Methods for sequencing GC-rich and CCT repeat DNA templates

• US 20050053948A1
• Filed: 09/04/2003
• Published: 03/10/2005
• Est. Priority Date: 09/04/2003
• Status: Active Grant

• Current Assignee: Los Alamos National Security LLC (Government of the United States of America)
• Original Assignee: Los Alamos National Security LLC (Government of the United States of America)
• Inventors: Robinson, Donna L.
• Granted Patent: US 7,179,602 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2525/151 repeat or repeated sequences, e.g. VNTR, microsatellite, concatemer
• CPC Class Codes: C12Q 2527/101 Temperature
• CPC Class Codes: C12Q 2535/113 Cycle sequencing

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.

Claims

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.

2. The method according to claim 1, wherein the number of cycles is between about 30 and 50 cycles.

3. The method according to claim 1, wherein the number of cycles is between about 50 and 60 cycles.

4. The method according to claim 1, wherein the number of cycles is between about 60 and 70 cycles.

5. The method according to claim 1, wherein the the primers are complementary to a PUC18 vector containing the sample DNA and have the following nucleotide sequences:

6. The method according to claim 5, wherein primer annealing is achieved at 67°

C. for 30 seconds, and primer extension is achieved at 75°

C. for 4 minutes.

7. The method according to claim 1, wherein the thermostable DNA polymerase is a Taq polymerase.

8. The method according to claim 1, wherein the Taq polymerase contains a F667Y point mutation.

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 cycle

Step 2=30 sec @ 92°

C.

30 sec @ 67°

C.

4 min @ 75°

C.×

60 cycles

Step 3=soak @ 4°

C.

(b) determining the nucleotide sequence of the DNA sample.

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 cycle

Step2=15 sec@92°

C.

10 sec@ 54°

C.

4 min @ 65°

C.×

60 cycles

Step 3=soak @ 4°

C.

(b) determining the nucleotide sequence of the DNA sample.