Shot-gun sequencing and amplification without cloning

• US 6,521,428 B1
• Filed: 11/04/1999
• Issued: 02/18/2003
• Est. Priority Date: 04/21/1999
• Status: Expired due to Term

• Current Assignee: GenOne Technologies, LLC
• Original Assignee: GenOne Technologies, LLC
• Inventors: Senapathy, Periannan
• Primary Examiner(s): Whisenant, Ethan C.
• Assistant Examiner(s): LU, FRANK WEI MIN
• Application Number:
  US09/434,761
• Time in Patent Office: 1,202 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 435/183, 436/94, 536/23.1, 536/24.3, 536/24.33, 536/25.3
• US Class Current: 435/91.2
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2525/179 incorporating arbitrary or random nucleotide sequences
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

Disclosed is a method for sequencing and amplifying nucleic acid templates wherein a degenerate primer with a fixed sequence region and a random sequence region is utilized. By determining the statistical expectancy of the fixed sequence in the nucleic acid template, this determines the average length of a nucleic acid template that can be sequenced. During the annealing of such a primer with the nucleic acid template, the fixed sequence determines where the complete primer binds by binding to its complementary sequence on the nucleic acid template. The random sequence regions of the primers make it possible for the presence of a unique sequence adjacent to the fixed sequence to be present, thus providing a primer with full complementarity with the nucleic acid template. Thus, this procedure is able to provide a full-length primer with a fully complementary sequence capable of binding statistically once within an expected length of the nucleic acid template, even though the sequence of the template is unknown. The method can also be adopted for use in PCR amplification of a nucleic acid template.

Claims

1. A method of sequencing a of a nucleic acid template comprising:

(a) providing a plurality of first primers, each first primer comprising (i) a region of different fixed nucleotide sequence having a defined length of from about 5 to 15 bases long and (ii) a region of randomized nucleotide sequence having a defined length of from about 2 to 11 bases long located 5′

to or 3′

to the region of fixed nucleotide sequence;

(b) annealing the plurality of first primers to different locations on a nucleic acid template, wherein at least one primer from within the plurality of first primers anneals specifically to the template;

(c) extending the specifically annealed primer from step (b) with a mixture of dNTPs and ddNTPs to generate a series of nucleic acid fragments; and

(d) determining the nucleotide sequence of a first region of the template from the series of nucleic acid fragments.

2. The method of claim 1, wherein in step (a) is provided a plurality of first primers having a region of fixed nucleotide sequence of from about 10 to about 12 bases.

3. The method of claim 1, wherein in step (d) is determined about 500 bases of the first region of the nucleic acid template.

4. The method of claim 1, wherein in step (a) in each first primer the region of randomized nucleotide sequence contains only purine bases.

5. The method of claim 1, wherein in step (a) in each first primer the region of randomized nucleotide sequence contains only pyrmidine bases.

6. The method of claim 1, wherein in step (a) is provided a plurality of first primers wherein the region of randomized nucleotide sequence in the first primers has an unequal distribution of bases.

7. The method of claim 1, further comprising in step (b) cutting the template with a restriction enzyme prior to annealing.

8. A method of sequencing a first contig of a nucleic acid template comprising:

(a) providing a plurality of first primers, each first primer comprising (i) a region of different fixed nucleotide sequence having a defined length of from about 5 to 15 bases long and (ii) a region of randomized nucleotide sequence having a defied length of from about 2 to 11 bases long located 5′

to or 3′

to the region of fixed nucleotide sequence;

(b) annealing the plurality of first primers to different locations on a nucleic acid template, wherein at least one primer from within the plurality of first primers anneals specifically to the template;

(c) extending the specifically annealed primer from step (b) with a mixture of dNTPs and ddNTPs to generate a series of nucleic acid fragments; and

(d) determining the nucleotide sequence of the template from the series of nucleic acid fragments;

(e) providing a plurality of second primers, each second primer comprising (i) a region of different fixed nucleotide sequence of from about 5 to 15 bases long and (ii) a region of randomized nucleotide sequence of from about 2 to 11 bases long located 5′

to or 3′

to the region of fixed nucleotide sequence;

(f) repeating steps (b)-(d) for the second plurality of primers to thereby determine the nucleotide sequence of a second region of the template; and

(g) assembling sequenced regions and the second, sequenced regions of the template nucleic acid to form a first contig.

9. The method of claim 8, wherein in step (f) about 500 bases of the second region of the nucleic acid template are determined.

10. The method of claim 8, further comprising:

(h) repeating steps (e)-(g) to form a second contig;

(i) providing a plurality of third primers, each third primer comprising (i) a region of different fixed nucleotide sequence of from about 5 to 15 bases long and (ii) a region of randomized nucleotide sequence of from about 2 to 11 bases long located 5′

to or 3′

to the region of fixed nucleotide sequence;

(i) annealing the plurality of third primers to different locations on the nucleic acid template, wherein at least one primer from the third plurality annals to the template near a terminus of one of the first or second contigs;

(k) extending the annealed third primer with a mixture of dNTPs and ddNTPs to generate a series of nucleic acid fragments; and

(l) determining the sequence of the template between the first and second contigs from the series of nucleic acid fragments.

11. A method of sequencing a nucleic acid template comprising:

(a) providing a plurality of first primers, each first primer comprising (i) a region of different fixed nucleotide sequence having a defined length of from about 5 to 15 bases long and (ii) a region of randomized nucleotide sequence having a defined length of from about 2 to 11 bases long located 5′

to or 3′

to the region of fixed nucleotide sequence;

(b) annealing the plurality of first primers to different locations on a nucleic acid template, wherein at least one primer from within the plurality of first primers anneals specifically to the template;

(b)(i) adding a plurality of fixed-sequence primers, each fixed-sequence primer comprising (i) a shorter region of different fixed nucleotide sequence that is shorter than the region of fixed nucleotide sequence in the first plurality of primers and (ii) a region of randomized nucleotide sequence located 5′

to or 3′

to the region of fixed nucleotide sequence;

(b)(ii) annealing the plurality of fixed-sequence primers to different locations on the nucleic acid template, wherein at least one fixed-sequence primer anneals to the nucleic acid template; and

(b)(iii) amplifying the nucleic acid template with the annealed first and annealed fixed-sequence primers with a mixture of dNTPs to amplify copies of the nucleic acid template bounded by the annealed first and fixed-sequence primers;

(c) extending the specifically annealed primer from step (b) with a mixture of dNTPs and ddNTPs to generate a series of nucleic acid fragments; and

(d) determining the nucleotide sequence of the template from the series of nucleic acid fragments.

12. A method of sequencing a first region of a nucleic acid template comprising:

(a) providing a plurality of first primers, each first primer comprising (i) a region of different fixed nucleotide sequence having a defined length of from about 5 to 15 bases long and (ii) a region of randomized nucleotide sequence having a defined length of from about 2 to 11 bases long located 5′

to or 3′

to the region of fixed nucleotide sequence;

(b) annealing the plurality of first primers to different locations on a nucleic acid template, wherein at least one primer from within the plurality of first primers anneals specifically to the template and wherein a sequence corresponding to or complementary to the region of fixed nucleotide sequence of the first plurality of primers occurs within the nucleic acid template at a frequency that is different than statistically predicted based on a random distribution of bases throughout the template;

(c) extending the specifically annealed primer from step (b) with a mixture of dNTPs and ddNTPs to generate a series of nucleic acid fragments; and

(d) determining the nucleotide sequence of a first region of the template from the series of nucleic acid fragments.

13. The method of claim 12, wherein the frequency is greater than statistically predicted based on a random distribution of the template.

14. The method of claim 12, wherein the frequency is less than statistically predicted based on a random distribution of bases throughout the template.

15. The method of claim 12, wherein binding sites complementary to the region of fixed nucleotide sequence in each primer of the plurality of first primers are distributed uniformly throughout the template.

16. A method of sequencing a nucleic acid template comprising:

(a) providing a plurality of first primers, each first primer comprising (i) a region of different fixed nucleotide sequence having a defined length of from about 5 to 15 bases long and (ii) a region of randomized nucleotide sequence having a defined length of from about 2 to 11 bases long located 5′

to or 3′

to the region of fixed nucleotide sequence;

(a)(i) relaxing torsion, secondary structure, or tertiary structure in the template;

(b) annealing the plurality of first primers to different locations on a nucleic acid template, wherein at least one primer from within the plurality of first primers anneals specifically to the template;

(c) extending the specifically annealed primer from step (b) with a mixture of dNTPs and ddNTPs to generate a series of nucleic acid fragments; and

(d) determining the nucleotide sequence of a first region of the template from the series of nucleic acid fragments.

17. The method of claim 16, wherein in step (a)(i), the torsion, secondary structure, or tertiary is relaxed by shearing, nebulizing, nicking, or cutting the template.

18. The method of claim 16, wherein in step (a)(i), relaxing torsion, secondary structure, or tertiary structure in the template yields template nucleic acid fragments and the fragments remain commingled during steps (b) and (c).

19. A method of sequencing a nucleic acid template comprising:

(a) providing a plurality of first primers, each first primer comprising (i) a region of different fixed nucleotide sequence having a defined length of from about 5 to 15 bases long and (ii) a region of randomized nucleotide sequence having a defined length of from about 2 to 11 bases long located 5′

to or 3′

to the region of fixed nucleotide sequence and a handle at an end of each first primer;

(b) annealing the plurality of first primers to different locations on a nucleic acid template, wherein at least one primer from within the plurality of first primers anneals specifically to the template;

(c) extending the specifically annealed primer from step ho) with a mixture of dNTPs and ddNTPs to generate a series of nucleic acid fragments; and

(d) determining the nucleotide sequence of a first region of the template from the series of nucleic acid fragments.

20. The method of claim 19, wherein the handle is at the 5′

end of each first primer.

21. The method of claim 20, wherein the handle is one or more universal bases.

22. The method of claim 21, wherein the universal base is selected from the group consisting of inosine and 5-nitroindole.

23. The method of claim 20, wherein the handle is one or more purine bases.

24. The method of claim 20, wherein the handle is one or more pyrimidine bases.

25. A method for amplifying a nucleic acid template comprising:

(a) providing a plurality of fast primers, each first primer comprising (i) a region of different fixed nucleotide sequence having a defined length of from about 5 to 15 bases long and (ii) a region of randomized nucleotide sequence having a defined length of from about 2 to 11 bases long located 5′

to or 3′

to the region of fixed nucleotide sequence;

(b) providing a plurality of second primers, each second primer comprising (i) a region of different fixed nucleotide sequence having a defined length of from about 5 to 15 bases long and (ii) a region of randomized nucleotide sequence having a defined length of from about 2 to 11 bases long located 5′

to or 3 ′

to the region of fixed nucleotide sequence, wherein the regions of fixed nucleotide sequence in the second plurality of primers is shorter than the regions of fixed nucleotide sequence in the first plurality of primers; and

(c) amplifying a first region of the nucleic acid template with the plurality of first primers and the plurality of second primers, wherein at least one primer from within each of the plurality of first primers and the plurality of second primers anneals specifically to the template.

26. The method of claim 25, wherein in step (a) is provided a plurality of first primers that is labeled and further sequencing the nucleic acid template with at least one annealed, labeled first primer.

27. The method of claim 25, further wherein in step (a) is provided a plurality of first primers having a handle at an end of the plurality of first primers.

28. The method of claim 27, wherein the handle is located at the 5′

end of the plurality of first primers.

29. The method of claim 25, wherein in step (b) the distance between annealing sites of a first plurality of primers and a second plurality of primers is at least 1000 bp.