to give 1.53 g (2 mmole) of O,O-dibenzoyl tartaric 2-(O-dimethoxytrityl)hydroxyethyl-N-methyl,N-methyl-2-hydroxyethyldiamide.
This material was dissolved in 20 ml CH2C12 containing diisopropylethylamine (DIPEA) (3 mmole). After cooling to 10~C, 2.2 mmole methoxy-N,N-diisopropylamino chloro phosphine was added under argon. After 15 min, ethyl acetate was added, and the organic phase washed with 80~ saturated aqueous NaCl, dried over solid Na2SO4, and evaporated to dryness. After coevaporation with toluene .
1~40231 and dry CH3CN, the residue was dissolved in 10 ml dry CH3CN. Thi~ solution was aliquoted into l9 dry Weaton~
vials and the solvent removed in ~acuo. The vials were closed with septum screw caps and stored at -20~C.
The DMT-PCL-phosphoramidite was coupled to oligonucleotides using standard techniques. The following oligonucleotide was synthesized:

O O
5'-HO[BLA3c]O-~-O[PCL]O-~-O[T~LLA2'TT]-OH-3 b- o~

In the foregoing structure, "PCL" represents the periodate-cleavable linkage ,CH3 H3C~
-CH2CH2N NCH2CH2-, COCH-CHCO
OH OH
"BLA3c" represents the nucleotide sequence (5' to 3') GATGTGGll~lCGTACTT, and "T'LLA2~TT" represents the nucleotide sequence (5' to 3') TTGACACGGGTCCTATGCCTAAT.
After complete deprotection the oligonucleotide was purified by PAGE, the product band excised, eluted with MG
. buffer and desalted using a SEP-PAKD cartridge as described by Sanchez-Pescador et al., DNA 3:339-343 (1984).
Degradation was performed as follows:
-0.6 OD purified material in 6 A H2O was treated with 50 A 0.1M Na I04 and left for 1 hr at 20~C. 0.4 ml of a solution of glycerol in H20 (1~~/ml) was added. The combined solution was passed through a PD-10 column (Sephadex~G25), equilibrated with 0.lM triethylammonium acetate (TEAA) and eluted with the same buffer. 0.5 ml (*) Trademark ~ ~ .

~40- 1340231 fractions were collected and pooled, and the solvent removed in a Speed-Vac~. Analysis by 15% PAGE showed complete degradation of starting material into two new bands of the expected sizes.

VII. Sodium Periodate Release.
A. A 32P-labeled probe was prepared as described by Urdea et al. in Nucleic Acids Research 16 (1988), cited in the preceding section. The probe had the sequence (5' to 3') AAGTACGACAACCACATCGGATGACCTCGGATCGA
CCT*T-32P where * is the modified nucleotide represented by the structure CH

fH2 S
~ + Fluorescein - NHCNH(CH2)5CO~NHS
N~ ~ or ~ ~
O N Long chain biotin NHS
~ (from Pierce Chemical) O ~ OPO3 HO
as described in EP Publication No. 0225807, supra. A
synthetic oligonucleotide having the sequence (5' to 3') GATGTGGllGlCGTACllCllCTTTGGAGAAAGTGGTG was used as analyte. Microtiter capture wells were prepared using two different probes: (1) *CACCACTTTCTCCAAAGAAG (designated XTl~lca in Table 3, below); and (2) *TT-X-CACCACTTTCTCCAAAGAAG (* represting the alkylamine nucleotide above), where X represents the periodate-cleavable linkage as described in the preceding section, using the following procedure. The wells were prepared .
(*) Trademark from the Immulo~ II strips by adding, to each well, 200~1 of a 200~g/ml solution of poly-phenylalanyl-lysine (Sigma Chemical Inc.) in water. The covered strips were left at room temperature for 30 min to 2 hr, then washed as above.
A 10 OD260 sample of the oligonucleotide of 3B above in 60 ~1 of 1 x PBS was treated with 140~1 of DMF containing 10 mg of ethylene glycolbis(succinimidylsuccinate) (Pierce Chemicals Inc.). The mixture was vortexed and incubated in the dark at room temperature. After 15 min, the solution was passed over a Sephade~ G-25 column (PD-10 from Pharmacia), previously equilibrated with 30 ml of 1 x PBS. The void volume of the column was diluted to a final volume of 3S ml with 1 x PBS. To each well, a 50~1 aliquot of the capture probe solution was added. After covering with plastic wrap, the wells were incubated at room temperature in the dark for 30 min to overnight. The wells were not overcoated with hybridization mix.
Stock solutions containing 1 fmole, 100 amoles and 10 amoles of the analyte fragment were prepared in a hybridization buffer containing 4X SSC. A control solution was similarly prepared, containing no analyte.
Four sets of wells were then set up. To each well was added 40~1 of the selected solution, i.e., containing either 1 fmole, 100 amoles, 10 amoles or no analyte.
Hybridization was carried out at 55~C in a water bath for 1 hr.. Tubes were capped and wells were sealed with an adhesive Linbro~Titertek*membrane. After washing twice with 380~1 of 4X SSC, an additional 40~1 of 4X SSC
containing 10 fmoles of 32P-labeled probe was added. The wells were incubated for 1 hr, at 37~C, at which point they were again washed twice with 380~1 of 4X SSC.
Total counts P were then evaluated using an LKB 1214 Rackbeta*scintillation counter. Results are set forth under ~'Total Counts" in Table 3.

(*) Trademark . .

1~40231 To one set of wells containing the periodate-cleavable polynucleotide and one set of wells containing XTl*lca solution was added 100 ~l of 100 mM NaI04 in 4X
SSC. The wells were incubated at room temperature for one minute. The periodate solution was transferred to clean wells and counted. Results are set forth in the columns entitled "100 mM NaI04" and "4X SSC" in Table 3.
As a control, 100 ~l of 4X SSC was added to one set of wells containing the periodate-cleavable polynucleotide. The wells were then incubated at room temperature for one minute and the solution was transferred to clean wells. The transferred solution was counted and results are tabulated under the heading "XTl*lca wells" in Table 3.
Table 3 Analyte T o t a 1 1 0 0 m M XTl*lca Amount Counts NaIO44X SSC wells 1 fm 8378.80 563.4971.79 53.84 3440.30 465.7429.91 43.87 3368.20 638.2923.93 41.88 100 am 3130.60 53.8443.87 57.83 5661.70 66.8182.76 48.87 3068.50 47.8626.92 23.93 10 am 3119.60 17.9436.89 35.90 7161.20 52.8554.84 36.9 2408.61 20.9417.94 34.9 zero 3133.49 34.90129.64 32.91 5729.60 38.8922.93 43.88 3613.92 14.9532.90 58.84 S/N Ratios:
No ReleaseRelease 1 fmole 1.2 +/- 0.8 18.8 +/- 8.6 100 amoles 1.0 +/- 0.5 1.9 +/- 0.9 10 amoles 1.0 +/- 0.7 1.0 +/- 0.8 ~43~ 13 40 2 31 B. In a second experiment, the aforementioned procedure was repeated with the following variations: (1) the probe used was a 32P-labelled l9-mer having the sequence *CGTGTCAGGCATAGGACC (5' to 3', * as above); (2) the "analyte'~ was a synthetic oligonucleotide having the sequence GGTCCTATGCCTGACACGCTTCTTTGGAGAAAGTGGTG; (3) one analyte concentration was evaluated rather than three (1 fmole)i and (4) 100 fmoles rather than 10 fmoles 32p_ labelled probe were used. Results are summarized in Table 4.

Table 4 Anal yte Tot al lOOmM Na IO4 Amount counts in 4X SSC 4X SSC
1 fm 1885.95 582.44 93.97 1963.40 581.44 97.72 2130.60 346.05 119.66 1877.20 1692.90 1666.98 zero 710.60 50.85 64.81 762.70 36.89 39.88 731.60 31.91 55.84 1030.35 554.52 892.67 S/N Ratios:

No Release Release fmole 2.40 +/- 0.55 12.6 +/- 4.59 .

VIII. Strand Replacement.
An alkaline phosphatase probe was prepared as described by Urdea et al., Nucleic Acids Research 16, supra. The probe had the sequence (5' to 3') S AAGTACGACAACCACATCGGATGACCTCGGATCGACCT*T with * as above.
A synthetic oligonucleotide having the sequence (5' to 3') GATGTGGTTGTCGTACTTCTTCTTTGGAGAAAGTGGTG was used as analyte. A second synthetic oligonucleotide was prepared having the sequence CTTCTTTGGAGAAAGTGGTGTTCATAGAGAAACGATAT
ATAGAGACACGATATAGGGATA and was used as the specific release strand, i.e., the replacement strand enabling label release as discussed above. Plates were made using an oligonucleotide having the sequence *TATCCCTATATCGTGT
CTCTATATATCGTTTCTCTATGAACACCAC~llClCCAAAGAAG as capture probe. Wells were prepared as described in the preceding section, except that Microlite*I wells (Dynatech~ were used and, after the final incubation step, the wells were washed with lx PBS, then coated with H buffer, and washed again.
Three sets of capture wells were set up, each set having an analyte-containing well and a control well, i.e., containing no analyte. To each well was added 40~1 4X SSC with either 1 fmole or no analyte. Hybridization was carried out at 55~C for 1 hour. After washing twice with 380,~1 of 4X SSC, 40~1 of 4X SSC containing 100 fmoles of alkaline phosphatase probe were added to the wells. The wells were incubated at 37~C for 1 hr, at which point washing was carried out (1) twice with 380~1 of buffer containing O.lX SSC and 0.1% SDS, and (2) twice with 380~ul of 4X SSC.
Alkaline phosphatase activity was measured ~y incubation of the samples with dioxetane, a chemiluminescent substrate. Luminescence was recorded using a microtiter dish reading luminometer (Dynatech).
(*) Trademark 1~40231 To one set of wells, 20~1 4X SSC were added, followed by incubation at 37~C for 1 hr. 20~1 dioxetane were added, and the wells were again incubated at 37~C for 1 hr. Alkaline phosphatase activity was measured, and results are set forth in Table 5 under the heading ~'No Transfer".
20~1 4X SSC were added to a second set of wells, which were then incubated at 37~C for 1 hr. The individual solutions were transferred to Microlite I
wells, and 20~1 dioxetane was added. The wells were again incubated at 37~C for 1 hr. Alkaline phosphatase activity was measured as above, and results are tabulated in Table 5 under "SSC Release".
20~1 4X SSC were added to the third set of wells containing 30 pmoles specific release oligonucleotide. The wells were incubated at 37~C for 1 hr, and the solutions were then transferred to Microlite I
wells. 20~1 dioxetane were added, and the wells were again incubated at 37~C for 1 hr. Alkaline phosphatase activity was measured as above, and results are set forth in Table 5 under "Oligo Release".

-46- 13~02'~1 Table 5 Analyte No SSC Oligo Amount Transfer Release Release 1 fm 16.26 0.47 9.52 14.66 0.56 9.89 15.76 0.55 10.42 zero 0.33 0.04 0.08 , 0-33 0 04 ~.~S
0.28 0.04 0-07 S/N Ratios:
No SSC Oligo Transfer Release Release 1 fmole 49.66 +/- 13.17 +/-149.15 +/-5.27 1.23 34.84 2 5 B. The experiment of Section VIII A was repeated, with results set forth in Table 6.

.... . . . .

Table 6 Analyte No SSC Oligo Amount Transfer Release Release 1 fm 11.82 0.20 4.05 12.39 0.18 5.02 12.72 0.19 4.79 zero 0.98 0.07 0.10 1.09 0.06 0.11 1.11 0.08 0.10 S/N Ratios:
No SSC Oligo Transfer Release Release 1 fmole 11.6 +/- 2.7 +/- 46.2 +/-0.88 0.4 6.9 It is evident from the above results, that the subject method provides for a simple, rapid and accurate approach for detecting specific polynucleotide sequences from diverse sources. The method provides for high sensitivity and great flexibility in allowing for differ-ent types of labels which involve detectable signals which have been employed in immunoassays. Thus, the subject method can be readily adapted to use in conventional equipment for immunoassays which are capable of detecting radioactivity, light adsorption in spectrophotometers and light emission in fluorometers or scintillation counters.
The subject method is applicable to any DNA sequence and -48- 134~2~1 can use relatively small probes to reduce false positive and minimize undesirable heteroduplexing. By cleavage of the label from the support for measurements, background values can be greatly reduced, since the reading can occur away from the support. Also, there is a further redirection in background due to the necessity to cleave the label from the polynucleotide chain. The subject method can therefore provide for the accurate and economi-cal determination of DNA sequences for diagnosing disease, monitoring hybrid DNA manipulations, determining genetic traits, and the like.
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications maybe practiced within the scope of the appended claims.

Claims (11)

1. A method for detecting the presence of an oligonucleotide sequence of interest in a nucleic acid analyte present in a nucleic acid sample, said methodcomprising:
combining under hybridizing conditions in an aqueous medium, said nucleic acid sample with a polynucleotide reagent, where one of said sample or acomponent of said reagent is bound to a support and hybridization of said analyte and said polynucleotide reagent results in a label being bound to said support through a selectable cleavage site;
separating said support having bound polynucleotide reagent and nucleic acid analyte from said aqueous medium;
washing said support with a medium of different hybridizing stringency from said aqueous medium to remove label bound to said support other than through said selectable cleavage site;
cleaving said cleavage site; and detecting label free of said support.
2. A method according to claim 1, wherein said polynucleotide reagent comprises a first polynucleotide capture probe bound to a support and a second polynucleotide label probe, wherein said first and second probes have oligonucleotide sequences complementary to sequences present in said analyte to form duplexes therewith under said hybridizing conditions, at least one of said oligonucleotide sequences being a sequence of interest, wherein said capture probe contains saidselectable cleavage site.
3. A method for detecting the presence of an oligonucleotide sequence of interest in a nucleic acid analyte present in a nucleic acid sample, said methodcomprising:
combining under hybridizing conditions said nucleic acid with a first polynucleotide capture probe and a second polynucleotide label probe, wherein said first and second probes have oligonucleotide sequences complementary to sequences present in said analyte so as to form first and second duplexes therewith under said hybridizing conditions, wherein said capture probe is boundto a solid support;
introducing a replacement polynucleotide strand selected so as to form a duplex with said capture probe that is more stable than said first duplex, thereby substantially freeing said support of label bound to said support; and detecting label free of support.
4. An article useful for detecting the presence of an oligonucleotide sequence of interest in a nucleic acid analyte present in a nucleic acid sample,comprising a single stranded polynucleotide bound proximal to one end to a support and at its opposite end having a sequence complementary to said sequence of interest, said polynucleotide containing in addition a selectable cleavage site, wherein said cleavage site (a) is chemically cleavable; (b) is other than a restriction enzyme cleavable site; (c) is other than a phosphodiester linkage; and (d) provides for a complete break between adjacent nucleotides in the polynucleotide reagent upon cleavage.
5. A polynucleotide reagent having the structure where DNA, is a first strand of DNA, DNA2 is a second strand of DNA, and X
comprises a selectable cleavage site, wherein said cleavage site X (a) is chemically cleavable; (b) is other than a restriction enzyme cleavable site; (c) is other than a phosphodiester linkage; and (d) provides for a complete break between adjacent nucleotides in the polynucleotide reagent upon cleavage.
6. The polynucleotide reagent of claim 5, wherein X comprises a linkage cleavable by OH-, hydroxylamine, SH-, or periodate.
7. The polynucleotide reagent of claim 5, wherein X comprises a linkage selected from the group consisting of , , , -S-S-, and .
8. The polynucleotide reagent of claim 5, wherein X contains the moiety .
9. The polynucleotide reagent of claim 5, wherein X is
10. A reagent useful in polynucleotide synthesis, given by the structure Wherein R1 is an acid-sensitive, base-stable protecting group, and R2 is selected from the group consisting of H, phosphoramidite, phosphotriester, phosphodiester, phosphite, H-phosphonate and phosphonothioate.
11. The reagent of claim 10, wherein R1 is DMT and R2 is where DMT is mimethoxy and iPr is isopropyl.
CA000579309A 1984-10-16 1988-10-04 Polynucleotede dertermination with selectable cleavage sites Expired - Fee Related CA1340231C (en)

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EP88309203A EP0360940B1 (en) 1988-09-29 1988-10-03 Polynucleotide determination with selectable cleavage sites
ES88309203T ES2083955T3 (en) 1988-09-29 1988-10-03 DETERMINATION OF POLYNUCLEOTIDES WITH SELECTABLE SPLIT SITES.
CA000579309A CA1340231C (en) 1988-09-29 1988-10-04 Polynucleotede dertermination with selectable cleavage sites
JP63250726A JP2676535B2 (en) 1988-09-29 1988-10-04 Measurement of polynucleotides using selectable cleavage sites
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Families Citing this family (243)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5258506A (en) * 1984-10-16 1993-11-02 Chiron Corporation Photolabile reagents for incorporation into oligonucleotide chains
US5430136A (en) * 1984-10-16 1995-07-04 Chiron Corporation Oligonucleotides having selectably cleavable and/or abasic sites
US5714380A (en) 1986-10-23 1998-02-03 Amoco Corporation Closed vessel for isolating target molecules and for performing amplification
US5720928A (en) * 1988-09-15 1998-02-24 New York University Image processing and analysis of individual nucleic acid molecules
US6147198A (en) * 1988-09-15 2000-11-14 New York University Methods and compositions for the manipulation and characterization of individual nucleic acid molecules
US5324829A (en) * 1988-12-16 1994-06-28 Ortho Diagnostic Systems, Inc. High specific activity nucleic acid probes having target recognition and signal generating moieties
US6610256B2 (en) 1989-04-05 2003-08-26 Wisconsin Alumni Research Foundation Image processing and analysis of individual nucleic acid molecules
US5102784A (en) * 1990-05-04 1992-04-07 Oncor, Inc. Restriction amplification assay
US5677440A (en) * 1990-07-16 1997-10-14 Howard Florey Institute Of Experimental Physiology And Medicine Oligonucleotide-polyamide conjugates
EP0529070A1 (en) * 1991-02-27 1993-03-03 Amoco Corporation Methods for improving the sensitivity of hybridization assays
CA2110591A1 (en) * 1991-06-17 1992-12-23 Michael S. Urdea Polynucleotide determination with selectable cleavage sites
US5294534A (en) * 1991-08-13 1994-03-15 Miles, Inc. Amplification method for polynucleotide assays
US6872816B1 (en) * 1996-01-24 2005-03-29 Third Wave Technologies, Inc. Nucleic acid detection kits
US5846717A (en) * 1996-01-24 1998-12-08 Third Wave Technologies, Inc. Detection of nucleic acid sequences by invader-directed cleavage
US5994069A (en) * 1996-01-24 1999-11-30 Third Wave Technologies, Inc. Detection of nucleic acids by multiple sequential invasive cleavages
WO1993005184A1 (en) * 1991-09-10 1993-03-18 Love Jack D Dna/rna target and signal amplification
US6652808B1 (en) * 1991-11-07 2003-11-25 Nanotronics, Inc. Methods for the electronic assembly and fabrication of devices
US5632957A (en) * 1993-11-01 1997-05-27 Nanogen Molecular biological diagnostic systems including electrodes
US6017696A (en) 1993-11-01 2000-01-25 Nanogen, Inc. Methods for electronic stringency control for molecular biological analysis and diagnostics
US6569382B1 (en) * 1991-11-07 2003-05-27 Nanogen, Inc. Methods apparatus for the electronic, homogeneous assembly and fabrication of devices
GB9207380D0 (en) * 1992-04-03 1992-05-13 Ici Plc Compounds
GB9207381D0 (en) * 1992-04-03 1992-05-13 Ici Plc Synthesis of oligonucleotides
ES2168275T3 (en) * 1992-04-06 2002-06-16 Abbott Lab METHOD AND DEVICE FOR DETECTION OF NUCLEIC ACID OR ANALYTE THROUGH A TOTAL INTERNAL REFLECTION TECHNIQUE.
US5543292A (en) * 1992-06-16 1996-08-06 Hitachi, Ltd. Process for the measurement of nucleic acids
US5795714A (en) * 1992-11-06 1998-08-18 Trustees Of Boston University Method for replicating an array of nucleic acid probes
US6436635B1 (en) * 1992-11-06 2002-08-20 Boston University Solid phase sequencing of double-stranded nucleic acids
US5614402A (en) * 1992-12-07 1997-03-25 Third Wave Technologies, Inc. 5' nucleases derived from thermostable DNA polymerase
US6194144B1 (en) 1993-01-07 2001-02-27 Sequenom, Inc. DNA sequencing by mass spectrometry
WO1994016101A2 (en) 1993-01-07 1994-07-21 Koester Hubert Dna sequencing by mass spectrometry
US5605798A (en) * 1993-01-07 1997-02-25 Sequenom, Inc. DNA diagnostic based on mass spectrometry
ATE151816T1 (en) * 1993-01-08 1997-05-15 Hybridon Inc DETECTION OF SYNTHETIC OLIGONUCLEOTIDES EXTRACTED FROM BODY FLUIDS OR TISSUES:
US5679510A (en) * 1993-04-27 1997-10-21 Hybridon, Inc. Quantitative detection of specific nucleic acid sequences using lambdoid bacteriophages linked by oligonucleotides to solid support
JP2842758B2 (en) * 1993-05-10 1999-01-06 株式会社日立製作所 Automatic analyzer
US7314708B1 (en) * 1998-08-04 2008-01-01 Nanogen, Inc. Method and apparatus for electronic synthesis of molecular structures
US6331274B1 (en) 1993-11-01 2001-12-18 Nanogen, Inc. Advanced active circuits and devices for molecular biological analysis and diagnostics
US6225059B1 (en) 1993-11-01 2001-05-01 Nanogen, Inc. Advanced active electronic devices including collection electrodes for molecular biological analysis and diagnostics
WO1995016788A1 (en) * 1993-12-17 1995-06-22 Cubicciotti Roger S Nucleotide-directed assembly of bimolecular and multimolecular drugs and devices
US6986985B1 (en) 1994-01-13 2006-01-17 Enzo Life Sciences, Inc. Process for producing multiple nucleic acid copies in vivo using a protein-nucleic acid construct
US20050123926A1 (en) * 1994-01-13 2005-06-09 Enzo Diagnostics, Inc., In vitro process for producing multiple nucleic acid copies
US20110097791A1 (en) * 1999-04-16 2011-04-28 Engelhardt Dean L Novel process, construct and conjugate for producing multiple nucleic acid copies
US5484702A (en) * 1994-01-27 1996-01-16 Research Foundation Of The State University Of New York At Buffalo Method for preselecting recombinant clones containing a specific nucleic acid sequence and subsequent transformation with preselected clones
AU2187295A (en) * 1994-03-18 1995-10-09 General Hospital Corporation, The Cleaved amplified rflp detection methods
US6110709A (en) * 1994-03-18 2000-08-29 The General Hospital Corporation Cleaved amplified modified polymorphic sequence detection methods
US5831065A (en) * 1994-04-04 1998-11-03 Lynx Therapeutics, Inc. Kits for DNA sequencing by stepwise ligation and cleavage
US5714330A (en) * 1994-04-04 1998-02-03 Lynx Therapeutics, Inc. DNA sequencing by stepwise ligation and cleavage
US5552278A (en) * 1994-04-04 1996-09-03 Spectragen, Inc. DNA sequencing by stepwise ligation and cleavage
US6589736B1 (en) * 1994-11-22 2003-07-08 The Trustees Of Boston University Photocleavable agents and conjugates for the detection and isolation of biomolecules
US6593086B2 (en) 1996-05-20 2003-07-15 Mount Sinai School Of Medicine Of New York University Nucleic acid amplification methods
US20070269799A9 (en) * 1994-06-22 2007-11-22 Zhang David Y Nucleic acid amplification methods
US5942391A (en) * 1994-06-22 1999-08-24 Mount Sinai School Of Medicine Nucleic acid amplification method: ramification-extension amplification method (RAM)
USRE38442E1 (en) * 1994-06-22 2004-02-24 Mount Sinai School Of Medicine Nucleic acid amplification method hybridization signal amplification method (HSAM)
US5876924A (en) * 1994-06-22 1999-03-02 Mount Sinai School Of Medicine Nucleic acid amplification method hybridization signal amplification method (HSAM)
US5552471A (en) * 1994-08-17 1996-09-03 The Perkin-Elmer Corporation Solid support reagents for the synthesis of 3'-Nitrogen containing polynucleotides
US5580731A (en) * 1994-08-25 1996-12-03 Chiron Corporation N-4 modified pyrimidine deoxynucleotides and oligonucleotide probes synthesized therewith
US5597909A (en) * 1994-08-25 1997-01-28 Chiron Corporation Polynucleotide reagents containing modified deoxyribose moieties, and associated methods of synthesis and use
US5990300A (en) * 1994-09-02 1999-11-23 Andrew C. Hiatt Enzyme catalyzed template-independent creation of phosphodiester bonds using protected nucleotides
US5763594A (en) * 1994-09-02 1998-06-09 Andrew C. Hiatt 3' protected nucleotides for enzyme catalyzed template-independent creation of phosphodiester bonds
US5808045A (en) * 1994-09-02 1998-09-15 Andrew C. Hiatt Compositions for enzyme catalyzed template-independent creation of phosphodiester bonds using protected nucleotides
US6232465B1 (en) 1994-09-02 2001-05-15 Andrew C. Hiatt Compositions for enzyme catalyzed template-independent creation of phosphodiester bonds using protected nucleotides
US6214987B1 (en) * 1994-09-02 2001-04-10 Andrew C. Hiatt Compositions for enzyme catalyzed template-independent formation of phosphodiester bonds using protected nucleotides
US5872244A (en) * 1994-09-02 1999-02-16 Andrew C. Hiatt 3' protected nucleotides for enzyme catalyzed template-independent creation of phosphodiester bonds
USRE43097E1 (en) 1994-10-13 2012-01-10 Illumina, Inc. Massively parallel signature sequencing by ligation of encoded adaptors
US6013445A (en) * 1996-06-06 2000-01-11 Lynx Therapeutics, Inc. Massively parallel signature sequencing by ligation of encoded adaptors
US6974666B1 (en) 1994-10-21 2005-12-13 Appymetric, Inc. Methods of enzymatic discrimination enhancement and surface-bound double-stranded DNA
US5660979A (en) * 1994-11-04 1997-08-26 Akzo Nobel N.V. Detection of human retrovirus infection
US20030104361A1 (en) * 1997-09-29 2003-06-05 Susan Weininger Method of detection of nucleic acids with a specific sequence composition
US5871902A (en) * 1994-12-09 1999-02-16 The Gene Pool, Inc. Sequence-specific detection of nucleic acid hybrids using a DNA-binding molecule or assembly capable of discriminating perfect hybrids from non-perfect hybrids
EP1260593A3 (en) * 1994-12-23 2003-12-03 Dade Behring Inc. Detection of nucleic acids by nuclease-catalyzed product formation
EP0724015B1 (en) * 1995-01-12 2004-03-31 Toyo Boseki Kabushiki Kaisha Novel alkaline phosphatase, its production and use
US5559000A (en) * 1995-01-18 1996-09-24 The Scripps Research Institute Encoded reaction cassette
US6428955B1 (en) 1995-03-17 2002-08-06 Sequenom, Inc. DNA diagnostics based on mass spectrometry
EP0871640A4 (en) 1995-04-03 2002-04-17 Univ New York Methods for measuring physical characteristics of nucleic acids by microscopic imaging
US7775368B2 (en) * 1995-04-03 2010-08-17 Wisconsin Alumni Research Foundation Micro-channel long molecule manipulation system
US8142708B2 (en) * 1995-04-03 2012-03-27 Wisconsin Alumni Research Foundation Micro fluidic system for single molecule imaging
US20060063193A1 (en) * 1995-04-11 2006-03-23 Dong-Jing Fu Solid phase sequencing of double-stranded nucleic acids
US7803529B1 (en) 1995-04-11 2010-09-28 Sequenom, Inc. Solid phase sequencing of biopolymers
US5750341A (en) * 1995-04-17 1998-05-12 Lynx Therapeutics, Inc. DNA sequencing by parallel oligonucleotide extensions
US5830655A (en) 1995-05-22 1998-11-03 Sri International Oligonucleotide sizing using cleavable primers
US5700642A (en) * 1995-05-22 1997-12-23 Sri International Oligonucleotide sizing using immobilized cleavable primers
US6027879A (en) * 1995-08-09 2000-02-22 The Regents Of The University Of California Detection and isolation of nucleic acid sequences using a bifunctional hybridization probe
US6146854A (en) * 1995-08-31 2000-11-14 Sequenom, Inc. Filtration processes, kits and devices for isolating plasmids
US5747255A (en) * 1995-09-29 1998-05-05 Lynx Therapeutics, Inc. Polynucleotide detection by isothermal amplification using cleavable oligonucleotides
US6849398B1 (en) 1996-01-18 2005-02-01 The Scripps Research Institute Use of encoded reaction cassette
US6613508B1 (en) 1996-01-23 2003-09-02 Qiagen Genomics, Inc. Methods and compositions for analyzing nucleic acid molecules utilizing sizing techniques
US6027890A (en) * 1996-01-23 2000-02-22 Rapigene, Inc. Methods and compositions for enhancing sensitivity in the analysis of biological-based assays
CZ218498A3 (en) * 1996-01-23 1998-12-16 Rapigene, Inc. Processes and composition for determining molecule sequence of nucleic acids
US6312893B1 (en) 1996-01-23 2001-11-06 Qiagen Genomics, Inc. Methods and compositions for determining the sequence of nucleic acid molecules
WO1997027325A2 (en) * 1996-01-23 1997-07-31 Rapigene, Inc. Methods and compositions for analyzing nucleic acid molecules utilizing sizing techniques
EP0992511B1 (en) * 1996-01-23 2009-03-11 Operon Biotechnologies, Inc. Methods and compositions for determining the sequence of nucleic acid molecules
CN1515541A (en) * 1996-01-23 2004-07-28 ռ�˹ Method for detecting ligand pair combination by using non-fluorescent marker and its composite
US6875572B2 (en) 1996-01-24 2005-04-05 Third Wave Technologies, Inc. Nucleic acid detection assays
US7195871B2 (en) * 1996-01-24 2007-03-27 Third Wave Technologies, Inc Methods and compositions for detecting target sequences
US7432048B2 (en) * 1996-11-29 2008-10-07 Third Wave Technologies, Inc. Reactions on a solid surface
US6706471B1 (en) * 1996-01-24 2004-03-16 Third Wave Technologies, Inc. Detection of nucleic acid sequences by invader-directed cleavage
US20080160524A1 (en) * 1996-01-24 2008-07-03 Third Wave Technologies, Inc. Methods and Compositions for Detecting Target Sequences
US7122364B1 (en) * 1998-03-24 2006-10-17 Third Wave Technologies, Inc. FEN endonucleases
US7527928B2 (en) * 1996-11-29 2009-05-05 Third Wave Technologies, Inc. Reactions on a solid surface
AU731062B2 (en) * 1996-01-24 2001-03-22 Third Wave Technologies, Inc. Invasive cleavage of nucleic acids
US6090606A (en) * 1996-01-24 2000-07-18 Third Wave Technologies, Inc. Cleavage agents
US5985557A (en) * 1996-01-24 1999-11-16 Third Wave Technologies, Inc. Invasive cleavage of nucleic acids
EP0885236A1 (en) * 1996-01-26 1998-12-23 Codon Pharmaceuticals, Inc. Oligonucleotide analogs
US5824478A (en) * 1996-04-30 1998-10-20 Vysis, Inc. Diagnostic methods and probes
US20040266706A1 (en) * 2002-11-05 2004-12-30 Muthiah Manoharan Cross-linked oligomeric compounds and their use in gene modulation
CA2260361A1 (en) * 1996-07-08 1998-01-15 Burstein Laboratories, Inc Cleavable signal element device and method
US6342349B1 (en) * 1996-07-08 2002-01-29 Burstein Technologies, Inc. Optical disk-based assay devices and methods
US6780982B2 (en) * 1996-07-12 2004-08-24 Third Wave Technologies, Inc. Charge tags and the separation of nucleic acid molecules
US5858665A (en) * 1996-07-25 1999-01-12 Navix, Inc. Homogeneous diagnostic assay method utilizing simultaneous target and signal amplification
US5853990A (en) 1996-07-26 1998-12-29 Edward E. Winger Real time homogeneous nucleotide assay
US6352827B1 (en) * 1996-08-28 2002-03-05 President And Fellows Of Harvard College Detection of multiple nucleic acid sequences in a fluid sample
US5777324A (en) 1996-09-19 1998-07-07 Sequenom, Inc. Method and apparatus for maldi analysis
WO1998020020A2 (en) 1996-11-06 1998-05-14 Sequenom, Inc. High density immobilization of nucleic acids
US7285422B1 (en) * 1997-01-23 2007-10-23 Sequenom, Inc. Systems and methods for preparing and analyzing low volume analyte array elements
US5900481A (en) * 1996-11-06 1999-05-04 Sequenom, Inc. Bead linkers for immobilizing nucleic acids to solid supports
DE69738206T2 (en) 1996-11-06 2008-07-17 Sequenom, Inc., San Diego DNA diagnostics by mass spectrometry
US6024925A (en) 1997-01-23 2000-02-15 Sequenom, Inc. Systems and methods for preparing low volume analyte array elements
US6140053A (en) * 1996-11-06 2000-10-31 Sequenom, Inc. DNA sequencing by mass spectrometry via exonuclease degradation
US6133436A (en) 1996-11-06 2000-10-17 Sequenom, Inc. Beads bound to a solid support and to nucleic acids
EP2055781B1 (en) 1996-11-29 2011-10-26 Third Wave Technologies, Inc. FEN-1 endonucleases, mixtures and cleavage methods
US6706473B1 (en) 1996-12-06 2004-03-16 Nanogen, Inc. Systems and devices for photoelectrophoretic transport and hybridization of oligonucleotides
CA2274587A1 (en) * 1996-12-10 1998-06-18 Genetrace Systems Inc. Releasable nonvolatile mass-label molecules
US5905024A (en) * 1996-12-17 1999-05-18 University Of Chicago Method for performing site-specific affinity fractionation for use in DNA sequencing
TR199902473T2 (en) * 1997-02-21 2000-07-21 Burstein Laboratories, Inc. Gene sequencer and methods.
US5846726A (en) * 1997-05-13 1998-12-08 Becton, Dickinson And Company Detection of nucleic acids by fluorescence quenching
AU737771B2 (en) * 1997-05-21 2001-08-30 Gesellschaft Fur Biotechnologische Forschung Mbh Method and kit for the detection of mutations in DNA's using restriction enzymes
US5928869A (en) * 1997-05-30 1999-07-27 Becton, Dickinson And Company Detection of nucleic acids by fluorescence quenching
DE69814629T2 (en) * 1997-07-22 2004-03-25 Qiagen Genomics, Inc., Bothell METHOD AND CONNECTIONS FOR ANALYZING NUCLEIC ACIDS BY MASS SPECTROMETRY
US6365731B1 (en) 1997-08-06 2002-04-02 Ambion, Inc. Stripping nucleic acids with iodine and sodium thiosulfate
US5955378A (en) * 1997-08-20 1999-09-21 Challener; William A. Near normal incidence optical assaying method and system having wavelength and angle sensitivity
US6207370B1 (en) 1997-09-02 2001-03-27 Sequenom, Inc. Diagnostics based on mass spectrometric detection of translated target polypeptides
US8182991B1 (en) 1997-11-26 2012-05-22 Third Wave Technologies, Inc. FEN-1 endonucleases, mixtures and cleavage methods
US6268131B1 (en) 1997-12-15 2001-07-31 Sequenom, Inc. Mass spectrometric methods for sequencing nucleic acids
GB9815163D0 (en) * 1998-07-13 1998-09-09 Brax Genomics Ltd Compounds
US6326479B1 (en) 1998-01-27 2001-12-04 Boston Probes, Inc. Synthetic polymers and methods, kits or compositions for modulating the solubility of same
WO1999040173A1 (en) * 1998-02-09 1999-08-12 Toyo Kohan Co., Ltd. Substrates for immobilizing and amplifying dna, dna-immobilized chips having dna immobilized on the substrates, and method for amplifying dna
US6723564B2 (en) 1998-05-07 2004-04-20 Sequenom, Inc. IR MALDI mass spectrometry of nucleic acids using liquid matrices
US6607888B2 (en) 1998-10-20 2003-08-19 Wisconsin Alumni Research Foundation Method for analyzing nucleic acid reactions
US6221592B1 (en) 1998-10-20 2001-04-24 Wisconsin Alumi Research Foundation Computer-based methods and systems for sequencing of individual nucleic acid molecules
US20020025519A1 (en) 1999-06-17 2002-02-28 David J. Wright Methods and oligonucleotides for detecting nucleic acid sequence variations
US6201112B1 (en) * 1999-07-22 2001-03-13 Agilent Technologies Inc. Method for 3′ end-labeling ribonucleic acids
US6528319B1 (en) * 1999-09-02 2003-03-04 Amersham Biosciences Corp Method for anchoring oligonucleotides to a substrate
US7824859B2 (en) * 1999-10-29 2010-11-02 Cytyc Corporation Methods for detection of a target nucleic acid by forming a cleavage structure using an RNA polymerase
US6893819B1 (en) * 2000-11-21 2005-05-17 Stratagene California Methods for detection of a nucleic acid by sequential amplification
US7838225B2 (en) * 1999-10-29 2010-11-23 Hologic, Inc. Methods for detection of a target nucleic acid by forming a cleavage structure using a reverse transcriptase
US7118860B2 (en) 1999-10-29 2006-10-10 Stratagene California Methods for detection of a target nucleic acid by capture
JP2001204463A (en) * 2000-01-27 2001-07-31 Toyo Kohan Co Ltd Support for immobilizing nucleotide
JP2004501869A (en) 2000-03-22 2004-01-22 ソルリンク・インコーポレイテッド Hydrazine-based and carbonyl-based bifunctional crosslinkers
US20040029113A1 (en) * 2000-04-17 2004-02-12 Ladner Robert C. Novel methods of constructing libraries of genetic packages that collectively display the members of a diverse family of peptides, polypeptides or proteins
US8288322B2 (en) 2000-04-17 2012-10-16 Dyax Corp. Methods of constructing libraries comprising displayed and/or expressed members of a diverse family of peptides, polypeptides or proteins and the novel libraries
WO2001096607A2 (en) 2000-06-13 2001-12-20 The Trustees Of Boston University Use of nucleotide analogs in the analysis of oligonucleotide mixtures and in highly multiplexed nucleic acid sequencing
US20060057565A1 (en) * 2000-09-11 2006-03-16 Jingyue Ju Combinatorial fluorescence energy transfer tags and uses thereof
US6627748B1 (en) 2000-09-11 2003-09-30 The Trustees Of Columbia University In The City Of New York Combinatorial fluorescence energy transfer tags and their applications for multiplex genetic analyses
US7829348B2 (en) * 2000-09-22 2010-11-09 Iowa State University Research Foundation, Inc. Raman-active reagents and the use thereof
JP4624644B2 (en) 2000-09-25 2011-02-02 ピコリター インコーポレイテッド Acoustic emission of fluid from multiple reservoirs
DE60138916D1 (en) 2000-09-26 2009-07-16 Boston Probes Inc Probes, probes, methods and kits for detection, identification and / or counting of bacteria
US9708358B2 (en) 2000-10-06 2017-07-18 The Trustees Of Columbia University In The City Of New York Massive parallel method for decoding DNA and RNA
JP2004510433A (en) 2000-10-06 2004-04-08 ザ・トラスティーズ・オブ・コランビア・ユニバーシティー・イン・ザ・シティー・オブ・ニューヨーク Massively parallel methods for decoding DNA and RNA
CA2426686A1 (en) 2000-10-30 2002-07-18 Sequenom, Inc. Method and apparatus for delivery of submicroliter volumes onto a substrate
WO2002070755A2 (en) * 2000-11-15 2002-09-12 Third Wave Technologies, Inc. Fen endonucleases
AU2002249854B2 (en) 2000-12-18 2007-09-20 Dyax Corp. Focused libraries of genetic packages
KR20020063359A (en) * 2001-01-27 2002-08-03 일렉트론 바이오 (주) nucleic hybridization assay method and device using a cleavage technique responsive to the specific sequences of the complementary double strand of nucleic acids or oligonucleotides
EP2295971B1 (en) * 2001-03-09 2016-09-07 TrovaGene, Inc. Conjugate probes and optical detection of analytes
AU2013205033B2 (en) * 2001-04-17 2016-06-09 Takeda Pharmaceutical Company Limited Novel Methods of Constructing Libraries Comprising Displayed and/or Expressed Members of a Diverse Family of Peptides, Polypeptides or Proteins and the Novel Libraries
US6534646B2 (en) 2001-06-04 2003-03-18 Barrskogen, Inc. Oligonucleotide labeling reagents
US6893822B2 (en) 2001-07-19 2005-05-17 Nanogen Recognomics Gmbh Enzymatic modification of a nucleic acid-synthetic binding unit conjugate
US20050147984A1 (en) * 2001-08-31 2005-07-07 Clondiag Chip Technologies Gmbh Interaction detection on several probe arrays
AU2002364945A1 (en) * 2001-10-25 2003-07-09 Neurogenetics, Inc. Genes and polymorphisms on chromosome 10 associated with alzheimer's disease and other neurodegenerative diseases
US20030170678A1 (en) * 2001-10-25 2003-09-11 Neurogenetics, Inc. Genetic markers for Alzheimer's disease and methods using the same
US20030224380A1 (en) * 2001-10-25 2003-12-04 The General Hospital Corporation Genes and polymorphisms on chromosome 10 associated with Alzheimer's disease and other neurodegenerative diseases
GB0129012D0 (en) 2001-12-04 2002-01-23 Solexa Ltd Labelled nucleotides
US7057026B2 (en) * 2001-12-04 2006-06-06 Solexa Limited Labelled nucleotides
DE10200374A1 (en) * 2002-01-08 2003-07-24 Friz Biochem Gmbh Method for the qualitative and quantitative comparative detection of chemical substances
DE10201463B4 (en) 2002-01-16 2005-07-21 Clondiag Chip Technologies Gmbh Reaction vessel for performing array method
DE10224825A1 (en) * 2002-06-05 2003-12-24 Eppendorf Ag Methods for the analysis of biomolecules
US7074597B2 (en) * 2002-07-12 2006-07-11 The Trustees Of Columbia University In The City Of New York Multiplex genotyping using solid phase capturable dideoxynucleotides and mass spectrometry
US20040067532A1 (en) * 2002-08-12 2004-04-08 Genetastix Corporation High throughput generation and affinity maturation of humanized antibody
AU2003259350A1 (en) 2002-08-23 2004-03-11 Solexa Limited Modified nucleotides for polynucleotide sequencing
US7414116B2 (en) 2002-08-23 2008-08-19 Illumina Cambridge Limited Labelled nucleotides
US11008359B2 (en) 2002-08-23 2021-05-18 Illumina Cambridge Limited Labelled nucleotides
AU2003273298B2 (en) * 2002-09-08 2008-03-06 Applera Corporation Methods, compositions and libraries pertaining PNA dimer and PNA oligomer synthesis
WO2004055160A2 (en) * 2002-12-13 2004-07-01 The Trustees Of Columbia University In The City Of New York Biomolecular coupling methods using 1,3-dipolar cycloaddition chemistry
KR20070121853A (en) 2003-03-31 2007-12-27 에프. 호프만-라 로슈 아게 Compositions and methods for detecting certain flaviviruses, including members of the japanese encephalitis virus serogroup
US7158353B2 (en) * 2003-11-06 2007-01-02 Seagate Technology Llc Magnetoresistive sensor having specular sidewall layers
JP2007534308A (en) * 2003-11-19 2007-11-29 アレロジック・バイオサイエンシズ・コーポレーション Oligonucleotides labeled with multiple fluorophores
BRPI0508100A (en) * 2004-02-28 2007-07-17 Chang Ning J Wang nucleic acid complexes as well as detection process and multiphase system
EP2436778A3 (en) * 2004-03-03 2012-07-11 The Trustees of Columbia University in the City of New York Photocleavable fluorescent nucleotides for DNA sequencing on chip constructed by site-specific coupling chemistry
WO2006073436A2 (en) * 2004-04-29 2006-07-13 The Trustees Of Columbia University In The City Of New York Mass tag pcr for multiplex diagnostics
US7939251B2 (en) 2004-05-06 2011-05-10 Roche Molecular Systems, Inc. SENP1 as a marker for cancer
US20080138801A1 (en) * 2004-05-28 2008-06-12 Arizona Board Of Regents, A Body Corporate Acting On Behalf Of Arizona State University Surface Plasmon Resonance Sensor for Detecting Changes in Polynucleotide Mass
US7828954B2 (en) * 2004-09-21 2010-11-09 Gamida For Life B.V. Electrode based patterning of thin film self-assembled nanoparticles
US20060234253A1 (en) * 2004-10-08 2006-10-19 Sysmex Corporation Method for detecting a target substance by using a nucleic acid probe
WO2006056443A2 (en) * 2004-11-24 2006-06-01 Aplagen Gmbh Method for solid-phase peptide synthesis and purification
CA2596496A1 (en) * 2005-02-01 2006-08-10 Agencourt Bioscience Corp. Reagents, methods, and libraries for bead-based sequencing
US7608433B2 (en) 2005-02-09 2009-10-27 Idexx Laboratories Method of detection of classical swine fever
US9169510B2 (en) 2005-06-21 2015-10-27 The Trustees Of Columbia University In The City Of New York Pyrosequencing methods and related compositions
GB0517097D0 (en) 2005-08-19 2005-09-28 Solexa Ltd Modified nucleosides and nucleotides and uses thereof
AU2006318462A1 (en) * 2005-11-21 2007-05-31 The Trustees Of Columbia University In The City Of New York Multiplex digital immuno-sensing using a library of photocleavable mass tags
KR100652903B1 (en) * 2005-12-21 2006-12-04 한국과학기술연구원 Manufacturing method of dehumidifying agent having superabsorbing polymer and manufacturing apparatus thereof
AU2007237909A1 (en) * 2006-04-19 2007-10-25 Applied Biosystems, Llc. Reagents, methods, and libraries for gel-free bead-based sequencing
US7674924B2 (en) * 2006-05-22 2010-03-09 Third Wave Technologies, Inc. Compositions, probes, and conjugates and uses thereof
WO2008042067A2 (en) 2006-09-28 2008-04-10 Illumina, Inc. Compositions and methods for nucleotide sequencing
GB2457402B (en) 2006-12-01 2011-10-19 Univ Columbia Four-color DNA sequencing by synthesis using cleavable fluorescent nucleotide reversible terminators
US20090017562A1 (en) * 2007-03-13 2009-01-15 Iowa State University Research Foundation, Inc. Raman-active reagents
JP5933894B2 (en) 2007-09-14 2016-06-15 アディマブ, エルエルシー Rationally designed synthetic antibody libraries and their use
US8877688B2 (en) * 2007-09-14 2014-11-04 Adimab, Llc Rationally designed, synthetic antibody libraries and uses therefor
WO2009039122A2 (en) 2007-09-17 2009-03-26 Sequenom, Inc. Integrated robotic sample transfer device
US20110014611A1 (en) * 2007-10-19 2011-01-20 Jingyue Ju Design and synthesis of cleavable fluorescent nucleotides as reversible terminators for dna sequences by synthesis
DK2725107T3 (en) 2007-10-19 2019-01-02 Univ Columbia DNA Sequencing with Non-Fluorescent Nucleotide Reversible Terminators and ddNTPs Modified by Split Label and Nucleic Acid comprising Inosine with Reversible Terminators
US20090215050A1 (en) 2008-02-22 2009-08-27 Robert Delmar Jenison Systems and methods for point-of-care amplification and detection of polynucleotides
WO2009114815A1 (en) 2008-03-13 2009-09-17 Dyax Corp Libraries of genetic packages comprising novel hc cdr3 designs
KR101419980B1 (en) * 2008-03-15 2014-07-15 홀로직, 인크. Compositions and methods for analysis of nucleic acid molecules during amplification reactions
WO2009132287A2 (en) * 2008-04-24 2009-10-29 Dyax Corp. Libraries of genetic packages comprising novel hc cdr1, cdr2, and cdr3 and novel lc cdr1, cdr2, and cdr3 designs
US8208909B2 (en) 2008-06-02 2012-06-26 West Corporation System, apparatus and method for availing a mobile call of address information
EP2478136A4 (en) * 2009-09-14 2013-09-25 Dyax Corp Libraries of genetic packages comprising novel hc cdr3 designs
WO2011100561A1 (en) * 2010-02-12 2011-08-18 Saint Louis University Molecular biosensors capable of signal amplification
CA2797057A1 (en) * 2010-04-23 2011-10-27 Australian Centre For Plant Functional Genomics Pty Ltd Tethered enzyme mediated nucleic acid detection
US9354228B2 (en) 2010-07-16 2016-05-31 Adimab, Llc Antibody libraries
JP5916732B2 (en) * 2010-09-20 2016-05-11 シージーン アイエヌシー Target nucleic acid sequence detection in solid phase using single-labeled immobilized probe and exonucleolytic activity
EA027558B1 (en) 2011-05-19 2017-08-31 Эйджена Байосайенс, Инк. Process for multiplex nucleic acid identification
JP6333297B2 (en) 2013-03-15 2018-05-30 イルミナ ケンブリッジ リミテッド Modified nucleoside or modified nucleotide
US10648026B2 (en) 2013-03-15 2020-05-12 The Trustees Of Columbia University In The City Of New York Raman cluster tagged molecules for biological imaging
TWI646230B (en) 2013-08-05 2019-01-01 扭轉生物科技有限公司 Re-synthesized gene bank
US10669304B2 (en) 2015-02-04 2020-06-02 Twist Bioscience Corporation Methods and devices for de novo oligonucleic acid assembly
US9981239B2 (en) 2015-04-21 2018-05-29 Twist Bioscience Corporation Devices and methods for oligonucleic acid library synthesis
CN114540470A (en) 2015-04-24 2022-05-27 基纳生物技术有限公司 Multiplexing method for identification and quantification of minor alleles and polymorphisms
CN107787371B (en) 2015-04-24 2022-02-01 基纳生物技术有限公司 Parallel method for detecting and quantifying minor variants
CA2998169A1 (en) 2015-09-18 2017-03-23 Twist Bioscience Corporation Oligonucleic acid variant libraries and synthesis thereof
CN108698012A (en) 2015-09-22 2018-10-23 特韦斯特生物科学公司 Flexible substrates for nucleic acid synthesis
US9895673B2 (en) 2015-12-01 2018-02-20 Twist Bioscience Corporation Functionalized surfaces and preparation thereof
AU2017315294B2 (en) 2016-08-22 2023-12-21 Twist Bioscience Corporation De novo synthesized nucleic acid libraries
JP6871364B2 (en) 2016-09-21 2021-05-12 ツイスト バイオサイエンス コーポレーション Nucleic acid-based data storage
WO2018066713A1 (en) * 2016-10-07 2018-04-12 地方独立行政法人神奈川県立産業技術総合研究所 Method for detecting nucleic acid, and kit for said method
GB2573069A (en) 2016-12-16 2019-10-23 Twist Bioscience Corp Variant libraries of the immunological synapse and synthesis thereof
KR20190119107A (en) 2017-02-22 2019-10-21 트위스트 바이오사이언스 코포레이션 Nucleic Acid Based Data Storage
CA3056388A1 (en) 2017-03-15 2018-09-20 Twist Bioscience Corporation Variant libraries of the immunological synapse and synthesis thereof
CN111566209A (en) 2017-06-12 2020-08-21 特韦斯特生物科学公司 Seamless nucleic acid assembly method
WO2018231864A1 (en) 2017-06-12 2018-12-20 Twist Bioscience Corporation Methods for seamless nucleic acid assembly
JP2020536504A (en) 2017-09-11 2020-12-17 ツイスト バイオサイエンス コーポレーション GPCR-coupled protein and its synthesis
GB2583590A (en) 2017-10-20 2020-11-04 Twist Bioscience Corp Heated nanowells for polynucleotide synthesis
EP3735459A4 (en) 2018-01-04 2021-10-06 Twist Bioscience Corporation Dna-based digital information storage
GB2590196A (en) 2018-05-18 2021-06-23 Twist Bioscience Corp Polynucleotides, reagents, and methods for nucleic acid hybridization
EP3930753A4 (en) 2019-02-26 2023-03-29 Twist Bioscience Corporation Variant nucleic acid libraries for glp1 receptor
WO2020176680A1 (en) 2019-02-26 2020-09-03 Twist Bioscience Corporation Variant nucleic acid libraries for antibody optimization
US11332738B2 (en) 2019-06-21 2022-05-17 Twist Bioscience Corporation Barcode-based nucleic acid sequence assembly

Family Cites Families (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS6091999A (en) * 1983-10-25 1985-05-23 Fujirebio Inc Measurement of polynucleotide
US4766064A (en) * 1984-05-07 1988-08-23 Allied Corporation Displacement polynucleotide assay employing polyether and diagnostic kit
US4775619A (en) * 1984-10-16 1988-10-04 Chiron Corporation Polynucleotide determination with selectable cleavage sites
CA1293938C (en) * 1985-03-28 1992-01-07 Thomas Horn Purification of synthetic oligomers
US4772691A (en) * 1985-06-05 1988-09-20 The Medical College Of Wisconsin, Inc. Chemically cleavable nucleotides
US4725537A (en) * 1985-09-19 1988-02-16 Allied Corporation Assay, reagent and kit employing nucleic acid strand displacement and restriction endonuclease cleavage
US4876187A (en) * 1985-12-05 1989-10-24 Meiogenics, Inc. Nucleic acid compositions with scissile linkage useful for detecting nucleic acid sequences
WO1987003911A1 (en) * 1985-12-17 1987-07-02 Genetics Institute, Inc. Displacement polynucleotide method and reagent complex
ATE88762T1 (en) * 1986-08-11 1993-05-15 Siska Diagnostics Inc METHODS AND COMPOSITIONS FOR TESTING WITH NUCLEIC ACID PROBES.
DE3681176D1 (en) * 1986-12-01 1991-10-02 Molecular Biosystems Inc METHOD FOR INCREASING THE SENSITIVITY OF NUCLEIC ACID HYBRIDIZATION TESTS.
CA1339351C (en) * 1987-10-15 1997-08-26 Michael S. Urdea Nucleic acid multimers and amplified nucleic acid hybridization assays using same

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JP2676535B2 (en) 1997-11-17
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