CA2180722A1 - A method of sequencing short oligonucleotides - Google Patents
A method of sequencing short oligonucleotidesInfo
- Publication number
- CA2180722A1 CA2180722A1 CA002180722A CA2180722A CA2180722A1 CA 2180722 A1 CA2180722 A1 CA 2180722A1 CA 002180722 A CA002180722 A CA 002180722A CA 2180722 A CA2180722 A CA 2180722A CA 2180722 A1 CA2180722 A1 CA 2180722A1
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- CA
- Canada
- Prior art keywords
- oligonucleotide
- primer
- nucleotides
- preparing
- target
- 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.)
- Abandoned
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING 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
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6862—Ligase chain reaction [LCR]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING 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
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6869—Methods for sequencing
-
- Y—GENERAL 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
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/81—Packaged device or kit
Abstract
Disclosed is a method for determining the nucleotide sequence of a target oligonucleotide. In this method a single-stranded ligation product is prepared which contains a target oligonucleotide-to-be-sequenced and an auxiliary oligonucleotide. A primer complementary to a portion of the auxiliary oligonucleotide and having a label covalently attached thereto is annealed to the auxiliary oligonucleotide portion of the ligation product. the primer is extended with chain-extending nucleoside triphosphates and chain-terminating nucleoside triphosphates in the presence of a polymerase to yield a plurality of primer extension products. These extension products are then separated on the basis of thenr base length; and the nucleoside sequence of the target oligonucleotide is determined from the mobilities of the primer extension products obtained during their separation.
Description
WO 95/20680 , 2 ~ 8 D 7 2 2 PCT/US95/0ll0 A METHOD OF ~ u ~:N~:lN~
S~ORT OLIGON~CLEOTIDES
FIELD QF T~ INVFNTION
The invention relates to the characterization of oligonucleotides, and more particularly, to methods of determining the nucleotide sequence of oligonucleotides and oligonucleotide analogs.
BACKGROUND OF THE INVENTION
Nucleotide sequence determination is an important step in the analysis of a short strand of an unknown oligonucleotide or oligonucleotide analog and to conf irm the specif ic sequence of oligonucleotides used as antisense drugs. At the present, most sequencing protocols use the chemical degradation approach of Maxam et al.
(Proc. Natl. Acad. Sci. (USA) (1977) 74:560) or the chain-termination method of Sanger et al. (Proc.
Natl. Acad. Sci. (USA) (1977) 74:5463) . In these methods, four separate r~t1-~n~ are performed to yield fragments dif~ering in length by only a single nucleotide which terminate at adenosine, cytosine, guanosine, or thymidine residues.
These seS[uencing products are generally resolved by electrophoresis on denaturing polyacrylamide gels (PAGE) . High performance capillary electrophoresis (HPCE) has also been used to separate oligonucleotide se(auencing WO 95/20680 r~ o~
S~ORT OLIGON~CLEOTIDES
FIELD QF T~ INVFNTION
The invention relates to the characterization of oligonucleotides, and more particularly, to methods of determining the nucleotide sequence of oligonucleotides and oligonucleotide analogs.
BACKGROUND OF THE INVENTION
Nucleotide sequence determination is an important step in the analysis of a short strand of an unknown oligonucleotide or oligonucleotide analog and to conf irm the specif ic sequence of oligonucleotides used as antisense drugs. At the present, most sequencing protocols use the chemical degradation approach of Maxam et al.
(Proc. Natl. Acad. Sci. (USA) (1977) 74:560) or the chain-termination method of Sanger et al. (Proc.
Natl. Acad. Sci. (USA) (1977) 74:5463) . In these methods, four separate r~t1-~n~ are performed to yield fragments dif~ering in length by only a single nucleotide which terminate at adenosine, cytosine, guanosine, or thymidine residues.
These seS[uencing products are generally resolved by electrophoresis on denaturing polyacrylamide gels (PAGE) . High performance capillary electrophoresis (HPCE) has also been used to separate oligonucleotide se(auencing WO 95/20680 r~ o~
2 ~ 8~7~
products (Cohen et al. (1988) J. Chromatogr. 458:323;
Cohen et al. (1988) Proc. Nat~. Acad. Sci. (USA) 85:9660; Guttman et al. (1990) Arlal. Chem. 62:137;
Cohen et al. (1990) J. Chromatogr. 516:49; Cohen et al., Anal. Chem. (in press) ), and can be readily coupled to mass spectrometry (Smith et al. (1988) Anal. Chem. 60:1948; Lee et al. (1988) J. Chromatogr.
457 :313) . E~owever, traditionally, the method of product vis~l~l i7~t;on has been autoradiography wherein 3~P or 35S i8 incorporated ir~to the oligonucleotide strand.
Recently, sequencing with laser-induced fluorescence (LIF) as a detection mode hax been used in a variety of Sanger et al.-related protocols. In the basic method, four unique f luoresce~t tags are attached either to the primer (Smith et al. (1986) Nature 321:674) or to each of the terminating dideoxynucleotides (Prober et al.
(1987) Science 238 :336) . In other Sanger et al . -related protocols, single-dye-based codin~ of bases with four diffe~ent peak heights has been used (Tabor et al. (1990) J.Biol. Chem. 265:8322-8326; Ausorge et al. (1990) Nucleic Acids Res.
18:3419-3420; Pentoney et al. (1992) EleL~r~,~
13 :461-474); Huang et al. (1992) Ar~al. Chem.
64 : 2149-2154), as well as single-dye-based coding of bases by peak height ratios plus one base coded by a gap (Ausorge et al. (1990) Nucleic Acids Res.
30 18:3419-3420; Pentoney et al- (1992) ElC.. ~r.r~ s 13 :461-474), and two-dye-binary coding of three WO95120fi80 2 ~ 2 PCT/US95l0l101 bases with one base coded by a gap or two optical channels (Carson et al., Anal. Chem. (in press) ) .
Unfortunately, many oligonucleotides and oligonucleotide analogs such as those useful for the antisense chemotherapeutic approach are too short to be sequenced by conventional sequencing methodologies. For example, if one uses the ~ -Sanger et al. approach to sequence a short (e.g., 15 to 17 bases in length), single-stranded DNA, the base sequence at its 3 ~ end is lost . The loss of information i8 primer size-dependent and normally 15 to 17 bases, i . e, sequence information will be provided right after the primer only.
Nevertheless, correct sequences are required for efficacy, and quality control procedures are needed to ensure that synthetic oligonucleotides have the desired nucleotide sequences. At present, the sequences of such oligonucleotides are often assumed to be correct based on the step-by-step synthesis itself since there is no convenient method available for their sequence analysis .
Enzymatic sequencing of short DNA analogs has been documented (Nordhoft et al. (1992) Rapid Comm.
Mass. Spectrom. 6:771; Wu et al. (1993) Rapid Comm.
Mass. Spectrom. 7:142; and Rile et al. (1993) Rapid Comm. Mass. Spectrom. 7:195) . This method uses exonucleases with phosphodiester-linked DNA as a substrate and MALDI-MS for detection. The current Wo 9sl20680 PcTll~s9slollo~
2 1 80~22 protocol is relatively 910w, as aliquots are taken every 15 minutes and directly analyzed ~y MA~DI-MS
(Tabor et al. (1990) J. Biol. Chem. 265:8322-8326) .
In addition, when DNA analogs are sequenced under these conditions, exonuclease digestion is very problematic and sometimes impossible.
An added level of complexity is the presence of modifications in oligonucleotides including non-phosphodiester linkages such as phosphorothioates or alkylphosphonates. Previous method of analyzing such oligonucleo'cide analogs have been laborious for commercial application.
For example, Agrawal et al . (J. Cl~rorn~togr. ( 19 9 0 ) 509 :396-399) discloses analysis of oligonucleotide phosphorothioates involving conversion of phosphorothioate linkages to phosphodiesters followed by digestion with snake venom phosphodiesterase, phosphatase treatment, and analysis of base composition on reYerEed phase HPl: C .
Thus, there remains a need for more simple and reliable methods of det~rTn;n;n~ the sequence of short oligonucleotides and oligonucleotide analogs from their very first to their very last ~ase .
W095/20680 2 ~ 8 ~ ~2 2 PCT/US9S101104 SUMMARY OF THE INVENTION
The present invention provides an ef f icient and reliable method for determining the nucleotide sequence of short oligonucleotides and o~igonucleotide analogs. Generally, conventional methods f or determining nucleotide sequences are difficult to use for many oligonucleotides and synthetic oligonucleotides because such molecules are too short to serve as an efficient template.
The method according to the invention OVt~
this problem by providing a sequencing- length oligonucleotide that includes the target oligonucleotide-to-be-sequenced which is long enough to serve as an efficient template.
Target oligonucleotides capable of being sequenced by the method of the invention are composed of ribonucleotide3, deoxyribonucleotides, 2 0 analogs of ribonucleotides, analogs of deoxyribonucleotides, and combinations thereof.
Thus, in some embodiments, the target oligonucleotides are synthetic or modif ied oligonucleotides or oligonucleotide analogs.
As used herein, the term "oligonucleotide~
includes polymers of two or more ribonucleotide and/or deoxyribonucleotide monomers covalently linked by at least one 5 ~ to 3 ~ internucleotide
products (Cohen et al. (1988) J. Chromatogr. 458:323;
Cohen et al. (1988) Proc. Nat~. Acad. Sci. (USA) 85:9660; Guttman et al. (1990) Arlal. Chem. 62:137;
Cohen et al. (1990) J. Chromatogr. 516:49; Cohen et al., Anal. Chem. (in press) ), and can be readily coupled to mass spectrometry (Smith et al. (1988) Anal. Chem. 60:1948; Lee et al. (1988) J. Chromatogr.
457 :313) . E~owever, traditionally, the method of product vis~l~l i7~t;on has been autoradiography wherein 3~P or 35S i8 incorporated ir~to the oligonucleotide strand.
Recently, sequencing with laser-induced fluorescence (LIF) as a detection mode hax been used in a variety of Sanger et al.-related protocols. In the basic method, four unique f luoresce~t tags are attached either to the primer (Smith et al. (1986) Nature 321:674) or to each of the terminating dideoxynucleotides (Prober et al.
(1987) Science 238 :336) . In other Sanger et al . -related protocols, single-dye-based codin~ of bases with four diffe~ent peak heights has been used (Tabor et al. (1990) J.Biol. Chem. 265:8322-8326; Ausorge et al. (1990) Nucleic Acids Res.
18:3419-3420; Pentoney et al. (1992) EleL~r~,~
13 :461-474); Huang et al. (1992) Ar~al. Chem.
64 : 2149-2154), as well as single-dye-based coding of bases by peak height ratios plus one base coded by a gap (Ausorge et al. (1990) Nucleic Acids Res.
30 18:3419-3420; Pentoney et al- (1992) ElC.. ~r.r~ s 13 :461-474), and two-dye-binary coding of three WO95120fi80 2 ~ 2 PCT/US95l0l101 bases with one base coded by a gap or two optical channels (Carson et al., Anal. Chem. (in press) ) .
Unfortunately, many oligonucleotides and oligonucleotide analogs such as those useful for the antisense chemotherapeutic approach are too short to be sequenced by conventional sequencing methodologies. For example, if one uses the ~ -Sanger et al. approach to sequence a short (e.g., 15 to 17 bases in length), single-stranded DNA, the base sequence at its 3 ~ end is lost . The loss of information i8 primer size-dependent and normally 15 to 17 bases, i . e, sequence information will be provided right after the primer only.
Nevertheless, correct sequences are required for efficacy, and quality control procedures are needed to ensure that synthetic oligonucleotides have the desired nucleotide sequences. At present, the sequences of such oligonucleotides are often assumed to be correct based on the step-by-step synthesis itself since there is no convenient method available for their sequence analysis .
Enzymatic sequencing of short DNA analogs has been documented (Nordhoft et al. (1992) Rapid Comm.
Mass. Spectrom. 6:771; Wu et al. (1993) Rapid Comm.
Mass. Spectrom. 7:142; and Rile et al. (1993) Rapid Comm. Mass. Spectrom. 7:195) . This method uses exonucleases with phosphodiester-linked DNA as a substrate and MALDI-MS for detection. The current Wo 9sl20680 PcTll~s9slollo~
2 1 80~22 protocol is relatively 910w, as aliquots are taken every 15 minutes and directly analyzed ~y MA~DI-MS
(Tabor et al. (1990) J. Biol. Chem. 265:8322-8326) .
In addition, when DNA analogs are sequenced under these conditions, exonuclease digestion is very problematic and sometimes impossible.
An added level of complexity is the presence of modifications in oligonucleotides including non-phosphodiester linkages such as phosphorothioates or alkylphosphonates. Previous method of analyzing such oligonucleo'cide analogs have been laborious for commercial application.
For example, Agrawal et al . (J. Cl~rorn~togr. ( 19 9 0 ) 509 :396-399) discloses analysis of oligonucleotide phosphorothioates involving conversion of phosphorothioate linkages to phosphodiesters followed by digestion with snake venom phosphodiesterase, phosphatase treatment, and analysis of base composition on reYerEed phase HPl: C .
Thus, there remains a need for more simple and reliable methods of det~rTn;n;n~ the sequence of short oligonucleotides and oligonucleotide analogs from their very first to their very last ~ase .
W095/20680 2 ~ 8 ~ ~2 2 PCT/US9S101104 SUMMARY OF THE INVENTION
The present invention provides an ef f icient and reliable method for determining the nucleotide sequence of short oligonucleotides and o~igonucleotide analogs. Generally, conventional methods f or determining nucleotide sequences are difficult to use for many oligonucleotides and synthetic oligonucleotides because such molecules are too short to serve as an efficient template.
The method according to the invention OVt~
this problem by providing a sequencing- length oligonucleotide that includes the target oligonucleotide-to-be-sequenced which is long enough to serve as an efficient template.
Target oligonucleotides capable of being sequenced by the method of the invention are composed of ribonucleotide3, deoxyribonucleotides, 2 0 analogs of ribonucleotides, analogs of deoxyribonucleotides, and combinations thereof.
Thus, in some embodiments, the target oligonucleotides are synthetic or modif ied oligonucleotides or oligonucleotide analogs.
As used herein, the term "oligonucleotide~
includes polymers of two or more ribonucleotide and/or deoxyribonucleotide monomers covalently linked by at least one 5 ~ to 3 ~ internucleotide
3 0 l inkage .
The terms "modified oligonucleotide" and "oligonucleotide analog, n as used herein, Wo gS/20680 PCT/US9S/0ll0~ ~
2~80722 encompass a molecule of ribonucleotides or deoxyribonucleotides which are covalently linked via at least one synthetic linkage. A ~synthetic internucleotide linkage" is a linkage other than a phosphodiester between the 5 ~ end of one nucleotide and the 3 ~ end of another nucleotide in which the 5 ' internucleotide phosphate has been replaced with any number of chemical groups.
Representative synthetic linkages include phosphorothioates, phosphorodithioates, alkylphosphonothioates, phosphoramidates, phosphate~esters, carbamates, carbonates, phosphate:~triesters, acetamidate, and carboxymethyl esters.
The term ~oligonucleotide analog" also en.~ 8PR oligonucleotides with a modified base and/or sugar. For example, a 3 ', 5 ~ -substituted oligonucleotide i8 a modif ied oligonucleotide having a sugar which, at both its 3~ and 5~
positions is attached to a chemical group other than a hydroxyl group (at its 3 ~ position) and other than a phosphate group (at its 5 ' position) .
A modif ied oligonucleoti~e may also be a capped species. Also .onr~nTnr~RRed by these terms are unoxidized oligonucleotides or oligomers having a substitution in one nonbridging oxygen per nucleotide in the molecule.
3 0 oligonucleotide analogs may also be synthetic oligonucleotides" which ~n~ RRe8 polymers of 3 to 5'-linked ribonucleosides, 2'-modified ribonucleosides and/or deoxyribonucleosides having W095l20680 2 1 8~7~ PCTIUS95/0ll0~
only as many nucleosides as are conveniently chemically synthesized (i . e., up to about 80 100). Also encompassed are those oligonucleotides having base or sugar modif ications as well as those having nuclease resistance-conferring bulky substituents at their 3' and/or 5' end(s), multiple ribonucleosides and/or deoxyribonucleosides linked via an internucleotide linkage not found in native DNA, i.e., linkages other than phosphodiester bonds, or having modif ied bases and/or sugars in various other structural modifications not found in vivo without human intervention.
In the method of the invention, a single-stranded ligation product is prepared which includes a target oligonucleotide-to-be-sequenced and an ~ r;l;Ary oligonucleotide, each having a 3' and 5 ~ end The auxiliary oligonucleotide has a nucleotide sequence complementary to the sequence of a primer-to-be-used. In one aspect of the invention, the ~ ry oligonucleotide also includes a si~n~ll;n~ sequence of at least four contiguous nucleotides at its 5 ' end, which become linked to the 3 ' end of the target ol; ~r~nll~l eotide in the ligation product. In some embodiments, the i~lll~; l; ~rV oligonucleotide is at least eight nucleotides in length.
3 0 The invention provides several pref erred methods of preparing the single-stranded ligation product depending in part upon the degree to which the sequence of the target oligonucleotide is Wo 95/20680 PCT/US95/OllOJ
2i~7~2 known, I~at least the last three nucleotidee, and preferably six, are known, a bridge oligonucleotide i6 prepared which is complementary to these known nucleotides at its 5 ' end and which further includes a nucleotide sequence that is complementary to the first at least four 5' nucleotides of the auxiliary nucleotide. This bridge is used to anneal to the 3 ' end of the target oligonucleotide and the 5 ' end of the auxiliar~v nucleotide, forming a double stranded construct. The target oli~nllrl o~tide can then be ligated to the ~ rry oligonucleotide via a ligase .
= A bridge is also used in another embodiment where the sequence of the target oligonucleotide is completely unknown. In this method a set of sixteen bridge oligonucleotides i8 prepared. Each bridge nl ;~nllt~ tide i5 identical in part in having about six nucleotides at its 3' end which are complementary to the six nucleotides at the 5 ' end of the auxiliary oligonucleotide. The bridge oligonucleotides differ in having at their 5' ends a unique dinucleotide sequence (i . e ., one of sixteen possible combinations of four nucleotides), one of which being complementary to the last two unknown 3 ' nucleotides of the target nli~r~nllrl~ tides). The one bridge ol;~-nl-~leotide which anneals to the auxiliary and target oligonucleotides provides the same function as described above in the first bridge ~mhori; t.
Wo 95/20680 2 1 ~ ~ 7 ~ 2 PCT/US9~/01104 _ g _ In another preferred embodiment, the method used for preparing the ligation product including a target oligonucleotide with a completely unknown sequence is as follows. An auxiliary oligonucleotide i5 prepared which, like those described above, is composed of a 3 ~ sequence Cornrl~ ti:lry to the gequence Qf a primer to be used. This sequence is linked at its 5 ~ end to a signalling se~uence of at least four known contiguous nucleotides . The 3 ' nucleotide of the ~llC; l; Ary oligonucleotide is protected in some cases (when using RNA ligase) . The 5 ' end oi this ;l ;Ary oligonucleotide is ligated directly to the 3 ' end of the target molecule. In preferred a3pects of the invention, ligation is accomplished with a blunt end ligase such as T4 RNA ligase.
In any case, to this ligation product is annealed a primer which has a nucleotide sequence complementary to a portion of the auxiliary oligonucleotide. In some aspects of the invention, the primer comprises a nucleotide sequence complementary to at least four, but pref erably at least eight, nucieotides of the All~; l; ;Iry oligonucleotide portion of the ligation product. This primer also has a label covalently attached theret4. Preferably, this label is a fluorescent, chemiluminescent, or radioactive tag.
Most preferably, the label is a fluorescent label which is excitable in the W or visible range and which fluoresces in the visible range.
Wo 95/20680 PCT/US95/0l lO~
2~8~722 Next, the primer is extended with chain-extending-nucleoside triphosphates and chain-terminating nucleoside triphosphates in the presence of a polymerase to yield a plurality of primer extension products of differing ~engths.
The~e products are then separated on the basis of their relative mobilities, from which the nucleotide se~uence of the target oligonucleotide can be derived. In preferred embodiments of the invention separation is achieved by slab gel or high performance capillary gel electrophoresis.
Depending on the primer label used, laser-induced f~uorescence, W absorption, or radiation are mea3ured to determine the relative mobilities of the primer extension products.
WO9s/20680 2 f 8 ~ ~ ~ 2 PCT/US95/0ll04 BRIEF D~SCRIPTIQN QF T~ DRAWINGS
The foregoing and other objects of the present invention, the various features thereof, as well as the invention itself may be more fully understood from the following description, when read together with the ;3 ~ nying drawings in which:
FIG. lA is a schematic representation of one embodiment of the invention by which a single-stranded ligation product is prepared from a target oligonucleotide whose sequence is partially known;
FIG. lB i9 a schematic representation of :
another e~nbodiment of the invention by which a single-stranded ligation product is prepared, with the aid of a bridge oligonucleotide, from a target ~ n~ l eotide whose sequence i9 unknown;
FIG. lC is a schematic representation of yet another embodiment of the invention by which a single-stranded ligation product is prepared, without the aid of a bridge oligonucleotide, from a target oligonucleotide whose sequence i8 unknown;
FIG. 2A is a W electropherogram of a reaction mixture r~r~nt~;n;n~ ATP, a 12mer bridge oligonucleotide, a 25mer target oligonucleotide whose sequence is partially known, and a 31mer All~ ry oligonucleotide;
W095/20680 2 1 807~2 PCr/l~S9510110~ ~
FIG. 2B is a W electropherogram of the reaction mixture described in FIG . 2A af ter ligation with TfDNA ligase, and shows the species in FIG. 2A as well as a 57mer ligation product;
FIG. ~3A i9 a W electropherogram of the HPCE
separation of the components of a TçRNA ligase reaction mixture including ATP, the target oligonucleotide, and a 31mer ~ ry oligonucleotide with an unprotected 3 ' hydroxyl group;
FIG. 3B is a W electropherogram of HPCE
l~ separation of the components of a TjRNA ligase reaction mixture as in FIG 3A, except using a 31mer auxiliary oligonucleotide with a 3 ' amino protected end, and resulting in a 57mer ligation product;
FIG. 4A is an LIF electropherogram of the HPCE separation of primer extension products made from, and complementary to, the 57mer ligation product using the ddA terminat~d sequencing reaction, and separated as in FIG. 2B;
FIG.--4B is an ~IF electropherogram of the HPCE separation of primer extension products made from, and complementary to, the 57mer ligation 3 0 product using the ddG terminated se~uencing reaction;
W095l20680 21 80722 r~ o4 FIG. 4C is an ~IF electropherogram of the HPCE separation of primer extension products made from, and complementary to, the 57mer ligation product using the ddT terminated sequencing reaction;
FIG. 4D i8 an bIF electropherogram of the HPCE separation of primer extension products made from, and complementary to, the 57mer T4 DNA
ligase ligation product using the ddC terminated sequencing reaction;
FIG. 5A is a computer overlay of the ~IF
electropherograms from FIGS. 4A-4C using two point re-size alignmenti FIG. 5B is a computer overly of the ~IF
electropherograms from FIGS. 4A-4D, using two point re-size alignment; and FIG . 6 is a plot of relative f ragment migration versus base number f or the data presented in FIGS. 5A and 5B, showing a linear relationship with R2=o . 999 .
W09s~20680 2 ~ 8Q722 r~ o~ ~
BRIEF ~ESCRIPTION ~F T~ ) EMBODIMENTS
The patent and scientific literature referred to herein establishes the knowledge that i8 available to those with skill in the art. The issued U. S . patent and allowed applications cited herein are hereby incorporated by ref erence .
This invention provides a new ser~uencing procedure which determines the se~uence of a target oligonucleotide from its very first 5' nucleotide to its very last 3 ' nucleotide, despite the shortness of its length or~ the fact that it may be an oligonucleotide analog with non-phosphodiester ;n~f~rnllrleotide linkages and/or other modif ications . In fact, any target oligonucleotide can, in principle, be se~uenced.
For e~cample, the target oligonucleotides-to-be-sequenced can range from about 4 to about 100 nucleotides in length, with oligonuclçotides having from about 8 to about 50 nucleotides in length being most common Furthermore, target oli~nnl~rl ~ntides can have any type of internucleotide l; nk;~r~c or even any combination of different types of ;ntf~rnl~rleotide linkages, as long as the target oligonucleotide can be ligated to the ~-l~; l; ;Iry oli~nni~rl Pntide and can be extended by a polymerase. For example, a target 3 0 oligonucleotide may have more than one non-phosphodiester linkage, and up to having all non-phosphodiester linkages. The non-~hosphodiester l; nk~ present in the target oligonucleotide may wo 9sno680 2 1 8 0 7 2 2 PCr/US95l0110~
include at least phosphorothioate, alkylphosphonate, phosphoramidate, alkylphosphonothioate, phosphodithioate, and sulfone, 8U lfate, keto, phosphate ester, bridged phosphorothioate and bridged phosphoramidate linkages, all of which are known in the art (see Uhlmann et al. (1990) (Chem. Re~. 90:543-584 for a review on the synthesis and characteristics of phosphodiester and non-phosphodiester-linked antisense oli~n~ tides).
The method of the invention requires the =
preparation of an 'IAllx;l ;~ry oligonucleotide"
which is used ior ligation to the 3 ~ end of the target oligonucleotide-to-be-sequenced, thereby forming a single-stranded ligation product. The auxiliary oligonucleotide is a single-stranded RNA, DNA, or RNA/DNA-r~nti~;n;n~ molecule with a known sequence that is complementary to a primer.
The ~3--,c; l; i~ry oligonucleotide may also include at its 5' end a region of at least four preselected, contiguous nucleotides which serves as a marker or .
"si~n~l 1 ;n~ 8equence. "
This ~]~; l; i~ry oligonucleotide is ligated to the target molecule to form a single-stranded ligation product, wherein base #1 of the target oligonucleotide-to-be-sequenced is located directly after the ~--~; l; ;Iry DNA.
The invention provides several protocols for preparing the ligation product, depenaing on the degree to which the sequence of the target WO 95/20680 PCTIUS95/0ll0~
2 ~ 8~7~2 oligonucleotide is known. If at least the laet three, and preferably six nucleotides at the 3 ' end of the target are known, a bridge oligonucleotide can be constructed which supports the target r~ nllrl eotide and facilitates the ligation reaction. The bridge includes ribonucleotides and/or deoxyribonucleotides linked via phosphodiester and other than phosphodiester lnt~rn~ leotide linkages. This molecule is complementary to these at least six nucleotides and further; n~ Pq a sequence that i5 complementary to the first at least four 5' nucleotides of the auxiliary nucleotide. The bridge oligonucleotide is annealed to the target oligonucleotide and to the All~; l; Ary oligonucleotide such that the f irst two 3 ' nucleotides of the bridge oligonucleotide are Annf~Al ed to the laet two 3 ~ nucleotides of the target oligonucleotide, and at least the Ilext 9ix nucleotides of the bridge oligonucleotide towards its 5~ end are annealed to the first six 5' nucleotides of the ~llr; l; Ary oligonucleotide, thereby yielding a partially double stranded construct . Then, the 3 ' end of the target oligonucleotide is ligated to the si~nAl l; n~
sequence at the 5 ' end of the ~ ; Ary oligonucleotide with a template-dependent enzyme such as T~ DNA ligase or Ta~ DNA ligase. Upon denaturation, a single-stranded ligation product 3 0 is obtained .
An example of this ligation protocol is shown schematically in FIG. lA, where at least 3 bases W095l20680 2 l ~2~ PCT/U595/0ll0.1 .
of a target oligonucleotide are already known. In this figure, the bases that are unknown are depicted as "?". A 12mer bridge (SEQ ID NO:2) is prepared to facilitate the ligation of the ~117r; 1; ~ry oligonucleotide to the target molecule.
This bridge consists of two regions of six bases, one region that is complementary to the last six bases of the auxiliary oligonucleotide (SEQ ID
NO: 3 ) at its 5 ' end and the other being complementary to a predetermined first six bases of the target oligonucleotide at its 3 ' end which are known. FIGS. 2A and 2B show the separation of varies species of oligonucleotides (target, auxiliary, and bridge) by capillary electrophoresis, followed by W detection, before and after ligation with T4 DNA ligase, respectively. Migration order of detected peaks in FIG. 2A is (1) the fast migrating 12mer bridge (SEQ ID NO:2); (2) the target oligonucleotide-to-be-sequenced; and (3) the auxiliary oligonucleotide. When T4 DNA ligase and ATP are added to the reaction mixture, after 30 minutes incubation at 37 C, a 57mer ligation product is observed (FIG. 2s).
If the entire sequence of the target oligonucleotide is unknown, two methods of preparing the ligation product be used. One, like the method above, also requires the support of a bridge oligonucleotide. In this method, a set of sixteen bridge oligonucleotides is prepared, all of which are identical at their 5 ~ ends because they include a sequence complementary to the same WO 95/20680 ~ PCT/lJS95/0ll0~
2~8~72~
four, but preferably six to eight or more nucleotides of an ;111~; 1; i~ry oligonucleotide to be used. In addition, the bridge oligonucleotides include at their 3 ' ends one of sixteen possible dinucleotides: AA, AC, AG, AT, CA, CC, CG, CT, GA, GC, GG, GT, TA, TC, TG, TT. One of these rlinll~ otideg Will be complementary to the two most 3 ' nucleotides of the target oligonucleotide .
Thus, when this set of sixteen bridge oligonucleotides is mixed with the target oligonucleotide of unknown sequence and with the auxiliary oligonucleotide described above, under conditions conducive for ~nn.sAI ;n~, the one bridge oligonucleotide having a dinucleotide complementary to the last two 3 ' nucleotides of the target molecule will hybridize to it as well as to the ~ll~i 1 i i~ry oligonucleotide. This method is shown schematically in FIG. lB, where the sixteen bridge oligonucleotides have SEQ ID ~OS: 6-21, and each unknown base in the target oligonucleotide is depicted shown as a "?".
Alternatively, a blunt end ligase such as T4 RNA ligase may be used which does not require the presence of a double-stranded construct to link two nucleotides together. In this case, an auxiliary oligonucleotide is prepared which is composed of a 3 ' -sequence complementary to the sequence of a primer to be used, linked to a 5 ' -f:i~ni~l ;ng~-sequence of at least four contiguous nucleotides. The 3~ end of this i~il~;7 ;~ry oligonucleotide is proFected with, for example, a dideoxynucleotide (ddA, ddC, ddG, ddT) or an amino WO95/20680 2 ~ PCTIUS9~/0l10l group . The 5 ~ end of the auxiliary oligonucleotide is then ligated to the 3 ~ end of the target oli~nnllrl .o-ntidel thereby forming a single-stranded ligation product.
An example of this ligation protocol i9 shown schematically in FIG. lC. T4 RNA ligase is used to ligate a target oligonucleotide of unknown seriuence to a 31mer ;ill-f; 1; ;iry oligonucleotide (S~Q
ID NO:3) without the presence of a bridge in the reaction mixture. The W electropherogram shown in FIGS. 3A and 3B demonstrates the synthesis of the 57mer ligation product . If an ;iil~; l; ;iry oligonucleotide is used having an unprotected 3 ' end, the enzyme forceæ the ligation process to :~
proceed in cycles and several cycles are observed (FIG. 3A) . This undesirable ~ onnm~nnn is prevented simply by uging an ~ ry oligonucleotide with a protecting dideoxy or an amino group at its 3 ' end, as shown in FIG . 3B
where only one ligation cycle is obtained. As in the T4 RNA ligase case, the ATP 25mer analog, auxiliary-31mer, and 57mer ligation product are observed .
Once the single-stranded ligation product rnnt~i;n;ng the target molecule is formed, a primer -is annealed to ligation product, f rom which strands complementary to the target molecule can be f~t,on~ by a polyinerase (i.e, primer extension products). The primer oligonucleotide can be any of the conv~ontinn~il types o~ RNA and/or DNA-rnnt;i;n;nr~ oligonucleotides that are well known Wo 95/20680 PCTIUS95/01l0~
2~ ~Q7~2 and commonly used for DNA or RNA sequencing or primer extension reactions. The primer oligonucleotide has a se~uence that is complementary to a 3 ' portion of the auxiliary oligonucleotide region of the ligation product which does not include the si~n~l l; n~ region .
At least one molecule of a label such as a luminescent, radioactive, or fluorescent label is attached to the primer. If the label is fluoresce~t it is excitable in the W or visible wavelength range, and fluoresces in the visible range. Such labels include fluorescein, or the N-sllfcin;m;de ester or other derivatives thereof, such as called "JOE" (Applied Biosystems, Foster City, CA), "FITC" (Applied Biosystems, Foster City, CA), and "FAM" (Applied Biosystems, Foster City, CA), and rhr~ m;n~, or derivatives thereof, such as tetramethylrh~ m;n~ ("TAMARA") (Applied Biosystems, Foster City, CA) and "Texas Red" or "ROX" (Applied Biosystems, Foster City, CA) (Smith ( 19 85) Nucl. Acid. ~es. 13: 23 9 9 -2412 ) . These labels can be covalently attached to the primer, for example, by using chemical DNA or RNA synthesis as described by Smith ~Am. Biolab. (1989) May:11-20), or by other methods which will not interfere with the ability of the primer to hybridize to the target molecule or to be ligated to the helper oligouucleotide An example of one such method includes covalently attaching an amino group onto the dye and then linking the amino group 5 ' end of oligonucleotide (Smith (1985) Nucl. Acid. Res.
WO 9S/20680 2 1 ~ ~ 7 2 2 PCTiUS9S/ollo~
13:2399-2412). Alternatively, the fragment may be f luorescently labelled with dideoxynucleotides .
~nn~l ;n~ of the bridge, ~ ry, and target oligonucleotides, and of the primer and ligation product is accomplished under conditions that are most conducive for the hybridization of a single-stranded species to a complementary, single-stranded oligonucleotide. These conditions include contact in ligation buffer (600 mM Tris-HCl, pH 7 . 6, 6 6 mM MgCl2, 10 0 mM DTT, 6 6 0 llm ATP ) at a temperature of from about 4C to 90C, but preferably at room temperature (i.e., 19C to 25C) .
Upon annealing of the primer to the ligation product, the primer extension reaction can take place in the presence of a polymerase. Many polymerases are known in the art and all are suitable in principle. Usually, a DNA polymerase will be used such as Taq DNA polymerase or T4 DNA
polymerase. If the very well known Sanger et al.
(ibid. ) eequencing method is to be followed, nucleotides and dideoxynucleotides are used to synthesize the primer extensions.
Finally, the dideoxy-terminated extension products are separated according to any number of well-known standard procedures that separate such molecules on the basie of size. Such protocols include polyacrylamide slab gel electrophoresis or high performance capillary gel electrophoresis.
Depending on the primer label used, laser-induced Wo g~/20680 PCTNS9S/OllO~
2~722 fluorescence, W absorption, or radiation are measured to deterlrLine the relative mobilities of the primer extension products.
For example, an auxiliary oligonucleotide is prepared from 17 bases at its 3 ~ end which are complementary to the sequence Qf the M13mpl8 (-21) primer. Next is a signalling region o ten T
bases. Then, base #l of the target 0 oligonucle-otide to be se~uenced is located directly 5 ~ to the ~ ry oligonucleotide .
In developing an automated single-stranded oligonucleotide se~uencer for routine antlsense analysis, a working strategy was developed to examine the enzymatic sequencing of single-stranded oligonucleotide analogs which ;nflll~l the ligation products described above. The f ollowing strategy was used to develop an expression for the electrophoretic migration of sequencing fragments which is an P~Pnt;~l element of automated data processing.
Over~a narrow range of molecular size, a linear relationship between relative migration time (T' ) and base number can be established using two ;ntPrn~l standards in what amounts to a two-point calibration. These two point~ are the primer (17mer) and the 58mer iragment which is one 3 0 base longer than the ligation product due to the endonuclease a~tivity of sequenase 2 . O . This linear relationship i8 described as follows:
Wo 9~/20680 PCTIUS9~l01104 T ' = TD - Ib~
5 Tf in - Tp r where "Tp" is the migration time of sequencing fragment; "Tpr" is the migration time of primer;
and "Tfin" iB the migration time of the last peak.
This relation8hip i9 linear for T' which is only fragment size-dependent.
To validate this term, the expression was tested under experimental cr~nf~;tl~ln~ as follows.
A 57mer ligation product (SEQ ID NO:4) (e.g., a 25mer target oligonucleotide analog (SEQ ID NO :1) + a 32mer ;~lll~;li~ry oligonucleotide (SEQ ID NO:3)) was subjected to enzymatic chain termination reaction for four different bases ;n~1~r~n~n~1y (i.e., A, G, C, T) . Each of the four reaction mixtures were run separately on different days and different gel columns. LIF electropherograms of the separation of the four sets of primer extensio~ products are shown in FIGS. 4A-4D.
~Y~nci r~n productg were separated by HPCE using a gel containing 12~ T acrylamide, 6.5 M urea, and 4096 (weight:weight) formamide. These figures are the computer two point alignments for T, G, A and C reactions . The f irst point iB the 17mer primer and the 3econd point i9 the 57mer latest migrating fragment. T' was calculated individually for each of the detected fragments between 17 and 57 bases in length. Fragment 33 corresponds to the 25mer target oligonucleotide. The obtained values are then rearranged in order form low to high, according to the occurrence in the four runs for the four individual bases in FIGS. 4A-4D. The Wo 9s/20680 PCT/US9510110~
2~72~
results are su~nmarized in TABLE l, which is a computer printout of relative migration obtained using two point re-size alignment for the data obtained from ~IGS. 4A- 4D.
T~3~ l Reading Base A T C G S~ nci Real Targe~ 01igo 3' 33 1 0.370 T
34 2 0.391 C
5 35 3 0.421 T
36 4 0.441 T
37 5 0.460 C
38 6 0.485 C
39 7 0.512 T
2 0 40 8 0.537 C
41 9 0.570 T
42 10 0.586 C
43 11 0.620 T
44 12 0.636 C
25 45 13 0.671 T
46 14 0.680 A
47 15 0.707 C
48 16 0.735 C
49 17 0.763 C
3 0 50 18 0.786 A
51 19 0.812 C
52 20 0.828 G
53 21 0.856 C
54 22 0.896 T
35 55 23 0.904 C
56 24 0.948 T
57 25 0.953 C
5' WO 95/20680 PCT/US95/0ll0~
2~80722 A6 indicated above, the sequence of the target oligonucleotide is determined in the right-most column of Table 1 from 3 ' to 5 ' end. This sequencing format is the result of computer software capable of ~-~t( t1ng the sequencing system and performing data processing.
When this software is interfaced with a commercial software (e.g., Turbochrome~ III, P.E.
Nelson, Cupertino, CA), the data obtained can be manipulated such that the f inal electropherogram of the sequencing can be plotted, as illustrated in FIGS. 5A and 5B. The results are summarized in FIG. 6 which demonstrates the linear relati"n~Th1E' between fragment migration and base number.
Thus, a mathematical expression has been ~:
derived and successfully used for an automated single-stranded oligonucleotide sequence determination. The strength of this expression is that "T' '~, relates only to the fragment length expressed as base number. Moreover, this expression is independent of the experimental conditions given that all sequencing fragments are separated. Separation is not a problem, since gel capillary can separate sequencing fragments with very high efficiency and resolution (Cohen et al.
(1988) J. Chrom~togr. 458:323; Swerdlow et al. (1990) Nucl. Acidsl~es. 18:1415-1419; Pentoney et al. (1992) EleT~r~,T,J,Tsis 13:461--74; Cohen et al. (1993) TRAC
12: 195-202) .
WO 9sno680 2 1 8 ~ 7 2 2 PCr/US95l0ll0~ ~
The following examples illustrate the pre~erred modes of making and practicing the present invention, but are not meant to limit the 3cope of the invention since alternative methods may be utilized to obtain similar results.
EXAMPLES
1. Preparation of Target, Auxiliary, Bridge, and Primer Oligonucleotides Phosphodiester-linked target, i31ly;l;Ary, and primer oli~onucleotides were synthesized by the phosphoramidite method (see McBride et al. (1983) TetrahedronLett. 24:245) using an Oligo lOO~M
automated DNA synthesizer (Beckman, Fullerton, CA) . Target , ~l~Y; l; ~ry, and primer oligonucleotide analogs were synthesized by known methods (Uhlmann et al. Analyt. Chem. (1990) 90:543-583), and then desalted, lyophilized, and reconstituted in buffer for the sequencing protocol or in sterile water for HPCE (Lyphomed Deerfield, IL) .
An ~Y~ ry oligonucleotide is prepared from 1~ bases at its 3 ' end which are complementary to the sequence of the M13mpl8 (-21) primer. Next is a signalling region of ten T bases. Then, base ~1 of the tar~et oligonucleotide to be sequenced is 3 0 located directly 5 ~ of the AllY; l; ;Iry oligonucleotide .
WO95/20680 2 l 8 0 722 PCT/US95/01104 A 12mer bridge is prepared which consists of two regions of six bases, one region being complementary to the last six bases of the ry oligonucleotide at its 5 ' end and the other being complementary to a predetermined f irst six bases of the target oligonucleotide at its 3 ' end .
The primer is labelled by covalently attaching a label to its 5' end. This is accomplished by phosphoramidite chemistry using an automated oligonucleotide synthesizer.
Alternatively, the primer is labelled by covalently attaching a fluorescent tag such as derivatized fluorescein ("~AM") to its 5~ end by using chemical DNA or RNA synthesis as described by Smith (Am. Biolab. (1989) May: 11-20) .
2 0 2 . Preparation of ~igation Product Including an oligonucleotide With a Known Sequence About 6 ~g of target oligonucleotide are mixed with 3 ~g of 5 ~ -phosphorylated All~ I'ry D~la, 4 llg of bridge oli~nnl~rl ~ntide, and 5 ~11 lOX
ligation buffer (USB 70087) . The firlal volume is about 15 ~1. This mixture is incubated at 37 C
for 15 minutes and then cooled in an ice bath at 4C for 20 minutes. 1 ~1 T4-DNA ligase (USB
70005, 300 units/~l~ is then added to the mixture .
and kept at 37C for 1 hour. The ligase is inactivated at 70C for 5 minutes.
wo g5/20680 ~ 2 ~ 8 ~ 7 ~ 2 PCT/US95/0ll0~ ~
3. Preparation of Ligation Product Including an Oligonucleotide With an Unknown Sequence A. No Bridge Method 5 ~ =
This protocol is based on the method of Tessioer (AnaIyt. Biochem. (1986) 158 :171-178) . 3 ~Ll 5X ligation buffer (50 mM MgCl~, 5 mM Co(NH3) 6C13, 250 mM Tri~ Cl, p~ 8, 50 mg/ml bovine serum albumin (USB 10848) ) is mixed with 6 ~g target oligonucleotide, 1 ~g Ai~ iAry DNA, 5 ~Ll 509;
polyethylene glycol (USB 19959), and 2 ~1 T4- RNA
ligase (USB 21245,20,000 ,untml) . The final volume is about 15 ~Ll . ~111~;1; Ary DNA i3 phosphorylated from 5' ena; and amino-linked from the 3' end to eliminate the formation o~ side ligation reaction products including the All~; 1 ;Al-y oligonucleotide and the primer. This mixture iE incubated at 25C
overnight .
B. Bridge Method A set of sixteen bridge oligonucleotides are synthesized by phosphoramidite chemistry uæing a Beckman Oligo 100 ' (Fullerton, CA) .
Then, about 6 ,ug of target oli~nnll~ ntide are mixed ~ith 3 l~g of 5 ' -phosphorylated AlllC'; l; ::lry DNA, 4 x 16 = 32 ~g of bridge oligonucleotide, and 5 ILl lQX ligation buffer (USB 70087) . The final volume is about 15 1ll. This mixture is incubated at 3 7 C f or 15 minutes and then cooled in an ice bath at 4C for 20 minutes. 1 111 T4-DNA ligase WO95/20680 2 ~ 22 PCTIUS95l011W
(USB 70005,300 units/lll) is then added to the mixture and kept at 37C for 1 hour The ligase is inactivated at 70C for 5 minutes.
The terms "modified oligonucleotide" and "oligonucleotide analog, n as used herein, Wo gS/20680 PCT/US9S/0ll0~ ~
2~80722 encompass a molecule of ribonucleotides or deoxyribonucleotides which are covalently linked via at least one synthetic linkage. A ~synthetic internucleotide linkage" is a linkage other than a phosphodiester between the 5 ~ end of one nucleotide and the 3 ~ end of another nucleotide in which the 5 ' internucleotide phosphate has been replaced with any number of chemical groups.
Representative synthetic linkages include phosphorothioates, phosphorodithioates, alkylphosphonothioates, phosphoramidates, phosphate~esters, carbamates, carbonates, phosphate:~triesters, acetamidate, and carboxymethyl esters.
The term ~oligonucleotide analog" also en.~ 8PR oligonucleotides with a modified base and/or sugar. For example, a 3 ', 5 ~ -substituted oligonucleotide i8 a modif ied oligonucleotide having a sugar which, at both its 3~ and 5~
positions is attached to a chemical group other than a hydroxyl group (at its 3 ~ position) and other than a phosphate group (at its 5 ' position) .
A modif ied oligonucleoti~e may also be a capped species. Also .onr~nTnr~RRed by these terms are unoxidized oligonucleotides or oligomers having a substitution in one nonbridging oxygen per nucleotide in the molecule.
3 0 oligonucleotide analogs may also be synthetic oligonucleotides" which ~n~ RRe8 polymers of 3 to 5'-linked ribonucleosides, 2'-modified ribonucleosides and/or deoxyribonucleosides having W095l20680 2 1 8~7~ PCTIUS95/0ll0~
only as many nucleosides as are conveniently chemically synthesized (i . e., up to about 80 100). Also encompassed are those oligonucleotides having base or sugar modif ications as well as those having nuclease resistance-conferring bulky substituents at their 3' and/or 5' end(s), multiple ribonucleosides and/or deoxyribonucleosides linked via an internucleotide linkage not found in native DNA, i.e., linkages other than phosphodiester bonds, or having modif ied bases and/or sugars in various other structural modifications not found in vivo without human intervention.
In the method of the invention, a single-stranded ligation product is prepared which includes a target oligonucleotide-to-be-sequenced and an ~ r;l;Ary oligonucleotide, each having a 3' and 5 ~ end The auxiliary oligonucleotide has a nucleotide sequence complementary to the sequence of a primer-to-be-used. In one aspect of the invention, the ~ ry oligonucleotide also includes a si~n~ll;n~ sequence of at least four contiguous nucleotides at its 5 ' end, which become linked to the 3 ' end of the target ol; ~r~nll~l eotide in the ligation product. In some embodiments, the i~lll~; l; ~rV oligonucleotide is at least eight nucleotides in length.
3 0 The invention provides several pref erred methods of preparing the single-stranded ligation product depending in part upon the degree to which the sequence of the target oligonucleotide is Wo 95/20680 PCT/US95/OllOJ
2i~7~2 known, I~at least the last three nucleotidee, and preferably six, are known, a bridge oligonucleotide i6 prepared which is complementary to these known nucleotides at its 5 ' end and which further includes a nucleotide sequence that is complementary to the first at least four 5' nucleotides of the auxiliary nucleotide. This bridge is used to anneal to the 3 ' end of the target oligonucleotide and the 5 ' end of the auxiliar~v nucleotide, forming a double stranded construct. The target oli~nllrl o~tide can then be ligated to the ~ rry oligonucleotide via a ligase .
= A bridge is also used in another embodiment where the sequence of the target oligonucleotide is completely unknown. In this method a set of sixteen bridge oligonucleotides i8 prepared. Each bridge nl ;~nllt~ tide i5 identical in part in having about six nucleotides at its 3' end which are complementary to the six nucleotides at the 5 ' end of the auxiliary oligonucleotide. The bridge oligonucleotides differ in having at their 5' ends a unique dinucleotide sequence (i . e ., one of sixteen possible combinations of four nucleotides), one of which being complementary to the last two unknown 3 ' nucleotides of the target nli~r~nllrl~ tides). The one bridge ol;~-nl-~leotide which anneals to the auxiliary and target oligonucleotides provides the same function as described above in the first bridge ~mhori; t.
Wo 95/20680 2 1 ~ ~ 7 ~ 2 PCT/US9~/01104 _ g _ In another preferred embodiment, the method used for preparing the ligation product including a target oligonucleotide with a completely unknown sequence is as follows. An auxiliary oligonucleotide i5 prepared which, like those described above, is composed of a 3 ~ sequence Cornrl~ ti:lry to the gequence Qf a primer to be used. This sequence is linked at its 5 ~ end to a signalling se~uence of at least four known contiguous nucleotides . The 3 ' nucleotide of the ~llC; l; Ary oligonucleotide is protected in some cases (when using RNA ligase) . The 5 ' end oi this ;l ;Ary oligonucleotide is ligated directly to the 3 ' end of the target molecule. In preferred a3pects of the invention, ligation is accomplished with a blunt end ligase such as T4 RNA ligase.
In any case, to this ligation product is annealed a primer which has a nucleotide sequence complementary to a portion of the auxiliary oligonucleotide. In some aspects of the invention, the primer comprises a nucleotide sequence complementary to at least four, but pref erably at least eight, nucieotides of the All~; l; ;Iry oligonucleotide portion of the ligation product. This primer also has a label covalently attached theret4. Preferably, this label is a fluorescent, chemiluminescent, or radioactive tag.
Most preferably, the label is a fluorescent label which is excitable in the W or visible range and which fluoresces in the visible range.
Wo 95/20680 PCT/US95/0l lO~
2~8~722 Next, the primer is extended with chain-extending-nucleoside triphosphates and chain-terminating nucleoside triphosphates in the presence of a polymerase to yield a plurality of primer extension products of differing ~engths.
The~e products are then separated on the basis of their relative mobilities, from which the nucleotide se~uence of the target oligonucleotide can be derived. In preferred embodiments of the invention separation is achieved by slab gel or high performance capillary gel electrophoresis.
Depending on the primer label used, laser-induced f~uorescence, W absorption, or radiation are mea3ured to determine the relative mobilities of the primer extension products.
WO9s/20680 2 f 8 ~ ~ ~ 2 PCT/US95/0ll04 BRIEF D~SCRIPTIQN QF T~ DRAWINGS
The foregoing and other objects of the present invention, the various features thereof, as well as the invention itself may be more fully understood from the following description, when read together with the ;3 ~ nying drawings in which:
FIG. lA is a schematic representation of one embodiment of the invention by which a single-stranded ligation product is prepared from a target oligonucleotide whose sequence is partially known;
FIG. lB i9 a schematic representation of :
another e~nbodiment of the invention by which a single-stranded ligation product is prepared, with the aid of a bridge oligonucleotide, from a target ~ n~ l eotide whose sequence i9 unknown;
FIG. lC is a schematic representation of yet another embodiment of the invention by which a single-stranded ligation product is prepared, without the aid of a bridge oligonucleotide, from a target oligonucleotide whose sequence i8 unknown;
FIG. 2A is a W electropherogram of a reaction mixture r~r~nt~;n;n~ ATP, a 12mer bridge oligonucleotide, a 25mer target oligonucleotide whose sequence is partially known, and a 31mer All~ ry oligonucleotide;
W095/20680 2 1 807~2 PCr/l~S9510110~ ~
FIG. 2B is a W electropherogram of the reaction mixture described in FIG . 2A af ter ligation with TfDNA ligase, and shows the species in FIG. 2A as well as a 57mer ligation product;
FIG. ~3A i9 a W electropherogram of the HPCE
separation of the components of a TçRNA ligase reaction mixture including ATP, the target oligonucleotide, and a 31mer ~ ry oligonucleotide with an unprotected 3 ' hydroxyl group;
FIG. 3B is a W electropherogram of HPCE
l~ separation of the components of a TjRNA ligase reaction mixture as in FIG 3A, except using a 31mer auxiliary oligonucleotide with a 3 ' amino protected end, and resulting in a 57mer ligation product;
FIG. 4A is an LIF electropherogram of the HPCE separation of primer extension products made from, and complementary to, the 57mer ligation product using the ddA terminat~d sequencing reaction, and separated as in FIG. 2B;
FIG.--4B is an ~IF electropherogram of the HPCE separation of primer extension products made from, and complementary to, the 57mer ligation 3 0 product using the ddG terminated se~uencing reaction;
W095l20680 21 80722 r~ o4 FIG. 4C is an ~IF electropherogram of the HPCE separation of primer extension products made from, and complementary to, the 57mer ligation product using the ddT terminated sequencing reaction;
FIG. 4D i8 an bIF electropherogram of the HPCE separation of primer extension products made from, and complementary to, the 57mer T4 DNA
ligase ligation product using the ddC terminated sequencing reaction;
FIG. 5A is a computer overlay of the ~IF
electropherograms from FIGS. 4A-4C using two point re-size alignmenti FIG. 5B is a computer overly of the ~IF
electropherograms from FIGS. 4A-4D, using two point re-size alignment; and FIG . 6 is a plot of relative f ragment migration versus base number f or the data presented in FIGS. 5A and 5B, showing a linear relationship with R2=o . 999 .
W09s~20680 2 ~ 8Q722 r~ o~ ~
BRIEF ~ESCRIPTION ~F T~ ) EMBODIMENTS
The patent and scientific literature referred to herein establishes the knowledge that i8 available to those with skill in the art. The issued U. S . patent and allowed applications cited herein are hereby incorporated by ref erence .
This invention provides a new ser~uencing procedure which determines the se~uence of a target oligonucleotide from its very first 5' nucleotide to its very last 3 ' nucleotide, despite the shortness of its length or~ the fact that it may be an oligonucleotide analog with non-phosphodiester ;n~f~rnllrleotide linkages and/or other modif ications . In fact, any target oligonucleotide can, in principle, be se~uenced.
For e~cample, the target oligonucleotides-to-be-sequenced can range from about 4 to about 100 nucleotides in length, with oligonuclçotides having from about 8 to about 50 nucleotides in length being most common Furthermore, target oli~nnl~rl ~ntides can have any type of internucleotide l; nk;~r~c or even any combination of different types of ;ntf~rnl~rleotide linkages, as long as the target oligonucleotide can be ligated to the ~-l~; l; ;Iry oli~nni~rl Pntide and can be extended by a polymerase. For example, a target 3 0 oligonucleotide may have more than one non-phosphodiester linkage, and up to having all non-phosphodiester linkages. The non-~hosphodiester l; nk~ present in the target oligonucleotide may wo 9sno680 2 1 8 0 7 2 2 PCr/US95l0110~
include at least phosphorothioate, alkylphosphonate, phosphoramidate, alkylphosphonothioate, phosphodithioate, and sulfone, 8U lfate, keto, phosphate ester, bridged phosphorothioate and bridged phosphoramidate linkages, all of which are known in the art (see Uhlmann et al. (1990) (Chem. Re~. 90:543-584 for a review on the synthesis and characteristics of phosphodiester and non-phosphodiester-linked antisense oli~n~ tides).
The method of the invention requires the =
preparation of an 'IAllx;l ;~ry oligonucleotide"
which is used ior ligation to the 3 ~ end of the target oligonucleotide-to-be-sequenced, thereby forming a single-stranded ligation product. The auxiliary oligonucleotide is a single-stranded RNA, DNA, or RNA/DNA-r~nti~;n;n~ molecule with a known sequence that is complementary to a primer.
The ~3--,c; l; i~ry oligonucleotide may also include at its 5' end a region of at least four preselected, contiguous nucleotides which serves as a marker or .
"si~n~l 1 ;n~ 8equence. "
This ~]~; l; i~ry oligonucleotide is ligated to the target molecule to form a single-stranded ligation product, wherein base #1 of the target oligonucleotide-to-be-sequenced is located directly after the ~--~; l; ;Iry DNA.
The invention provides several protocols for preparing the ligation product, depenaing on the degree to which the sequence of the target WO 95/20680 PCTIUS95/0ll0~
2 ~ 8~7~2 oligonucleotide is known. If at least the laet three, and preferably six nucleotides at the 3 ' end of the target are known, a bridge oligonucleotide can be constructed which supports the target r~ nllrl eotide and facilitates the ligation reaction. The bridge includes ribonucleotides and/or deoxyribonucleotides linked via phosphodiester and other than phosphodiester lnt~rn~ leotide linkages. This molecule is complementary to these at least six nucleotides and further; n~ Pq a sequence that i5 complementary to the first at least four 5' nucleotides of the auxiliary nucleotide. The bridge oligonucleotide is annealed to the target oligonucleotide and to the All~; l; Ary oligonucleotide such that the f irst two 3 ' nucleotides of the bridge oligonucleotide are Annf~Al ed to the laet two 3 ~ nucleotides of the target oligonucleotide, and at least the Ilext 9ix nucleotides of the bridge oligonucleotide towards its 5~ end are annealed to the first six 5' nucleotides of the ~llr; l; Ary oligonucleotide, thereby yielding a partially double stranded construct . Then, the 3 ' end of the target oligonucleotide is ligated to the si~nAl l; n~
sequence at the 5 ' end of the ~ ; Ary oligonucleotide with a template-dependent enzyme such as T~ DNA ligase or Ta~ DNA ligase. Upon denaturation, a single-stranded ligation product 3 0 is obtained .
An example of this ligation protocol is shown schematically in FIG. lA, where at least 3 bases W095l20680 2 l ~2~ PCT/U595/0ll0.1 .
of a target oligonucleotide are already known. In this figure, the bases that are unknown are depicted as "?". A 12mer bridge (SEQ ID NO:2) is prepared to facilitate the ligation of the ~117r; 1; ~ry oligonucleotide to the target molecule.
This bridge consists of two regions of six bases, one region that is complementary to the last six bases of the auxiliary oligonucleotide (SEQ ID
NO: 3 ) at its 5 ' end and the other being complementary to a predetermined first six bases of the target oligonucleotide at its 3 ' end which are known. FIGS. 2A and 2B show the separation of varies species of oligonucleotides (target, auxiliary, and bridge) by capillary electrophoresis, followed by W detection, before and after ligation with T4 DNA ligase, respectively. Migration order of detected peaks in FIG. 2A is (1) the fast migrating 12mer bridge (SEQ ID NO:2); (2) the target oligonucleotide-to-be-sequenced; and (3) the auxiliary oligonucleotide. When T4 DNA ligase and ATP are added to the reaction mixture, after 30 minutes incubation at 37 C, a 57mer ligation product is observed (FIG. 2s).
If the entire sequence of the target oligonucleotide is unknown, two methods of preparing the ligation product be used. One, like the method above, also requires the support of a bridge oligonucleotide. In this method, a set of sixteen bridge oligonucleotides is prepared, all of which are identical at their 5 ~ ends because they include a sequence complementary to the same WO 95/20680 ~ PCT/lJS95/0ll0~
2~8~72~
four, but preferably six to eight or more nucleotides of an ;111~; 1; i~ry oligonucleotide to be used. In addition, the bridge oligonucleotides include at their 3 ' ends one of sixteen possible dinucleotides: AA, AC, AG, AT, CA, CC, CG, CT, GA, GC, GG, GT, TA, TC, TG, TT. One of these rlinll~ otideg Will be complementary to the two most 3 ' nucleotides of the target oligonucleotide .
Thus, when this set of sixteen bridge oligonucleotides is mixed with the target oligonucleotide of unknown sequence and with the auxiliary oligonucleotide described above, under conditions conducive for ~nn.sAI ;n~, the one bridge oligonucleotide having a dinucleotide complementary to the last two 3 ' nucleotides of the target molecule will hybridize to it as well as to the ~ll~i 1 i i~ry oligonucleotide. This method is shown schematically in FIG. lB, where the sixteen bridge oligonucleotides have SEQ ID ~OS: 6-21, and each unknown base in the target oligonucleotide is depicted shown as a "?".
Alternatively, a blunt end ligase such as T4 RNA ligase may be used which does not require the presence of a double-stranded construct to link two nucleotides together. In this case, an auxiliary oligonucleotide is prepared which is composed of a 3 ' -sequence complementary to the sequence of a primer to be used, linked to a 5 ' -f:i~ni~l ;ng~-sequence of at least four contiguous nucleotides. The 3~ end of this i~il~;7 ;~ry oligonucleotide is proFected with, for example, a dideoxynucleotide (ddA, ddC, ddG, ddT) or an amino WO95/20680 2 ~ PCTIUS9~/0l10l group . The 5 ~ end of the auxiliary oligonucleotide is then ligated to the 3 ~ end of the target oli~nnllrl .o-ntidel thereby forming a single-stranded ligation product.
An example of this ligation protocol i9 shown schematically in FIG. lC. T4 RNA ligase is used to ligate a target oligonucleotide of unknown seriuence to a 31mer ;ill-f; 1; ;iry oligonucleotide (S~Q
ID NO:3) without the presence of a bridge in the reaction mixture. The W electropherogram shown in FIGS. 3A and 3B demonstrates the synthesis of the 57mer ligation product . If an ;iil~; l; ;iry oligonucleotide is used having an unprotected 3 ' end, the enzyme forceæ the ligation process to :~
proceed in cycles and several cycles are observed (FIG. 3A) . This undesirable ~ onnm~nnn is prevented simply by uging an ~ ry oligonucleotide with a protecting dideoxy or an amino group at its 3 ' end, as shown in FIG . 3B
where only one ligation cycle is obtained. As in the T4 RNA ligase case, the ATP 25mer analog, auxiliary-31mer, and 57mer ligation product are observed .
Once the single-stranded ligation product rnnt~i;n;ng the target molecule is formed, a primer -is annealed to ligation product, f rom which strands complementary to the target molecule can be f~t,on~ by a polyinerase (i.e, primer extension products). The primer oligonucleotide can be any of the conv~ontinn~il types o~ RNA and/or DNA-rnnt;i;n;nr~ oligonucleotides that are well known Wo 95/20680 PCTIUS95/01l0~
2~ ~Q7~2 and commonly used for DNA or RNA sequencing or primer extension reactions. The primer oligonucleotide has a se~uence that is complementary to a 3 ' portion of the auxiliary oligonucleotide region of the ligation product which does not include the si~n~l l; n~ region .
At least one molecule of a label such as a luminescent, radioactive, or fluorescent label is attached to the primer. If the label is fluoresce~t it is excitable in the W or visible wavelength range, and fluoresces in the visible range. Such labels include fluorescein, or the N-sllfcin;m;de ester or other derivatives thereof, such as called "JOE" (Applied Biosystems, Foster City, CA), "FITC" (Applied Biosystems, Foster City, CA), and "FAM" (Applied Biosystems, Foster City, CA), and rhr~ m;n~, or derivatives thereof, such as tetramethylrh~ m;n~ ("TAMARA") (Applied Biosystems, Foster City, CA) and "Texas Red" or "ROX" (Applied Biosystems, Foster City, CA) (Smith ( 19 85) Nucl. Acid. ~es. 13: 23 9 9 -2412 ) . These labels can be covalently attached to the primer, for example, by using chemical DNA or RNA synthesis as described by Smith ~Am. Biolab. (1989) May:11-20), or by other methods which will not interfere with the ability of the primer to hybridize to the target molecule or to be ligated to the helper oligouucleotide An example of one such method includes covalently attaching an amino group onto the dye and then linking the amino group 5 ' end of oligonucleotide (Smith (1985) Nucl. Acid. Res.
WO 9S/20680 2 1 ~ ~ 7 2 2 PCTiUS9S/ollo~
13:2399-2412). Alternatively, the fragment may be f luorescently labelled with dideoxynucleotides .
~nn~l ;n~ of the bridge, ~ ry, and target oligonucleotides, and of the primer and ligation product is accomplished under conditions that are most conducive for the hybridization of a single-stranded species to a complementary, single-stranded oligonucleotide. These conditions include contact in ligation buffer (600 mM Tris-HCl, pH 7 . 6, 6 6 mM MgCl2, 10 0 mM DTT, 6 6 0 llm ATP ) at a temperature of from about 4C to 90C, but preferably at room temperature (i.e., 19C to 25C) .
Upon annealing of the primer to the ligation product, the primer extension reaction can take place in the presence of a polymerase. Many polymerases are known in the art and all are suitable in principle. Usually, a DNA polymerase will be used such as Taq DNA polymerase or T4 DNA
polymerase. If the very well known Sanger et al.
(ibid. ) eequencing method is to be followed, nucleotides and dideoxynucleotides are used to synthesize the primer extensions.
Finally, the dideoxy-terminated extension products are separated according to any number of well-known standard procedures that separate such molecules on the basie of size. Such protocols include polyacrylamide slab gel electrophoresis or high performance capillary gel electrophoresis.
Depending on the primer label used, laser-induced Wo g~/20680 PCTNS9S/OllO~
2~722 fluorescence, W absorption, or radiation are measured to deterlrLine the relative mobilities of the primer extension products.
For example, an auxiliary oligonucleotide is prepared from 17 bases at its 3 ~ end which are complementary to the sequence Qf the M13mpl8 (-21) primer. Next is a signalling region o ten T
bases. Then, base #l of the target 0 oligonucle-otide to be se~uenced is located directly 5 ~ to the ~ ry oligonucleotide .
In developing an automated single-stranded oligonucleotide se~uencer for routine antlsense analysis, a working strategy was developed to examine the enzymatic sequencing of single-stranded oligonucleotide analogs which ;nflll~l the ligation products described above. The f ollowing strategy was used to develop an expression for the electrophoretic migration of sequencing fragments which is an P~Pnt;~l element of automated data processing.
Over~a narrow range of molecular size, a linear relationship between relative migration time (T' ) and base number can be established using two ;ntPrn~l standards in what amounts to a two-point calibration. These two point~ are the primer (17mer) and the 58mer iragment which is one 3 0 base longer than the ligation product due to the endonuclease a~tivity of sequenase 2 . O . This linear relationship i8 described as follows:
Wo 9~/20680 PCTIUS9~l01104 T ' = TD - Ib~
5 Tf in - Tp r where "Tp" is the migration time of sequencing fragment; "Tpr" is the migration time of primer;
and "Tfin" iB the migration time of the last peak.
This relation8hip i9 linear for T' which is only fragment size-dependent.
To validate this term, the expression was tested under experimental cr~nf~;tl~ln~ as follows.
A 57mer ligation product (SEQ ID NO:4) (e.g., a 25mer target oligonucleotide analog (SEQ ID NO :1) + a 32mer ;~lll~;li~ry oligonucleotide (SEQ ID NO:3)) was subjected to enzymatic chain termination reaction for four different bases ;n~1~r~n~n~1y (i.e., A, G, C, T) . Each of the four reaction mixtures were run separately on different days and different gel columns. LIF electropherograms of the separation of the four sets of primer extensio~ products are shown in FIGS. 4A-4D.
~Y~nci r~n productg were separated by HPCE using a gel containing 12~ T acrylamide, 6.5 M urea, and 4096 (weight:weight) formamide. These figures are the computer two point alignments for T, G, A and C reactions . The f irst point iB the 17mer primer and the 3econd point i9 the 57mer latest migrating fragment. T' was calculated individually for each of the detected fragments between 17 and 57 bases in length. Fragment 33 corresponds to the 25mer target oligonucleotide. The obtained values are then rearranged in order form low to high, according to the occurrence in the four runs for the four individual bases in FIGS. 4A-4D. The Wo 9s/20680 PCT/US9510110~
2~72~
results are su~nmarized in TABLE l, which is a computer printout of relative migration obtained using two point re-size alignment for the data obtained from ~IGS. 4A- 4D.
T~3~ l Reading Base A T C G S~ nci Real Targe~ 01igo 3' 33 1 0.370 T
34 2 0.391 C
5 35 3 0.421 T
36 4 0.441 T
37 5 0.460 C
38 6 0.485 C
39 7 0.512 T
2 0 40 8 0.537 C
41 9 0.570 T
42 10 0.586 C
43 11 0.620 T
44 12 0.636 C
25 45 13 0.671 T
46 14 0.680 A
47 15 0.707 C
48 16 0.735 C
49 17 0.763 C
3 0 50 18 0.786 A
51 19 0.812 C
52 20 0.828 G
53 21 0.856 C
54 22 0.896 T
35 55 23 0.904 C
56 24 0.948 T
57 25 0.953 C
5' WO 95/20680 PCT/US95/0ll0~
2~80722 A6 indicated above, the sequence of the target oligonucleotide is determined in the right-most column of Table 1 from 3 ' to 5 ' end. This sequencing format is the result of computer software capable of ~-~t( t1ng the sequencing system and performing data processing.
When this software is interfaced with a commercial software (e.g., Turbochrome~ III, P.E.
Nelson, Cupertino, CA), the data obtained can be manipulated such that the f inal electropherogram of the sequencing can be plotted, as illustrated in FIGS. 5A and 5B. The results are summarized in FIG. 6 which demonstrates the linear relati"n~Th1E' between fragment migration and base number.
Thus, a mathematical expression has been ~:
derived and successfully used for an automated single-stranded oligonucleotide sequence determination. The strength of this expression is that "T' '~, relates only to the fragment length expressed as base number. Moreover, this expression is independent of the experimental conditions given that all sequencing fragments are separated. Separation is not a problem, since gel capillary can separate sequencing fragments with very high efficiency and resolution (Cohen et al.
(1988) J. Chrom~togr. 458:323; Swerdlow et al. (1990) Nucl. Acidsl~es. 18:1415-1419; Pentoney et al. (1992) EleT~r~,T,J,Tsis 13:461--74; Cohen et al. (1993) TRAC
12: 195-202) .
WO 9sno680 2 1 8 ~ 7 2 2 PCr/US95l0ll0~ ~
The following examples illustrate the pre~erred modes of making and practicing the present invention, but are not meant to limit the 3cope of the invention since alternative methods may be utilized to obtain similar results.
EXAMPLES
1. Preparation of Target, Auxiliary, Bridge, and Primer Oligonucleotides Phosphodiester-linked target, i31ly;l;Ary, and primer oli~onucleotides were synthesized by the phosphoramidite method (see McBride et al. (1983) TetrahedronLett. 24:245) using an Oligo lOO~M
automated DNA synthesizer (Beckman, Fullerton, CA) . Target , ~l~Y; l; ~ry, and primer oligonucleotide analogs were synthesized by known methods (Uhlmann et al. Analyt. Chem. (1990) 90:543-583), and then desalted, lyophilized, and reconstituted in buffer for the sequencing protocol or in sterile water for HPCE (Lyphomed Deerfield, IL) .
An ~Y~ ry oligonucleotide is prepared from 1~ bases at its 3 ' end which are complementary to the sequence of the M13mpl8 (-21) primer. Next is a signalling region of ten T bases. Then, base ~1 of the tar~et oligonucleotide to be sequenced is 3 0 located directly 5 ~ of the AllY; l; ;Iry oligonucleotide .
WO95/20680 2 l 8 0 722 PCT/US95/01104 A 12mer bridge is prepared which consists of two regions of six bases, one region being complementary to the last six bases of the ry oligonucleotide at its 5 ' end and the other being complementary to a predetermined f irst six bases of the target oligonucleotide at its 3 ' end .
The primer is labelled by covalently attaching a label to its 5' end. This is accomplished by phosphoramidite chemistry using an automated oligonucleotide synthesizer.
Alternatively, the primer is labelled by covalently attaching a fluorescent tag such as derivatized fluorescein ("~AM") to its 5~ end by using chemical DNA or RNA synthesis as described by Smith (Am. Biolab. (1989) May: 11-20) .
2 0 2 . Preparation of ~igation Product Including an oligonucleotide With a Known Sequence About 6 ~g of target oligonucleotide are mixed with 3 ~g of 5 ~ -phosphorylated All~ I'ry D~la, 4 llg of bridge oli~nnl~rl ~ntide, and 5 ~11 lOX
ligation buffer (USB 70087) . The firlal volume is about 15 ~1. This mixture is incubated at 37 C
for 15 minutes and then cooled in an ice bath at 4C for 20 minutes. 1 ~1 T4-DNA ligase (USB
70005, 300 units/~l~ is then added to the mixture .
and kept at 37C for 1 hour. The ligase is inactivated at 70C for 5 minutes.
wo g5/20680 ~ 2 ~ 8 ~ 7 ~ 2 PCT/US95/0ll0~ ~
3. Preparation of Ligation Product Including an Oligonucleotide With an Unknown Sequence A. No Bridge Method 5 ~ =
This protocol is based on the method of Tessioer (AnaIyt. Biochem. (1986) 158 :171-178) . 3 ~Ll 5X ligation buffer (50 mM MgCl~, 5 mM Co(NH3) 6C13, 250 mM Tri~ Cl, p~ 8, 50 mg/ml bovine serum albumin (USB 10848) ) is mixed with 6 ~g target oligonucleotide, 1 ~g Ai~ iAry DNA, 5 ~Ll 509;
polyethylene glycol (USB 19959), and 2 ~1 T4- RNA
ligase (USB 21245,20,000 ,untml) . The final volume is about 15 ~Ll . ~111~;1; Ary DNA i3 phosphorylated from 5' ena; and amino-linked from the 3' end to eliminate the formation o~ side ligation reaction products including the All~; 1 ;Al-y oligonucleotide and the primer. This mixture iE incubated at 25C
overnight .
B. Bridge Method A set of sixteen bridge oligonucleotides are synthesized by phosphoramidite chemistry uæing a Beckman Oligo 100 ' (Fullerton, CA) .
Then, about 6 ,ug of target oli~nnll~ ntide are mixed ~ith 3 l~g of 5 ' -phosphorylated AlllC'; l; ::lry DNA, 4 x 16 = 32 ~g of bridge oligonucleotide, and 5 ILl lQX ligation buffer (USB 70087) . The final volume is about 15 1ll. This mixture is incubated at 3 7 C f or 15 minutes and then cooled in an ice bath at 4C for 20 minutes. 1 111 T4-DNA ligase WO95/20680 2 ~ 22 PCTIUS95l011W
(USB 70005,300 units/lll) is then added to the mixture and kept at 37C for 1 hour The ligase is inactivated at 70C for 5 minutes.
4 . Primer AnnP;31 ;n~
The single-stranded ligation product 3 ~Ll 0.1 pM/~l was mixed with 8 1ll 0 4 pM//ll primer (ABI
401131 - 21M13 primer) was mixed with 3 ~l 0.1 pM/~Ll ligation product and 4 ,ul 5X sequencing buffer (USB 70702) The mixture is heated at 65C
for 10 minutes and cooled slowly to room temperature for 30 minutes to allow for ~nnP;Il in~
The single-stranded ligation product 3 ~Ll 0.1 pM/~l was mixed with 8 1ll 0 4 pM//ll primer (ABI
401131 - 21M13 primer) was mixed with 3 ~l 0.1 pM/~Ll ligation product and 4 ,ul 5X sequencing buffer (USB 70702) The mixture is heated at 65C
for 10 minutes and cooled slowly to room temperature for 30 minutes to allow for ~nnP;Il in~
5. Sequencing the Primer Extension Products by the Chain Termination Method Primer extension/termination is accomplished as follows: 15 ILl annealed mixture is mixed with 5 ~ n~lnP~e buffer (USB 72600), 2 ILl 0.1 M
dithiothreitol (USB 70726), 2 ~l sequenase version 2 . O (USB 70775), and 1 ~11 pyrophosphates (USB
70950) in the presence of either four different mixtures ~ nt~;n;ng 4 ~Ll dNTP (2 mM each), 2 ~l o . 5 mM dd~TP or ddCTP or ddTNP or ddGTP, or a single mixture c~nt~;n;ng a specific ratio of dideoxynucleotide~ ddATP, ddCTP, ddTNP, and ddGTP
(8:4:2:1) as described by Tabor and Richardson (J
Biol. Chem. (1990) 265:8322-8328) The mixture is incubated at 37C for 15 minutes and precipitated with 70~6 EtOH twice.
WO 95/20680 2 1 8 ~ 7 ~ 2 PCT/US95/0110~ ~
dithiothreitol (USB 70726), 2 ~l sequenase version 2 . O (USB 70775), and 1 ~11 pyrophosphates (USB
70950) in the presence of either four different mixtures ~ nt~;n;ng 4 ~Ll dNTP (2 mM each), 2 ~l o . 5 mM dd~TP or ddCTP or ddTNP or ddGTP, or a single mixture c~nt~;n;ng a specific ratio of dideoxynucleotide~ ddATP, ddCTP, ddTNP, and ddGTP
(8:4:2:1) as described by Tabor and Richardson (J
Biol. Chem. (1990) 265:8322-8328) The mixture is incubated at 37C for 15 minutes and precipitated with 70~6 EtOH twice.
WO 95/20680 2 1 8 ~ 7 ~ 2 PCT/US95/0110~ ~
6. Separ~tion of the Primer Extension Products by HPOE
Gel-filled capillaries are prepared as follows. Fused-silica capillary tubing (Polymicro Technologies, Phoenix, AZ, USA) with an inner diameter of 75 ~m, an outer diameter of 375 llm, an effective lRngth of 10-15 cm, and a total length of 3 0 - 60 cm wa8 treated with (methylacryloxypropyl) trimethoxysilane (Petrach Systems, Bristol, E'A, USA), and then filled with a de-gassed solution of 14~6 T polymerizing acrylamide, 7 M urea in aqueous or f ormamide media (TBE buffer: 0.1 M to 0.3 M Tris-borate, 2-6 mM
EDTA pH 8 . 3, 7 M urea) . Polymerization was achieved by adding ;3~mnnlllm per8ulfate solution and ~, N, N', N',-tetramethyl-ethyl~n,~ m;n~
(TEMED). This protocol is based on the method of Smith et a~. (Nature (1986) 321:674). The running buffer was:~0.2 M TBE, and the applied field was 400 V/cm.
The capillary electrophoresis apparatus with W and laser-induced fluorescence detection for the separation of oligonucleotide sequencing fragments is the same a8 that de8cri~ed by Smith et al (Nature (1986) 321:674). Briefly, a 30 kV, 5 o o ~LA direct current high voltage power supply (Model ER/DM; Glassman, Whitehouse Station, NJ, 3 0 USA) is used to generate the potential across the capillary. W detection of phosphorothioates and other analogs at 270 nm was accomplished with a spectrophotometer (Spectra 110, Spectra-Physic8, ~, WO95/20680 ~ 1 8~72~ PCT/I~S95/0ll0 San Jose, CA) . For laser induced ~luorescence detection an argon ion la~er (Model 543 lOOBS, omnichrom, Chino, CA, USA) wa~ employed. The data were acquired and ~tored on an AcerPower 486/33 computer (Acer American Corp., San Jose, Q, USA) through an analog-to-digital converter (Model 970, Nelson Analytical, Cupertino, CA, USA) .
WO95/20680 21 80722 r~l~.n s llo~ ~
SEQUENCE LISTING
( 1 ) GENERAL INFORMATION:
i) APPLICANT: Cohen, S. Aharon, Belenky, Alexei and Ott, Christopher M.
(ii) TITLE OF L~V~;N'l'lON: A Method of Se~[uenci Short Ol igonucleotides (iii) NUMBER OF SEQUENCES: 21 (iv) CORRESPONDENCE ADDRESS:
(A) AnnRF~ sFl~ Lapp~n h Kusmer (B) STREET: 200 State Street ( C) CITY: Boston (D) STATE: Massachusetts ( E ) COUNTRY: USA
(F) ZIP: 02109 (v) COMPUTER RT'~n~RT,T~ FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
( D ) SOFTWARE: Patentln Release #1 .0 , Version #1 .2 (vi) CURRENT APPLICATION DATA:
(A) APPLICATIO~ NUMBER:
(B) FILING DATE:
( C) CLASS IFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Kerner, Ann-Loui8e (B) REGISTRATIO~ NUMBER: 33,523 (C) X~;~;~;NC~;/DOCKET NUMBER- HYZ-013PCT
( ix) TELECOMMUNI CATION- I~FORMATION:
( A ) TELEPXONE: 617 -13 D -13 0 0 (B) TELEFAX: 617-330-1311 ( 2 ) INFORMATION FOR SEQ ID NO :1:
U~N ~ T~ ~ ~Tl;~R T C T I CS:
(A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) STR~n~nl~ S: single (D) TOPOLOGY: l; near (ii) MOLECULE TYPE: cDNA
( iii ) ~Y ~ L CAL: NO
(iv) A-NTI-SENSE: YES
WO95/20680 2 ~ 8 ] 72 2 PCTIUS9510110'1 (Xi) ~;UU~N~ DESCRIPTION: SEQ ID NO:1: -CTCTCGCACC CA~ L~ l C CTTCT 2 5 ( 2 ) INFORMATIO~ FOR SEQ ID NO: 2:
.?U~;N~ RA(~T~RT~TIcs (A) LENGTH: 12 base pairs (B) TYPE: nucleic acid ( C ) STR ~Nn~nN~ : E ingl e ( D ) TOPOLOGY: l inear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
( iv ) ANTI - SENS E: NO
(xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
(2) INFORMATION FOR SEQ ID NO:3:
U~;N~ ; CHARACTERISTICS:
(A) LENGTH: 32 base pairs (B) TYPE: nucleic acid (C) STR~Nn~nN~ : single ( D ) TOPOLOGY: l inear ( i i ) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(Xi) ~ U~N~:~ DESCRIPTION: SEQ ID NO:3:
CTCCATTTTT TTTTTACTGG C~ jL11"1' AC . 32 ( 2 ) INFORMATION FOR SEQ ID NO: g:
1U~;N(:~; CHARACTERISTICS:
(A) LENGTH: 57 base pairs (B) TYPE: nucleic acid (C) STR~Nn~nN~ : 8ingle (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
( i i i ) HYPOTHETI CAL: NO
(iv) ANTI-SENSE: NO
WO9s/20680 2 1 8 Q 7 ~ 2 PCTIUS9VOI10~ ~
(Xi ) ~ UU~;NO~; DESCRIPTI~N: SEQ ID NO: 4:
CTCTCGCACC CATCTCTCTC ~ LC~ ~LLLll~LLlLL ACTGGCCGTC 50 GTTTTAC _ 5 7 (2) INFORM;~TION FOR SEQ ID NO:~:
U~NC~ CHARACTERISTICS:
(A) LENGTH: 16 base pairs (B) TYPE: nucl~ic acid (C) STR~Nn~nM~ ingle (D) TOPOLOGY: li~ear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) ~;UU~;N~:~; DESCRIPTION: SEQ ID NO:5:
TGCCGCCAGC AAaATG 16 ( 2 ) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE t~ 7T.CTICS:
(A) LENGTH: 8 base pair3 (B) TYPE: nucleic acid (C) STR~NDEDNESS: ~ingle ( D ) TOPO~OGY: l inear (ii) MOLECULE TYPE: cDNA
( i i i ) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) ~;~;UU~;N~; DESCRIPTION: SEQ ID NO:6:
AAGAGGTA ~ 8 ( 2 ) INFORMATION FOR SEQ ID NO: 7:
;5,)U~:N(~; CHARACTERISTICS:
(A) LENGTH: 8 base pairs (B) TYPE: nucleic acid (C) sTR~Nn~nNF~c~ ~ingle (D) TOP3LOGY: linear (ii) MOLECULE TYPE: cDNA
( i i i ) ~POTHET~CAL: ~o ... . ..
wogs/20680 2 ~ 22 PCT/US95/0110 (iv) ANTI-SENSE: NO
(xi) ~ U~:N~; DESCRIPTION SEQ ID NO:7:
AGGAGGTA ~ 8 (2) INFORMATION FOR SEQ ID NO:8:
;S.?U~;~; CH~RACTERISTICS:
(A) LENGTH: 8 base pairs (B) TYPE: nucleic acid (C) STR~NDEDNESS: single ( D ) TOPOLOGY: 1 inear (ii) MOLECULE TYPE: cDNA
( i i i ) HYPOTHETI CAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
( 2 ) INFORMATION FOR SEQ ID NO: 9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 base pairs (B) TYPE: nucleic acid (C) STRANn~nNF~c single ( D ) TOPOLOGY: 1 inear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: S13Q ID NO:9:
(2) INFORMATION FOR SEQ ID NO:10:
;5.?U~ rT~ARArT~RT,~TICS
(A) LENGTH: 8 base pairs (B) TYPE: nucleic acid (C~ sTRANn~nN~ single ( D ) TOPOLOGY: l inear ( ii ) MOLECULE TYPE: cDNA
( i i i ) ~ Y ~ CAL: NO
WO 95/20680 2 1 8 0 7 2 2 PCT/US95/0ll0~ ~
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
GAGAGGTA ~ 8 ( 2 ) INFORMATION FOR SEQ ID NO :11:
;UU~;N(~ C~RACTERISTICS:
(A) ~.ENGTX: 8 ba6e pairs (B) TYPE: nucleic acid (C) sTR~ND~nl~cc single (D) TOPOLOGY: linear ( ii ) MOI.ECULE TYPE: cDNA
( i i i ) XYPOTXET I CAL: ~O
(iv) ANTI-SENSE: NO
(Xi) ~ )U~;N~:~; DESCRIPTION: SEQ ID NO:11:
( 2 ) INFORMATION FOR SEQ ID NO :12:
( i ) ~i~; U~N~'~; CBARA
U CTERISTICS
(B) TYPE: nucleic acid ( C ) STR ~Nn~n~.~.q 8 ingle (D) TOPOLOGY: linear ( ii ) MO~ECU~E TYPE: cDNA
(iii) XYPOTXETICA~: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
(2) INFORMATION FOR SEQ ID NO:13:
(i) ~i~;uU~;N(:~ t'TThRi~('T~RT~TICS:
-(A) ~ENGTX: 8 base pair8 (B) TYPE: nucleic acid (C) STRAt~D~nN~ : 6ingle (D) TOPOLOGY: linear (ii) MOLECU~E TYPE: cDNA
(iii) XYPOTXETICA~: NO
... . . _ . ... _ W0 95/20680 Z ~ ~ a 7~ 2 PCTIUS9~/01l0.~
(iv) ANTI-SENSE: NO ~~
(Xi) ~ Ul:~;N(:~; DESCRIPTION: SEQ ID NO:13:
( 2 ) INFORMATION FOR SEQ ID NO :14:
U~;N~'~' C~RACTERISTICS:
(A) LENGTH: 8 }~a8e pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOSY: linear ( i i ) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
( iv) ANTI -SENSE: NO
(xi) SEQUENCE DESCRIPTION: 'SEQ ID NO:14:
(2) INFORMATION FOR SEQ ID NO:15:
U~N(:~; C~ARACTERISTICS:
(A) LENGTH: 8 base pairs (B) TYPE: nucleic acid (C) STRz~Nn~nNl; qs: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHET~CAL: ~0 (iv) ANTI-SENSE: NO
(Xi) ~ U~;N(~ DESCRIPTION: SEQ ID NO:15:
(2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENOE t~T~R~('T~RT.qTICS:
(A) LENGTH: 8 base pairs (B) TYPE: nucleic acid (C) STR~Nn~nN~q~q single ( D ) TOPOLOGY: l inear ( i i ) MOLECULE TYPE: cDNA
( i i i ) HYPOTHET I CAL: NO
WO 95/20680 2 1 8 ~ 7 2 2 PCT/US95/01l0 1 ( iv ) ANTI - SENSE: NO
(Xi) ~ U~;Nt~; DESCRIPTION: SEQ ID NO:16:
( 2 ) INFORMATION FOR SEQ ID NO :17:
( i ) SEQUENOE CE~RACTERISTICS:
(A) LENGTH: 8 base pairs (B) TYPE: nucleic acid (C) STR~N~ : single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTXETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
(2) INFORMATION FOR SEQ ID NO:18:
;uu~;N~:~ rTTz~Rz~rTF~RT~TIcs:
(A) LENGTH: 8 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear .
(ii) MOLECULE TYPE~ cDNA
(iii) HYPOTEETICAL: NO
(iv) ANTI-SENSE: NO
(xi) ~i~;UU~;N~; DESCRIPTION: SEQ ID NO:18:
(2) INFORMATION FOR SEQ ID NO:19:
(i) ~i~UU~;N~ r~l~R~rT~RT~TIcs:
(A) LENGTE: 8 base pairs (B) TYPE: nucleic acid (C) STR~ND~nNE~: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
.. . . . . . .. ..
WO9S/20680 2 1 8~72~ PCr/US9Sl01104 (iv~ ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:
(2) INFORMATION FOR SEQ ID NO:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 base pairs (B) TYPE: nucleic acid (C) sTR~ nNR~qs single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
( iii ) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(Xi) ~ U~:N~:~; DESCRIPTION: SEQ ID NO:20:
(2) INFORMATION FOR SEQ ID NO:21:
U~ ; CHARACTERISTICS:
(A) LENGTH: 8 base pairs (B) TYPE: nucleic acid ( C) STR ~ .q.q: single ( D ) TOPOLOGY: l inear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) ~ u~ ; DESCRIPTIOl~: SEQ ID NO:21:
Gel-filled capillaries are prepared as follows. Fused-silica capillary tubing (Polymicro Technologies, Phoenix, AZ, USA) with an inner diameter of 75 ~m, an outer diameter of 375 llm, an effective lRngth of 10-15 cm, and a total length of 3 0 - 60 cm wa8 treated with (methylacryloxypropyl) trimethoxysilane (Petrach Systems, Bristol, E'A, USA), and then filled with a de-gassed solution of 14~6 T polymerizing acrylamide, 7 M urea in aqueous or f ormamide media (TBE buffer: 0.1 M to 0.3 M Tris-borate, 2-6 mM
EDTA pH 8 . 3, 7 M urea) . Polymerization was achieved by adding ;3~mnnlllm per8ulfate solution and ~, N, N', N',-tetramethyl-ethyl~n,~ m;n~
(TEMED). This protocol is based on the method of Smith et a~. (Nature (1986) 321:674). The running buffer was:~0.2 M TBE, and the applied field was 400 V/cm.
The capillary electrophoresis apparatus with W and laser-induced fluorescence detection for the separation of oligonucleotide sequencing fragments is the same a8 that de8cri~ed by Smith et al (Nature (1986) 321:674). Briefly, a 30 kV, 5 o o ~LA direct current high voltage power supply (Model ER/DM; Glassman, Whitehouse Station, NJ, 3 0 USA) is used to generate the potential across the capillary. W detection of phosphorothioates and other analogs at 270 nm was accomplished with a spectrophotometer (Spectra 110, Spectra-Physic8, ~, WO95/20680 ~ 1 8~72~ PCT/I~S95/0ll0 San Jose, CA) . For laser induced ~luorescence detection an argon ion la~er (Model 543 lOOBS, omnichrom, Chino, CA, USA) wa~ employed. The data were acquired and ~tored on an AcerPower 486/33 computer (Acer American Corp., San Jose, Q, USA) through an analog-to-digital converter (Model 970, Nelson Analytical, Cupertino, CA, USA) .
WO95/20680 21 80722 r~l~.n s llo~ ~
SEQUENCE LISTING
( 1 ) GENERAL INFORMATION:
i) APPLICANT: Cohen, S. Aharon, Belenky, Alexei and Ott, Christopher M.
(ii) TITLE OF L~V~;N'l'lON: A Method of Se~[uenci Short Ol igonucleotides (iii) NUMBER OF SEQUENCES: 21 (iv) CORRESPONDENCE ADDRESS:
(A) AnnRF~ sFl~ Lapp~n h Kusmer (B) STREET: 200 State Street ( C) CITY: Boston (D) STATE: Massachusetts ( E ) COUNTRY: USA
(F) ZIP: 02109 (v) COMPUTER RT'~n~RT,T~ FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
( D ) SOFTWARE: Patentln Release #1 .0 , Version #1 .2 (vi) CURRENT APPLICATION DATA:
(A) APPLICATIO~ NUMBER:
(B) FILING DATE:
( C) CLASS IFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Kerner, Ann-Loui8e (B) REGISTRATIO~ NUMBER: 33,523 (C) X~;~;~;NC~;/DOCKET NUMBER- HYZ-013PCT
( ix) TELECOMMUNI CATION- I~FORMATION:
( A ) TELEPXONE: 617 -13 D -13 0 0 (B) TELEFAX: 617-330-1311 ( 2 ) INFORMATION FOR SEQ ID NO :1:
U~N ~ T~ ~ ~Tl;~R T C T I CS:
(A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) STR~n~nl~ S: single (D) TOPOLOGY: l; near (ii) MOLECULE TYPE: cDNA
( iii ) ~Y ~ L CAL: NO
(iv) A-NTI-SENSE: YES
WO95/20680 2 ~ 8 ] 72 2 PCTIUS9510110'1 (Xi) ~;UU~N~ DESCRIPTION: SEQ ID NO:1: -CTCTCGCACC CA~ L~ l C CTTCT 2 5 ( 2 ) INFORMATIO~ FOR SEQ ID NO: 2:
.?U~;N~ RA(~T~RT~TIcs (A) LENGTH: 12 base pairs (B) TYPE: nucleic acid ( C ) STR ~Nn~nN~ : E ingl e ( D ) TOPOLOGY: l inear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
( iv ) ANTI - SENS E: NO
(xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
(2) INFORMATION FOR SEQ ID NO:3:
U~;N~ ; CHARACTERISTICS:
(A) LENGTH: 32 base pairs (B) TYPE: nucleic acid (C) STR~Nn~nN~ : single ( D ) TOPOLOGY: l inear ( i i ) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(Xi) ~ U~N~:~ DESCRIPTION: SEQ ID NO:3:
CTCCATTTTT TTTTTACTGG C~ jL11"1' AC . 32 ( 2 ) INFORMATION FOR SEQ ID NO: g:
1U~;N(:~; CHARACTERISTICS:
(A) LENGTH: 57 base pairs (B) TYPE: nucleic acid (C) STR~Nn~nN~ : 8ingle (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
( i i i ) HYPOTHETI CAL: NO
(iv) ANTI-SENSE: NO
WO9s/20680 2 1 8 Q 7 ~ 2 PCTIUS9VOI10~ ~
(Xi ) ~ UU~;NO~; DESCRIPTI~N: SEQ ID NO: 4:
CTCTCGCACC CATCTCTCTC ~ LC~ ~LLLll~LLlLL ACTGGCCGTC 50 GTTTTAC _ 5 7 (2) INFORM;~TION FOR SEQ ID NO:~:
U~NC~ CHARACTERISTICS:
(A) LENGTH: 16 base pairs (B) TYPE: nucl~ic acid (C) STR~Nn~nM~ ingle (D) TOPOLOGY: li~ear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) ~;UU~;N~:~; DESCRIPTION: SEQ ID NO:5:
TGCCGCCAGC AAaATG 16 ( 2 ) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE t~ 7T.CTICS:
(A) LENGTH: 8 base pair3 (B) TYPE: nucleic acid (C) STR~NDEDNESS: ~ingle ( D ) TOPO~OGY: l inear (ii) MOLECULE TYPE: cDNA
( i i i ) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) ~;~;UU~;N~; DESCRIPTION: SEQ ID NO:6:
AAGAGGTA ~ 8 ( 2 ) INFORMATION FOR SEQ ID NO: 7:
;5,)U~:N(~; CHARACTERISTICS:
(A) LENGTH: 8 base pairs (B) TYPE: nucleic acid (C) sTR~Nn~nNF~c~ ~ingle (D) TOP3LOGY: linear (ii) MOLECULE TYPE: cDNA
( i i i ) ~POTHET~CAL: ~o ... . ..
wogs/20680 2 ~ 22 PCT/US95/0110 (iv) ANTI-SENSE: NO
(xi) ~ U~:N~; DESCRIPTION SEQ ID NO:7:
AGGAGGTA ~ 8 (2) INFORMATION FOR SEQ ID NO:8:
;S.?U~;~; CH~RACTERISTICS:
(A) LENGTH: 8 base pairs (B) TYPE: nucleic acid (C) STR~NDEDNESS: single ( D ) TOPOLOGY: 1 inear (ii) MOLECULE TYPE: cDNA
( i i i ) HYPOTHETI CAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
( 2 ) INFORMATION FOR SEQ ID NO: 9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 base pairs (B) TYPE: nucleic acid (C) STRANn~nNF~c single ( D ) TOPOLOGY: 1 inear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: S13Q ID NO:9:
(2) INFORMATION FOR SEQ ID NO:10:
;5.?U~ rT~ARArT~RT,~TICS
(A) LENGTH: 8 base pairs (B) TYPE: nucleic acid (C~ sTRANn~nN~ single ( D ) TOPOLOGY: l inear ( ii ) MOLECULE TYPE: cDNA
( i i i ) ~ Y ~ CAL: NO
WO 95/20680 2 1 8 0 7 2 2 PCT/US95/0ll0~ ~
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
GAGAGGTA ~ 8 ( 2 ) INFORMATION FOR SEQ ID NO :11:
;UU~;N(~ C~RACTERISTICS:
(A) ~.ENGTX: 8 ba6e pairs (B) TYPE: nucleic acid (C) sTR~ND~nl~cc single (D) TOPOLOGY: linear ( ii ) MOI.ECULE TYPE: cDNA
( i i i ) XYPOTXET I CAL: ~O
(iv) ANTI-SENSE: NO
(Xi) ~ )U~;N~:~; DESCRIPTION: SEQ ID NO:11:
( 2 ) INFORMATION FOR SEQ ID NO :12:
( i ) ~i~; U~N~'~; CBARA
U CTERISTICS
(B) TYPE: nucleic acid ( C ) STR ~Nn~n~.~.q 8 ingle (D) TOPOLOGY: linear ( ii ) MO~ECU~E TYPE: cDNA
(iii) XYPOTXETICA~: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
(2) INFORMATION FOR SEQ ID NO:13:
(i) ~i~;uU~;N(:~ t'TThRi~('T~RT~TICS:
-(A) ~ENGTX: 8 base pair8 (B) TYPE: nucleic acid (C) STRAt~D~nN~ : 6ingle (D) TOPOLOGY: linear (ii) MOLECU~E TYPE: cDNA
(iii) XYPOTXETICA~: NO
... . . _ . ... _ W0 95/20680 Z ~ ~ a 7~ 2 PCTIUS9~/01l0.~
(iv) ANTI-SENSE: NO ~~
(Xi) ~ Ul:~;N(:~; DESCRIPTION: SEQ ID NO:13:
( 2 ) INFORMATION FOR SEQ ID NO :14:
U~;N~'~' C~RACTERISTICS:
(A) LENGTH: 8 }~a8e pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOSY: linear ( i i ) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
( iv) ANTI -SENSE: NO
(xi) SEQUENCE DESCRIPTION: 'SEQ ID NO:14:
(2) INFORMATION FOR SEQ ID NO:15:
U~N(:~; C~ARACTERISTICS:
(A) LENGTH: 8 base pairs (B) TYPE: nucleic acid (C) STRz~Nn~nNl; qs: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHET~CAL: ~0 (iv) ANTI-SENSE: NO
(Xi) ~ U~;N(~ DESCRIPTION: SEQ ID NO:15:
(2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENOE t~T~R~('T~RT.qTICS:
(A) LENGTH: 8 base pairs (B) TYPE: nucleic acid (C) STR~Nn~nN~q~q single ( D ) TOPOLOGY: l inear ( i i ) MOLECULE TYPE: cDNA
( i i i ) HYPOTHET I CAL: NO
WO 95/20680 2 1 8 ~ 7 2 2 PCT/US95/01l0 1 ( iv ) ANTI - SENSE: NO
(Xi) ~ U~;Nt~; DESCRIPTION: SEQ ID NO:16:
( 2 ) INFORMATION FOR SEQ ID NO :17:
( i ) SEQUENOE CE~RACTERISTICS:
(A) LENGTH: 8 base pairs (B) TYPE: nucleic acid (C) STR~N~ : single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTXETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
(2) INFORMATION FOR SEQ ID NO:18:
;uu~;N~:~ rTTz~Rz~rTF~RT~TIcs:
(A) LENGTH: 8 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear .
(ii) MOLECULE TYPE~ cDNA
(iii) HYPOTEETICAL: NO
(iv) ANTI-SENSE: NO
(xi) ~i~;UU~;N~; DESCRIPTION: SEQ ID NO:18:
(2) INFORMATION FOR SEQ ID NO:19:
(i) ~i~UU~;N~ r~l~R~rT~RT~TIcs:
(A) LENGTE: 8 base pairs (B) TYPE: nucleic acid (C) STR~ND~nNE~: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
.. . . . . . .. ..
WO9S/20680 2 1 8~72~ PCr/US9Sl01104 (iv~ ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:
(2) INFORMATION FOR SEQ ID NO:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 base pairs (B) TYPE: nucleic acid (C) sTR~ nNR~qs single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
( iii ) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(Xi) ~ U~:N~:~; DESCRIPTION: SEQ ID NO:20:
(2) INFORMATION FOR SEQ ID NO:21:
U~ ; CHARACTERISTICS:
(A) LENGTH: 8 base pairs (B) TYPE: nucleic acid ( C) STR ~ .q.q: single ( D ) TOPOLOGY: l inear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) ~ u~ ; DESCRIPTIOl~: SEQ ID NO:21:
Claims (25)
1. A method for determining the nucleotide sequence of a target oligonucleotide, comprising the steps of:
(a) preparing a single-stranded ligation product comprising:
(i) a target oligonucleotide-to-be-sequenced having a 3'- and 5' -end; and (ii) an auxiliary oligonucleotide having a 3' end and 5' end and a nucleotide sequence complementary to the sequence of a primer-to-be-used;
(b) annealing a primer to the auxiliary oligonucleotide portion of the ligation product, the primer having a nucleotide sequence complementary to a portion of the auxiliary oligonucleotide and having a label covalently attached thereto;
(c) extending the primer with chain-extending nucleoside triphosphates and chain-terminating nucleoside triphosphates in the presence of a polymerase to yield a plurality of primer extension products;
(d) separating the primer extension products on the basis of their base length; and (e) determining the nucleotide sequence of the target oligonucleotide from mobilities of the primer extension products obtained during their separation.
(a) preparing a single-stranded ligation product comprising:
(i) a target oligonucleotide-to-be-sequenced having a 3'- and 5' -end; and (ii) an auxiliary oligonucleotide having a 3' end and 5' end and a nucleotide sequence complementary to the sequence of a primer-to-be-used;
(b) annealing a primer to the auxiliary oligonucleotide portion of the ligation product, the primer having a nucleotide sequence complementary to a portion of the auxiliary oligonucleotide and having a label covalently attached thereto;
(c) extending the primer with chain-extending nucleoside triphosphates and chain-terminating nucleoside triphosphates in the presence of a polymerase to yield a plurality of primer extension products;
(d) separating the primer extension products on the basis of their base length; and (e) determining the nucleotide sequence of the target oligonucleotide from mobilities of the primer extension products obtained during their separation.
2. The method of claim 1 wherein the target oligonucleotide includes nucleotides selected from the group consisting of ribonucleotides, deoxyribonucleotides, analogs of ribonucleotides, analogs of deoxyribonucleotides, and combinations thereof.
3. The method of claim 1 wherein the target oligonucleotide comprises internucleotide linkages selected from the group consisting of a phosphodiester, phosphorothioate, alkylphosphonothioate, phosphorodithioate, phosphoramidate, phosphate ester, phosphate triesters, carbamates, carbonates, acetamidate, carboxymethyl esters, and combinations thereof.
4. The method of claim 1 wherein preparing step (a) comprises preparing an auxiliary oligonucleotide including nucleotides selected from the group consisting of ribonucleotides, deoxyribonucleotides, analogs of ribonucleotides, analogs of deoxyribonucleotides, and combinations thereof.
5. The method of claim 1 wherein preparing step (a) comprises preparing an auxiliary oligonucleotide composed of at least 8 contiguous nucleotides.
6. The method of claim 1 wherein preparing step (a) comprises preparing an auxiliary oligonucleotide further comprising a signalling sequence of at least four contiguous nucleotides at its 5' end, linked to the 3' end of the target oligonucleotide.
7. The method of claim 6 wherein preparing step (a) comprises preparing an auxiliary oligonucleotide having a signalling sequence composed from 4 to 20 nucleotides long.
8. The method of claim 1 wherein preparing step (a) comprises:
(i) preparing an auxiliary oligonucleotide composed of a 3' -sequence complementary to the sequence of a primer to be used, linked to a 5' -signalling sequence of at least four contiguous nucleotides, the nucleotide at the 3' end being protected; and (ii) ligating the 5' end of the auxiliary oligonucleotide to the 3' end of the target oligonucleotide, thereby forming a single-stranded ligation product.
(i) preparing an auxiliary oligonucleotide composed of a 3' -sequence complementary to the sequence of a primer to be used, linked to a 5' -signalling sequence of at least four contiguous nucleotides, the nucleotide at the 3' end being protected; and (ii) ligating the 5' end of the auxiliary oligonucleotide to the 3' end of the target oligonucleotide, thereby forming a single-stranded ligation product.
9. The method of claim 8 wherein preparing step (a) comprises preparing an auxiliary oligonucleotide including a dideoxy or amino group protecting the 3' end.
10. The method of claim 8 wherein preparing step (a) comprises ligating the auxiliary oligonucleotide to the target oligonucleotide with a blunt end ligase.
11. The method of claim 10 wherein preparing step (a) comprises ligating the auxiliary oligonucleotide to the target oligonucleotide with T4 RNA ligase.
12. The method of claim 1 wherein preparing step (a) comprises:
(i) preparing an auxiliary oligonucleotide composed of a sequence complementary to the sequence of a primer to be used; and (ii) preparing a set of sixteen bridge oligonucleotides, each bridge oligonucleotide having a 3' end and a 5' end, and each including the same six nucleotides at its 5'-end which are complementary to at six nucleotides at the 5' -end of the auxiliary oligonucleotide and two nucleotides at its 3'-end which have one of sixteen different possible sequences, one of which is complementary to the last two 3' nucleotides of the target oligonucleotide;
(iii) annealing the auxiliary and target oligonucleotides to the bridge oligonucleotide with the two 3' nucleotides complementary to the last two 3' nucleotides of the target molecule, auch that the first two 3' nucleotides of the bridge oligonucleotide hybridize to the last two 3' nucleotides of the target oligonucleotide, and the next six nucleotides of the bridge ollgonucleotide at its 5' end hybridize to the first six 5' nucleotides of the auxiliary oligonucleotide; and (iv) ligating the 3' end of the target oligonucleotide to the signalling sequence at the 5' end of the ayxiliary oligonucleotide, thereby forming a ligation product.
(i) preparing an auxiliary oligonucleotide composed of a sequence complementary to the sequence of a primer to be used; and (ii) preparing a set of sixteen bridge oligonucleotides, each bridge oligonucleotide having a 3' end and a 5' end, and each including the same six nucleotides at its 5'-end which are complementary to at six nucleotides at the 5' -end of the auxiliary oligonucleotide and two nucleotides at its 3'-end which have one of sixteen different possible sequences, one of which is complementary to the last two 3' nucleotides of the target oligonucleotide;
(iii) annealing the auxiliary and target oligonucleotides to the bridge oligonucleotide with the two 3' nucleotides complementary to the last two 3' nucleotides of the target molecule, auch that the first two 3' nucleotides of the bridge oligonucleotide hybridize to the last two 3' nucleotides of the target oligonucleotide, and the next six nucleotides of the bridge ollgonucleotide at its 5' end hybridize to the first six 5' nucleotides of the auxiliary oligonucleotide; and (iv) ligating the 3' end of the target oligonucleotide to the signalling sequence at the 5' end of the ayxiliary oligonucleotide, thereby forming a ligation product.
13. The method of claim 12 wherein preparing step (a) comprises preparing a bridge oligonucleotide including nucleotides selected from the group consisting of ribonucleotides, deoxyribonucleotides, analogs of ribonucleotides, analogs of deoxyribonucleotides, and combinations thereof.
14. The method of claim 12 wherein preparing step (a) comprises preparing a bridge oligonucleotide comprising internucleotide linkages selected from the group consisting of phosphodiester, phosphorothioate, alkylphosphonothioate, phosphorodithioate, phosphoramidate, phosphate ester, phosphate triesters, carbamates, carbonates, acetamidate, carboxymethyl esters, and combinations thereof.
15. The method of claim 12 wherein preparing step (a) comprises preparing a bridge oligonucleotide comprising a dinucleotide at its 3' -end selected from the group consisting of AA, AT, AC, AG, TA, TT, TC, TG, CA, CT, CC, CG, GA, GT, GC, and GG.
16. The method of claim 5 wherein preparing step (a) comprises ligating the auxiliary oligonucleotide to the target oligonucleotide with T4 DNA ligase or Taq DNA ligase.
17. The method of claim 1 wherein annealing step (b) comprises annealing a primer complementary to the ligation product, the primer comprising a nucleotide sequence complementary to at least four nuclcotides of the auxiliary oligonucleotide portion of the ligation products.
18. The method of claim 1 wherein annealing step (c) comprises annealing a primer to the ligation product, the primer including nucleotides selected from the group consisting of ribonucleotides, deoxyribonucleotides, analogs of ribonucleotides, analogs of deoxyribonucleotides, and combinations thereof.
19. The method of claim 12 wherein annealing step (c) comprises annealing a primer to the ligation product, the primer including internucleotide linkages selected from the group consisting of phosphodiester, phosphorothioate, alkylphosphonothioate, phosphorodithioate, phosphoramidate, phosphate ester, phosphate triesters, carbamates, carbonates, acetamidate, carboxymethyl esters, and combinations thereof.
20. The method of claim 1 wherein annealing step (c) comprises annealing a primer to the ligation product, the primer having a label selected from the group consisting of a fluorescent, chemiluminescent, or radioactive tag.
21. The method of claim 20 wherein annealing step (c) comprises annealing a primer having a fluorescent label which is excitable in the UV or visible range and which fluoresces in the visible range.
22. The method of claim 1 wherein extending step (c) comprises extending the primer with a polymerase selected from the group consisting of T4 DNA ligase, T4 RNA ligase, and Taq DNA ligase.
23. The method of claim 1 wherein extending step (c) comprises extending the primer with chain terminating nucleotide triphosphates selected from the group consisting of dideoxyadenine, dideoxyguanidine, dideoxythymidine, and dideoxycytosine.
24. The method of claim 1 wherein separating step (d) comprises separating the primer extension products by slab gel or high performance capillary gel electrophoresis.
25. The method of claim 1 wherein determining step (e) comprises the use of laser induced fluorescence, UV absorption, or radiation detection to measure the relative mobilities of the primer extension products.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US08/187,749 US5525470A (en) | 1994-01-26 | 1994-01-26 | Method of sequencing [short] oligonucleotides |
US08/187,749 | 1994-01-26 |
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CA2180722A1 true CA2180722A1 (en) | 1995-08-03 |
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CA002180722A Abandoned CA2180722A1 (en) | 1994-01-26 | 1995-01-24 | A method of sequencing short oligonucleotides |
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US (1) | US5525470A (en) |
EP (1) | EP0741799B1 (en) |
JP (1) | JPH09508282A (en) |
AT (1) | ATE153078T1 (en) |
AU (1) | AU1734695A (en) |
CA (1) | CA2180722A1 (en) |
DE (1) | DE69500303T2 (en) |
WO (1) | WO1995020680A1 (en) |
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WO1997025444A1 (en) * | 1996-01-16 | 1997-07-17 | Hybridon, Inc. | Method of monitoring pharmacokinetics of oligonucleotide pharmaceuticals |
US5851804A (en) * | 1996-05-06 | 1998-12-22 | Apollon, Inc. | Chimeric kanamycin resistance gene |
US6291164B1 (en) * | 1996-11-22 | 2001-09-18 | Invitrogen Corporation | Methods for preventing inhibition of nucleic acid synthesis by pyrophosphate |
AU6553498A (en) * | 1997-03-14 | 1998-09-29 | Hybridon, Inc. | Method for sequencing of modified nucleic acids using electrospray ionization-fourier transform mass spectrometry |
US5888778A (en) * | 1997-06-16 | 1999-03-30 | Exact Laboratories, Inc. | High-throughput screening method for identification of genetic mutations or disease-causing microorganisms using segmented primers |
US20040038206A1 (en) * | 2001-03-14 | 2004-02-26 | Jia Zhang | Method for high throughput assay of genetic analysis |
JP2006500959A (en) * | 2002-09-30 | 2006-01-12 | パラレル バイオサイエンス, インコーポレイテッド | Polynucleotide synthesis and labeling by dynamic sampling binding |
US20040219565A1 (en) | 2002-10-21 | 2004-11-04 | Sakari Kauppinen | Oligonucleotides useful for detecting and analyzing nucleic acids of interest |
US7902160B2 (en) * | 2002-11-25 | 2011-03-08 | Masafumi Matsuo | ENA nucleic acid drugs modifying splicing in mRNA precursor |
US20090186343A1 (en) * | 2003-01-28 | 2009-07-23 | Visigen Biotechnologies, Inc. | Methods for preparing modified biomolecules, modified biomolecules and methods for using same |
US8192937B2 (en) * | 2004-04-07 | 2012-06-05 | Exiqon A/S | Methods for quantification of microRNAs and small interfering RNAs |
WO2006047787A2 (en) | 2004-10-27 | 2006-05-04 | Exact Sciences Corporation | Method for monitoring disease progression or recurrence |
US9777314B2 (en) | 2005-04-21 | 2017-10-03 | Esoterix Genetic Laboratories, Llc | Analysis of heterogeneous nucleic acid samples |
US20080045472A1 (en) * | 2006-03-31 | 2008-02-21 | Council Of Scientific And Industrial Research Bharat Biotech | Targets for human micro rnas in avian influenza virus (h5n1) genome |
WO2020239822A1 (en) * | 2019-05-27 | 2020-12-03 | The European Molecular Biology Laboratory | Nucleic acid construct binding to influenza polymerase pb1 rna synthesis active site |
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JP3042626B2 (en) * | 1988-05-24 | 2000-05-15 | ゲゼルシャフト フュア バイオテクノロギッシェ フォーシュング エムベーハー(ゲーベーエフ) | Determination of DNA base sequence using oligonucleotide bank |
US5403709A (en) * | 1992-10-06 | 1995-04-04 | Hybridon, Inc. | Method for sequencing synthetic oligonucleotides containing non-phosphodiester internucleotide linkages |
-
1994
- 1994-01-26 US US08/187,749 patent/US5525470A/en not_active Expired - Fee Related
-
1995
- 1995-01-24 AU AU17346/95A patent/AU1734695A/en not_active Abandoned
- 1995-01-24 JP JP7520189A patent/JPH09508282A/en active Pending
- 1995-01-24 AT AT95909358T patent/ATE153078T1/en active
- 1995-01-24 EP EP95909358A patent/EP0741799B1/en not_active Expired - Lifetime
- 1995-01-24 CA CA002180722A patent/CA2180722A1/en not_active Abandoned
- 1995-01-24 DE DE69500303T patent/DE69500303T2/en not_active Expired - Fee Related
- 1995-01-24 WO PCT/US1995/001104 patent/WO1995020680A1/en active IP Right Grant
Also Published As
Publication number | Publication date |
---|---|
WO1995020680A1 (en) | 1995-08-03 |
JPH09508282A (en) | 1997-08-26 |
EP0741799B1 (en) | 1997-05-14 |
AU1734695A (en) | 1995-08-15 |
ATE153078T1 (en) | 1997-05-15 |
DE69500303T2 (en) | 1997-10-30 |
DE69500303D1 (en) | 1997-06-19 |
US5525470A (en) | 1996-06-11 |
EP0741799A1 (en) | 1996-11-13 |
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