Methods for sequencing GC-rich and CCT repeat DNA templates
First Claim
Patent Images
1. A method of fluorescence-based cycle sequencing of a sample DNA, comprising, (a) preparing a reaction mixture containing:
- (i) the sample DNA, (ii) a primer set complementary to DNA primer sites flanking or interspersed within the sample DNA, wherein the Td of the primers in the primer set are between about 72°
C. and 75°
C., (iii) a thermostable polymerase, (iv) a mixture of dNTPs and fluorescently-labeled ddNTPs, and (v) a suitable buffer (b), dissociating the sample DNA to create single stranded templates, wherein said dissociation is achieved by heating the DNA to between about 92°
C. and 95°
C. for at least about 3 minutes;
(c) annealing the primers to the primer sites, wherein said annealing is achieved at a temperature of between about 65°
C. and 67°
C. for at least about 30 seconds;
(d) extending the annealed primers to generate a series of fluorescently-labeled dideoxynucleic acid fragments, wherein said primer extension is achieved at a temperature of between about 75°
C. and 78°
C. for between about 3 to 4 minutes;
(e) heating the reaction mixture to between about 92°
C. and 95°
C. in order to dissociate double stranded DNA;
(f) repeating the steps c through e for a plurality of cycles; and
(e) determining the nucleotide sequence of the sample DNA from the series of fluorescently-labeled dideoxynucleic acid fragments present in the reaction mixture.
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Abstract
The present invention is directed to a PCR-based method of cycle sequencing DNA and other polynucleotide sequences having high CG content and regions of high GC content, and includes for example DNA strands with a high Cytosine and/or Guanosine content and repeated motifs such as CCT repeats.
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Citations
10 Claims
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1. A method of fluorescence-based cycle sequencing of a sample DNA, comprising,
(a) preparing a reaction mixture containing: -
(i) the sample DNA, (ii) a primer set complementary to DNA primer sites flanking or interspersed within the sample DNA, wherein the Td of the primers in the primer set are between about 72°
C. and 75°
C.,(iii) a thermostable polymerase, (iv) a mixture of dNTPs and fluorescently-labeled ddNTPs, and (v) a suitable buffer (b), dissociating the sample DNA to create single stranded templates, wherein said dissociation is achieved by heating the DNA to between about 92°
C. and 95°
C. for at least about 3 minutes;
(c) annealing the primers to the primer sites, wherein said annealing is achieved at a temperature of between about 65°
C. and 67°
C. for at least about 30 seconds;
(d) extending the annealed primers to generate a series of fluorescently-labeled dideoxynucleic acid fragments, wherein said primer extension is achieved at a temperature of between about 75°
C. and 78°
C. for between about 3 to 4 minutes;
(e) heating the reaction mixture to between about 92°
C. and 95°
C. in order to dissociate double stranded DNA;
(f) repeating the steps c through e for a plurality of cycles; and
(e) determining the nucleotide sequence of the sample DNA from the series of fluorescently-labeled dideoxynucleic acid fragments present in the reaction mixture. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8)
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9. A method of sequencing a GC-rich DNA sample on an automated fluorescence-based cycle sequencer, comprising
(a) providing primers having a Td of between about 73° - C. and 74°
C. in a dye-terminator sequencing reaction comprising the DNA sample, a Taq polymerase and dNTPs and flubrescently-labeled ddNTPs, in a suitable buffer, under substantially the following cycle conditions;
Step 1=3 min@ 92°
C.×
1 cycleStep 2=30 sec @ 92°
C.30 sec @ 67°
C.4 min @ 75°
C.×
60 cyclesStep 3=soak @ 4°
C.(b) determining the nucleotide sequence of the DNA sample.
- C. and 74°
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10. A method of sequencing a DNA sample containing CCT repeats on an automated fluorescence-based cycle sequencer, comprising
(a) providing primers having a Td of between about 57° - C. and 75°
C. in a dye-terminator sequencing reaction comprising the DNA sample, a Taq polymerase and dNTPs and fluorescently-labeled ddNTPs, in a suitable buffer, under substantially the following cycle conditions;
Step 1=min @ 92°
C.×
1 cycleStep2=15 sec@92°
C.10 sec@ 54°
C.4 min @ 65°
C.×
60 cyclesStep 3=soak @ 4°
C.(b) determining the nucleotide sequence of the DNA sample.
- C. and 75°
Specification