USRE43096E1 - Tagged extendable primers and extension products - Google Patents
Tagged extendable primers and extension products Download PDFInfo
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- USRE43096E1 USRE43096E1 US10/389,663 US38966303A USRE43096E US RE43096 E1 USRE43096 E1 US RE43096E1 US 38966303 A US38966303 A US 38966303A US RE43096 E USRE43096 E US RE43096E
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- MEUQCPOTVNLCGK-UHFFFAOYSA-N [H]C1(C)OC([H])(COP(=O)(O)OC2([H])C([H])(COP(=O)(O)OC3([H])C([H])(COP(C)(=O)O)OC([H])(C)C3([H])[H])OC([H])(C)C2([H])[H])C([H])(OC)C1([H])[H] Chemical compound [H]C1(C)OC([H])(COP(=O)(O)OC2([H])C([H])(COP(=O)(O)OC3([H])C([H])(COP(C)(=O)O)OC([H])(C)C3([H])[H])OC([H])(C)C2([H])[H])C([H])(OC)C1([H])[H] MEUQCPOTVNLCGK-UHFFFAOYSA-N 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
- G01N27/447—Systems using electrophoresis
- G01N27/44704—Details; Accessories
- G01N27/44717—Arrangements for investigating the separated zones, e.g. localising zones
- G01N27/44721—Arrangements for investigating the separated zones, e.g. localising zones by optical means
- G01N27/44726—Arrangements for investigating the separated zones, e.g. localising zones by optical means using specific dyes, markers or binding molecules
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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
- C12Q1/6813—Hybridisation assays
- C12Q1/6816—Hybridisation assays characterised by the detection means
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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
- C12Q1/6869—Methods for sequencing
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
- G01N27/447—Systems using electrophoresis
- G01N27/44704—Details; Accessories
- G01N27/44717—Arrangements for investigating the separated zones, e.g. localising zones
- G01N27/44721—Arrangements for investigating the separated zones, e.g. localising zones by optical means
Definitions
- RNA ribonucleic acid
- nucleic acid sequencing allows dissection and analysis of animal, plant and viral genomes into discrete genes with defined chemical structure. Since the function of a biological molecule is determined by its structure, defining the structure of a gene is crucial to the eventual manipulation of this basic unit of hereditary information in useful ways. Once genes can be isolated and characterized, they can be modified to produce desired changes in their structure that allow the production of gene products—proteins—with different properties than those possessed by the original proteins.
- Microorganisms into which the natural or synthetic genes are placed can be used as chemical “factories” to produce large amounts of scarce human proteins such as interferon, growth hormone, and insulin. Plants can be given the genetic information to allow them to survive harsh environmental conditions or produce their own fertilizer.
- the method developed by Sanger is referred to as the dideoxy chain termination method.
- a DNA segment is cloned into a single-stranded DNA phage such as M13.
- M13 DNA phage
- These phage DNAs can serve as templates for the primed synthesis of the complementary strand by the Klenow fragment of DNA polymerase I.
- the primer is either a synthetic oligonucleotide or a restriction fragment isolated from the parental recombinant DNA that hybridizes specifically to a region of the M13 vector near the 3′′ end of the cloned insert.
- the primed synthesis is carried out in the presence of enough of the dideoxy analog of one of the four possible deoxynucleotides so that the growing chains are randomly terminated by the incorporation of these “dead-end” nucleotides.
- the relative concentration of dideoxy to deoxy forms is adjusted to give a spread of termination events corresponding to all the possible chain lengths that can be resolved by gel electrophoresis.
- the products from each of the four primed synthesis reactions are then separated on individuals tracks of polyacrylamide gels by the electrophoresis. Radioactive tags incorporated in the growing chains are used to develop an autoradiogram image of the pattern of the DNA in each electrophoresis track.
- the sequence of the deoxynucleotides in the cloned DNA is determined from an examination of the pattern of bands in the four lanes.
- the method developed by Maxam and Gilbert uses chemical treatment of purified DNA to generate size-nested sets of DNA fragments analogous to those produced by the Sanger method.
- cleavage is induced at one or two of the four nucleotide bases by chemical treatment.
- Cleavage involves a three-stage process: modification of the base, removal of the modified base from its sugar, and strand scission at that sugar. Reaction conditions are adjusted so that the majority of end-labeled fragments generated are in the size range (typically 1 to 400 nucleotides) that can be resolved by gel electrophoresis.
- the electrophoresis, autoradiography, and pattern analysis are carried out essentially as is done for the Sanger method. (Although the chemical fragmentation necessarily generates two pieces of DNA each time it occurs, only the piece containing the end label is detected on the autoradiogram.)
- the length of sequence that can be obtained from a single set of reactions is limited primarily by the resolution of the polyacrylamide gels used for electrophoresis. Typically, 200 to 400 bases can be read from a single set of gel tracks.
- both methods have serious drawbacks, problems associated primarily with the electrophoresis procedure.
- One problem is the requirement of the use of radiolabel as a tag for the location of the DNA bands in the gels.
- An oligonucleotide is a short polymer consisting of a linear sequence of four nucleotides in a defined order. The nucleotide subunits are joined by phosphodiester linkages joining the 3′ hydroxyl moiety of one nucleotide to the 5′ hydroxyl moiety of the next nucleotide.
- An example of an oligonucleotide is 5′ ApCpGpTpApTpGpGpCp 3′.
- the letters A, C, G and T refer to the nature of the purine of pyrimidine base coupled at the 1-position of deoxyribose.
- A, adenine; C cytosine; G, guanine; T, thymidine.
- P represents the phosphodiester bond.
- the structure of a section of an oligonucleotide is shown below.
- the single stranded oligonucleotides of this invention are further characterized by being homogenous with respect to the sequence of the nucleoside subunits and are of uniform molecular weight.
- Synthetic oligonucleotides are powerful tools in modern molecular biology and recombinant DNA work. There are numerous applications for these molecules, including a) as probes for the isolation of specific genes based on the protein sequence of the gene product, b) to direct the in vitro mutagenesis of a desired gene, c) as primers for DNA synthesis on a single-stranded template, d) as steps in the total synthesis of genes, and many more, reviewed in Wm. R. Bahl et al, Prog. Nucl. Acid Res. Mol. Biol., 21, 101 (1978).
- Fluorescent dyes attached to the oligonucleotides permits one to eliminate radioisotopes from the research, diagnostic and clinical procedures in which they are used, and improve shelf-life availability.
- a DNA sequencing machine Serial No. the synthesis of fluorescent-labeled oligonucleotides permits the automation of the DNA sequencing process.
- the invention of the present patent application addresses these and other problems associated with DNA sequencing procedures and is believed to represent a significant advance in the art.
- the preferred embodiment of the present invention represents a further and distinct improvement.
- this invention comprises a novel process for the electrophoetic analysis of DNA fragments produced in DNA sequencing operations wherein chromophores or fluorophores are used to tag the DNA fragments produced by the sequencing chemistry and permit the detection and characterization of the fragments as they are resolved by electrophoresis through a gel.
- the detection employs an absorption or fluorescent photometer capable of monitoring the tagged bands as they are moving through the gel.
- This invention further comprises a novel process for the electrophoretic analysis of DNA fragments produced in DNA sequencing operations wherein a set of four chromophores are used to tag the DNA fragments produced by the sequencing chemistry and permit the detection and characterization of the fragments as they are resolved by electrophoresis through a gel; the improvement wherein the four different fragment sets are tagged with the fluorophores fluorescein, Texas Red, tetramethyl rhodamine, and 7-nitrobenzofurazan.
- This invention also includes a novel system for the electrophoretic analysis of DNA fragments produced in DNA sequencing operations comprising:
- FIG. 1 is an illustration of one means of end-labeling a DNA fragment with a fluorescent tag.
- Pst. I and T4 DNA ligase are enzymes commonly used in recombinant DNA research.
- FIG. 2 is a block diagram of automated DNA sequencer, gel electrophoretic system.
- FIG. 3 is a comparison of the type of data produced by DNA sequencing of the sequence shown in FIG. 1 .
- FIG. 4 is a block diagram of a preferred DNA sequencer according to this invention.
- FIG. 5 shows the emission spectra for the four fluorophores used as tags in the preferred embodiment of this invention.
- FIG. 6 is a schematic diagram of a possible optical configuration in the detector unit.
- P lamp source
- L 1 objective lens
- L 2 collimating lens
- F 1 UV blocking filter
- F 2 heat blocking filter
- F 3 band pass excitation filter
- F 4 long pass emission filter
- DM dichroic mirror
- C polyacrylamide gel
- PMT photomultiplier tube.
- FIG. 7 is a schematic diagram of another possible optical configuration in the detector unit.
- F 1 to F 4 are bandpass filters centered at the emission maximum of the different dyes.
- P 1 to P 4 are photomultiplier tubes.
- the excitation light is of a wavelength such that it is not transmitted through any of the filters F 1 to F 4 .
- the primers must have the following characteristics. 1) They must have a free 3′ hydroxyl group to allow chain extension by the polymerase. 2) They must be complementary to a unique region 3′ of the cloned insert. 3) They must be sufficiently long to hybridize to form a unique, stable duplex. 4) The chromophore or fluorophore must not interfere with the hybridization or prevent 3′-end extension by the polymerase.
- Conditions 1, 2 and 3 above are satisfied by several synthetic oligonucleotide primers which are in general use for Sanger-type sequencing utilizing M13 vectors.
- One such primer is the 15 mer 5′ CCC AG TCA CGA CGT T 3′ where A, C, G and T represent the four different nucleoside components of DNA; A, adenosine; C, cytosine; G, guanosine; T, thymidine.
- each of these tags is coupled chemically to the primer used to initiate the synthesis of the fragment strands.
- each tagged primer is then paired with one of the dideoxynucleotides and used in the primed synthesis reaction with the Klenow fragment of DNA polymerase.
- ⁇ represents the wavelength in nanometers
- Ex excitation
- Em emission
- max maximum
- ⁇ the molar extinction coefficient
- This region will anneal with a complementary stretch of DNA, which may be covalently joined to the duplex DNA with the enzyme ligase. In this manner one of the strands is covalently linked to a detectable moiety.
- This moiety may be a dye, an amino group or a protected amino group (which could be deprotected and reacted with dye subsequent to the chemical reactions).
- the dideoxy sequencing reactions are performed in the standard fashion Smith, A. J. H., Methods in Enzymology 65, 560–580 (1980), except that the scale may be increased if necessary to provide an adequate signal intensity in each band for detection.
- the reactions are done using a different color primer for each different reaction. No radiolabeled nucleoside triphosphate need be included in the sequencing reaction.
- Maxam/Gilbert sequencing reactions are performed in the usual manner, Gil, S. F. Aldrichimica Acta 16(3), 59–61 (1983), except that the end label is either one or four colored dyes, or a free or protected amino group which may be reacted with dye subsequently.
- Detection There are many different ways in which the tagged molecules which have been separated by length using polyacrylamide gel electrophoresis may be detected. Four illustrative modes are described below. These are i) detection of the fluorescence excited by light of different wavelengths for the different dyes, ii) detection of fluorescence excited by light of the same wavelength for the different dyes, iii) elution of the molecules from the gel and detection by chemiluminescence, and iv) detection by the absorption of light by molecules. In modes i) and ii) the fluorescence detector should fulfill the following requirements. a) The excitation light beam should not have a height substantially greater than the height of a band.
- the excitation wavelength can be varied to match the absorption maxima of each of the different dyes or can be a single narrow, high intensity light band that excites all four fluorophores and does not overlap with any of the fluorescence emission.
- the optical configuration should minimize the flux of scattered and reflected excitation light to the photodetector 14 .
- the optical filters to block out scattered and reflected excitation light are varied as the excitation wavelength is varied.
- the photodetector 14 should have a fairly low noise level and a good spectral response and quantum efficiency throughout the range of the emission of the dyes (500 to 600 nm for the dyes listed above).
- the optical system for collection of the emitted fluorescence should have a high numerical aperture. This maximizes the fluorescence signal. Furthermore, the depth of field of the collection optics should include the entire width of the column matrix.
- FIGS. 6 and 7 Two illustrative fluorescence detection systems are diagrammed in FIGS. 6 and 7 .
- the system in FIG. 6 is compatible with either single wavelength excitation or multi wavelength excitation.
- the filter F 4 is one of four band pass filters centered at the peak emission wavelength of each of the dyes. This filter is switched every few seconds to allow continual monitoring of each of the four fluorophores.
- the optical elements F 3 (excitation filter), DM (dichroic mirror), and F 4 (barrier filter) are switched together. In this manner both the excitation light and the observed emission light are varied.
- the system in FIG. 7 is a good arrangement for the case of single wavelength excitation.
- This system has the advantage that no moving parts are required, and fluorescence from all four of the dyes may be simultaneously and continuously monitored.
- a third approach (iii above) to detection is to elute the labeled molecules at the bottom of the gel, combine them with an agent for excitation of chemiluminescence such as 1,2 dioxetane dione, Gill, S. K. Aldrichimica Acta 16(3), 59–61 (1983); Mellbin, G. J. Liq. Chrom. 6(9), 1603–1616 (1983), and flow the mixture directly into a detector which can measure the emitted light at four separate wavelengths.
- the background signal in chemiluminescence is much lower than in fluorescence, resulting in higher signal to noise ratios and increased sensitivity.
- the measurement may be made by measurements of light absorption (iv above).
- a light beam of variable wavelength is passed through the gel, and the decrease in the beam intensity due to absorption of light at the different wavelengths corresponding to the absorption maximum of the four dyes, it is possible to determine which dye molecule is in the light path.
- absorption measurements are inherently less sensitive than fluorescence measurements.
- the above-described detection system is interfaced to a computer 16 .
- the computer 16 receives a signal proportional to the measured signal intensity at that time for each of the four colored tags. This information tells which nucleotide terminates the DNA fragment of the particular length in the observation window at that time.
- the temporal sequence of colored bands gives the DNA sequence.
- FIG. 3 is shown the type of data obtained by conventional methods, as well as the type of data obtained by the improvements described in this invention.
- the bands of DNA are resolved from one another and pass through the detector 14 (more fully described above).
- the detector 14 detects the fluorescent or chromophoric bands of DNA in the gel and determines their color, and therefore to which nucleotide they correspond. This information yields the DNA sequence.
- FIG. 4 shows a block diagram of a DNA sequenator for use with one dye at a time.
- the beam (4880 A) from an argon ion laser 100 is passed into the polyacrylamide gel tube (sample) 102 by means of a beamsteerer 104 .
- Fluorescence exited by the beam is collected using a low f-number lens 106 , passed through an appropriate set of optical filters 108 and 110 to eliminate scattered excitation light and detected using a photomultiplier tube (PMT) 112 .
- PMT photomultiplier tube
- the signal is readily detected on a strip chart recorder.
- DNA sequencing reactions are carried out utlizing a fluorescein labeled oligonucletide primer.
- the peaks on the chart correspond to fragments to fluorescein labeled DNA of varying lengths synthesized in the sequencing reactions and separated in the gel tube by electrophoresis. Each peak contains on the order of 10 ⁇ 15 to 10 ⁇ 16 moles of fluorescein, which is approximately equal to the amount of DNA obtained per band in an equivalent sequencing gel utilizing radioisotope detection. This proves that the fluorescent tag is not removed or degraded from the oligonucleotide primer in the sequencing reactions. It also demonstrates that the detection sensitivity is quite adequate to perform DNA sequence analysis by this means.
- Fluorescein-5-isothiocyanate (FITC) and Texas Red were obtained from Molecular Probes, Inc. (Junction City, Oreg.). tetramethyl rhodamine isothiocyanate (TMRITC) was obtained from Research Organics, Inc. (Cleveland, Ohio.). 4-fluoro-7-nitro-benzofurazan (NBD-fluoride) was obtained from Sigma Chemical Co. (St. Louis, Mo.). Absorption spectra were obtained on a H/P 8491 spectrophotometer. High performance liquid chromatography was performed on a system composed of two Altex 110A pumps, a dual chamber gradient mixer, Rheodyne injector, Kratos 757 UV detector, and an Axxiom 710 controller.
- the protected 5′-aminothymidine phosphoramidites 5′-(N-9-fluorenylmethyloxycarbonyl)-5′-amino-5′-deoxy-3′-N, N-diisopropylaminomethoxyphosphinyl thymidine, is coupled to the 5′-hydroxyl of an oligonucleotide using well established DNA synthetic procedures.
- the solvents and reaction conditions used are identical to those used in oligonucleotide synthesis.
- the basic procedure used for the attachment of fluorescent dye molecules to the amino oligonucleotides is to combine the amino oligonucleotide and the dye in aqueous solution buffered to pH 9, to allow the reaction to stand at room temperature for several hours, and then to purify the product in two stages.
- the first purification step is to remove the bulk of the unreacted or hydrolyzed dye by gel filtration.
- the second purification stage is to separate the dye conjugate from unreacted oligonucleotide by reverse phase high performance liquid chromatography. Slight variations upon these conditions are employed for the different dyes, and the specific procedures and conditions used for four particular dyes are given below and in Table 1.
- the column was an Axxiom ODS 5 micron C 18 column #555-102 available from Cole Scientific, Calabasas, CA. This gradient is not optimized tor purification of PLP-15 and PLP-15-T-NH 2 , but the retention times are included for comparison with the dye primer conjugates.
- a PLP-15 is an oligonucleotide primer for DNA sequence analysis in the M13 vectors. Its sequence is 5′CCC AGT CAC GAC FTT 3′.
- the colored material is further purified by reverse phase high performance liquid chromatography on an Axxiom C 18 column (#555-102, Cole Scientific, Calabasas, Calif.) in a linear gradient of acetonitrile:0.1 M triethylammonium acetate, pH 7.0. It is convenient for this separation to run the column eluant through both a UV detector (for detecting the DNA absorbance) and a fluorescence detector (for detecting the dye moiety).
- the desired product is a peak on the chromatogram which is both strongly UV absorbing and strongly fluorescent.
- the dye oligonucleotide conjugates elute at higher acetonitrile concentrations than the oligonucleotides alone, as shown in Table 1.
- the oligonucleotide is obtained from the high performance liquid chromatographyin solution in a mixture of acetonitrile and 0.1 M triethylammonium acetate buffer. This is removed by lyophilization and the resulting material is redissolved by vortexing in 10 mM sodium hydroxzide (for a minimum amount of time) followed by neutralization with a five fold molar excess (to sodium hydroxide) of Tris buffer, pH 7.5.
- the conjugation with tetramethyl rhodamine isothiocyanate cyanate is identical to that for Texas Red except that the reaction-is carried out in 10 mM sodium carbonate/bicarbonate buffer, pH 9.0, and 50% dioxane. This increases solubility of the tetramethyl rhodamine and a much higher yield of dye oligonucleotide conjugate is obtained.
- the autoradiogram showed that the underivitized primer, amino-derivitized primer, and dye conjugated primers all give the same pattern of bands (corresponding to the DNA sequence), indicating that the derivitized primers retain their ability to hybridize specifically to the complementary strand.
- the bands generated using the different primers differ in their mobilities, showing that it is indeed the dye-primers which are responsible for the observed pattern, and not a contaminant of unreacted or underivitized oligonucleotide.
- the intensity of the bands obtained with the different primers is comparable, indicating that the strength of hybridization is not significantly perturbed by the presence of the dye molecules.
- the separations are again carried out in an acrylamide gel column.
- the beam from an argon ion laser is passed into the polyacrylamide gel tube (sample) by means of a beamsteerer. Fluorescence exited by the beam is collected using a low f-number lens, passed through an appropriate set of optical filters to eliminate scattered excitation light and detected using a photomultiplier tube (PMT).
- PMT photomultiplier tube
- the signal is monitored on a strip chart recorder.
- DNA sequencing reactions have been carried out utilizing each of the four different dye coupled oligonucleotide primers. In each case a series of peaks are observed on the chart paper.
- the peaks correspond to fragments of dye labeled DNA of varying lengths synthesized in the sequencing reactions and separated in the gel tube by electrophoresis.
- Each peak contains of the order of 10 ⁇ 14 to 10 ⁇ 16 moles of dye, which is approximately equal to the amount of DNA obtained per band in an equivalent sequencing gel utilizing radioisotope detection. This proves that the fluorescent tag is not removed or degraded from the oligonucleotide primer in the sequencing reactions. It also demonstrates that the detection sensitivity is quite adequate to perform DNA sequence analysis by this means, and that adequate resolution of the DNA fragments is obtained in a tube gel system.
Abstract
Description
-
- a source of chromophore or fluorescent tagged DNA fragments.
- a zone for containing an electrophoresis gel,
- means for introducing said tagged DNA fragments to said zone; and
- photometric means for monitoring or detecting said tagged DNA fragments as they move through and are separated by said gel.
TABLE 1 |
Reverse Phase HPLC Conditions for |
Dye-oligonucleotide Purification |
Sample | Retention time | |
PLP-15a | 18′ | |
PLP-15-T-NH2 b | 18′ | |
FITC PLP-15c | 27′ | |
NBD PLP-15 | 25′ | |
TMRITC PLP-15 | 32′ and 34′d | |
Texas Red PLP-15 | 42′ | |
Retention limes shown are for HPLC gradients of 20% solvent B/80% solvent A to 60% solvent B/40% solvent A in 40 min., where solvent A is 0.1 M triethylammonium acetate pH 7.0 and solvent B is 50% acelonitrile, 50% 0.1 M triethylammonium acetate pH 7.0. The column was an |
||
aPLP-15 is an oligonucleotide primer for DNA sequence analysis in the M13 vectors. Its sequence is 5′CCC AGT |
||
bPLP-15-T-NH2 is the oligonucleotide PLP-15 to which a 5′-amino-5′-deoxythymidine base has been added to==at the 5′ terminus. | ||
cThe nomenclature Dye PLP-15 signifies the conjugate of PLP-15-T-NH2 and the dye molecule. | ||
dTwo fluorescent oligonucleotide products were obtained with TMRITC. Both were equally effective in sequencing. This is presumed to be due to the two isomers of TMRITC which are present in the commercially available material. |
-
- a) prior to separation on Sephadex G-25 the reaction is made 1 M in ammonium acetate and kept at room temperature for 30 minutes, and
- b) the Sephadex G-25 column is run in 0.1 M ammonium acetate. This largely eliminates nonspecific binding of the dye molecule to the oligonucleotide.
Claims (158)
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US57097384A | 1984-01-16 | 1984-01-16 | |
US68901385A | 1985-01-02 | 1985-01-02 | |
US72274285A | 1985-04-11 | 1985-04-11 | |
US10623287A | 1987-10-07 | 1987-10-07 | |
US66016091A | 1991-02-21 | 1991-02-21 | |
US89801992A | 1992-06-12 | 1992-06-12 | |
US36117694A | 1994-12-21 | 1994-12-21 | |
US08/484,340 US6200748B1 (en) | 1984-01-16 | 1995-06-07 | Tagged extendable primers and extension products |
US10/389,663 USRE43096E1 (en) | 1984-01-16 | 2003-03-13 | Tagged extendable primers and extension products |
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