Coupled amplification and sequencing of DNA

• US 5,427,911 A
• Filed: 07/28/1993
• Issued: 06/27/1995
• Est. Priority Date: 05/01/1990
• Status: Expired due to Term

• Current Assignee: First Union Bank of Connecticut
• Original Assignee: Yale University
• Inventors: Ruano, Gualberto
• Primary Examiner(s): BICKEL, MINDY B
• Application Number:
  US08/098,748
• Time in Patent Office: 699 Days
• Field of Search: 435/6, 435/91.2, 536/24.33
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6869 Methods for sequencing

Abstract

A process for sequencing DNA segments having complementary strands comprising (a) synthesizing simultaneously truncated products and full-length products starting from both 3'"'"' ends of the complementary strands of the DNA segment, which serves as a template, by introducing specific oligonucleotide primers annealing to the 3'"'"' ends of both complementary strands of the DNA segment, a deoxyribonucleotide elongator for each of adenine, guanine, cytosine and thymine, a thermally stable DNA polymerase and a terminator for each of adenine, guanine, cytosine and thymine, (b) thermally cycling step (a), (c) separating out the resultant truncated products and full length products according to size, and (d) selectively detecting all truncated products and full length products according to size, and (e) selectively detecting all truncated products and full-length products generated from a given strand of template.

Claims

1. A process for sequencing DNA segments comprising:

(a) exponentially and simultaneously synthesizing both truncated products and full length products starting from 3'"'"' ends of each of two complementary strands of the DNA segment which serve as a template, Said exponential and simultaneous synthesis using the following reagents;

(i) a first primer for annealing to the 3'"'"' end of one of said two complementary strands of the DNA segment,

(ii) a second primer, which is different from the first primer, for annealing to the 3'"'"' end of the other of said two complementary strands of the DNA segment,

(iii) a reaction buffer,

(iv) a deoxyribonucleotide elongator for each of adenine, guanine, cytosine and thymine,

(v) a thermally stable DNA polymerase and

(vi) a dideoxynucleotide terminator for each of adenine, guanine, cytosine and thymine,

carrying out said synthesis by thermal cycling to provide a sufficiently readable signal,

separating out the resultant truncated products and full length products according to size, and

selectively detecting truncated products and full length products synthesized from one or the other of said two complementary strands of the DNA segments.

2. A process according to claim 1, wherein the thermally stable DNA polymerase is Taq DNA polymerase.

3. A process according to claim 1, wherein said thermal cycling comprises 10 to 20 cycles of denaturation at 90°

to 95°

C., annealing at 45°

to 65°

C. and extension at 65°

to 75°

C.

4. A process according to claim 1, wherein the separating out is conducted by high resolution gel electrophoresis.

5. A process according to claim 1, wherein the molar ratio of the dideoxynucleotide terminators the deoxyribonucleotide elongators are as follows:

##EQU2##

11. A process according to claim 1, wherein the DNA segment is a genomic DNA fragment of less than 2 kb.

12. A process according to claim 11, wherein the DNA is amplified in vitro to form said template.

13. A process according to claim 12, wherein the DNA polymerase is Taq DNA polymerase;

the thermally cycling comprises 10 to 20 cycles of denaturation at 90°

to 95°

C., annealing at 45°

to 65°

C. and extension at 65°

to 75°

C.;

the amplifying comprising introducing a thermally stable Taq DNA polymerase enzyme, salts and a deoxyribonucleotide elongator for each of adenine, guanine, cytosine and thymine and conducting 15 to 25 cycles of denaturation at 90°

to 95°

C., annealing at 45°

to 65°

C. and extension at 65°

to 75°

C.;

the separating out is conducted by high resolution gel electrophoresis; and

the molar ratios of the dideoxynucleotide terminators to the deoxyribonucleotide elongators are as follows;

##EQU4##

15. The process according to claim 1 wherein the detection is accomplished by using a labeled primer as said first or second primer.

16. The process according to claim 1 wherein detection is accomplished by using differentially labelled primers for both said first and second primers.

20. A process according to claim 1, wherein the DNA segment is a single stranded DNA of less than 2 kb.

21. A process according to claim 1, wherein the DNA segment is an oligonucleotide of less than 100 nucleotides.

6. A process for determining a base sequence of a DNA segment comprising

(a) amplifying in vitro said DNA, to obtain at least 10 femtomoles of amplification products, to form a template,

(b) aliquoting the resultant template from (a) into two aliquots,

(c) adding to each aliquot from (b) a labeled primer specific for each end of the template,

(d) subaliquoting each of the two aliquots from (c) into two sets of five aliquots,

(e) for each set of aliquots from (d), adding a dideoxynucleotide terminator for guanine to one aliquot, adding a dideoxynucleotide terminator for adenine to one aliquot, adding a dideoxynucleotide terminator for cytosine to one aliquot and adding a dideoxynucleotide terminator for thymine to one aliquot and maintaining one aliquot as a control,

(f) thermally cycling each of the aliquots from (e), and

(g) separating out the resultant molecules according to size and selectively detecting products synthesized from one or the other of said strands of said amplification products to determine the sequence.

7. A process according to claim 6, wherein said amplifying comprises introducing a thermally stable enzyme, salts and a deoxyribonucleotide elongator for each of adenine, guanine, cytosine, and thymine and conducting 15 to 25 cycles of denaturation at 90°

to 95°

C. annealing at 45°

to 65°

C. and extension at 65°

to 75°

C.

8. A process according to claim 6, wherein the molar ratios of the dideoxynucleotide terminators to the deoxyribonucleotide elongators are as follows:

##EQU3##

9. A process according to claim 6, wherein thermal cycling is carried out with Taq DNA polymerase.

10. A process according to claim 6, wherein the separating out is conducted by high resolution gel electrophoresis.

14. A process according to claim 7, wherein the amplification using thermally stable Taq DNA polymerase enzyme, salts and a deoxyribonucleotide elongator for each of adenine, guanine, cytosine and thymine and conducting 15 to 25 cycles of denaturation at 90°

to 95°

C., annealing at 45°

to 65°

C. and extension at 65°

to 75°

C.;

the separating out is conducted by high resolution gel electrophoresis; and

the molar ratios of the dideoxynucleotide terminators to the deoxyribonucleotide elongators are as follows;

##EQU5##

17. A process according to claim 6 wherein the DNA segment is a genomic DNA fragment of less than 2 kb.

18. A process according to claim 6 wherein the DNA segment is a single stranded DNA of less than 2 kb.

19. A process according to claim 6 wherein the DNA segment is an oligonucleotide of less than 100 nucleotides.