Targeted rolling circle amplification

• US 9,290,800 B2
• Filed: 03/13/2014
• Issued: 03/22/2016
• Est. Priority Date: 03/15/2013
• Status: Active Grant

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA), Regents of the University of California (University of California)
• Original Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA), Regents of the University of California (University of California)
• Inventors: Turner, Stephen W., Pham, Thang, Hagerman, Paul
• Primary Examiner(s): Strzelecka, Teresa E
• Application Number:
  US14/208,451
• Publication Number:
  US 20140295498A1
• Time in Patent Office: 740 Days
• US Class Current: 1/1
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2525/131 incorporating a restriction site
• CPC Class Codes: C12Q 2531/125 Rolling circle

Abstract

Methods for amplifying a desired target region of a nucleic acid through rolling circle amplification with a strand-displacing polymerase are provided. Concatameric hairpin products are resolved with endonuclease digestion, and the resulting amplified product hairpins or fragments can be circularized and employed as templates in a subsequent round of amplification. The methods are effective for targeted amplification of even highly repetitive sequences. Compositions, kits, and systems related to or useful in the methods are also described.

Claims

1. A method of amplifying a nucleic acid target region, the method comprising:

a) providing a nucleic acid template, which template is a circular nucleic acid having a double-stranded central region and two single-stranded hairpin end regions;

wherein the double-stranded central region comprises a first polynucleotide sequence and a second polynucleotide sequence complementary to the first polynucleotide sequence, which first and second polynucleotide sequences collectively comprise the target region, a recognition site for a first restriction endonuclease, and a recognition site for a second restriction endonuclease, which recognition sites flank the target region; and

wherein the circular nucleic acid comprises a first primer binding sequence;

b) binding a first primer to the first primer binding sequence;

c) performing polymerase-mediated template-directed primer extension of the first primer with a polymerase comprising strand displacement activity, thereby producing a first nucleic acid product comprising at least two copies of the first polynucleotide sequence and the second polynucleotide sequence, at least one copy of each of which is not base paired to the template;

d) digesting the first product with the first and second restriction endonucleases and releasing at least one first product fragment having a double-stranded region that comprises the target region, and

e) ligating a first hairpin adapter to one end of the first product fragment and ligating a second hairpin adapter to the other end of the first product fragment, thereby producing at least one first circular progeny nucleic acid.

2. The method of claim 1, wherein step d) is performed concurrently with step c).

3. The method of claim 1, wherein step e) is performed concurrently with step d).

4. The method of claim 1, wherein steps c), d), and e) are performed concurrently.

5. The method of claim 1, comprising performing template-directed sequencing by synthesis on at least a portion of the target region in the first circular progeny nucleic acid.

16. The method of claim 1, wherein the first circular progeny nucleic acid comprises a second primer binding sequence, a recognition site for a third restriction endonuclease, and a recognition site for a fourth restriction endonuclease, which recognition sites flank the target region, the method comprising:

without denaturing the first circular progeny nucleic acid, binding a second primer to the second primer binding sequence;

performing polymerase-mediated template-directed primer extension of the second primer with a polymerase comprising strand displacement activity, thereby producing a second nucleic acid product comprising at least two copies of the first polynucleotide sequence and the second polynucleotide sequence, at least one copy of each of which is not base paired to the first circular progeny nucleic acid; and

digesting the second product with the third and fourth endonucleases and releasing at least one second product fragment having a double-stranded region that comprises the target region.

17. The method of claim 16, wherein the first or second hairpin adapter comprises the second primer binding sequence.

18. The method of claim 16, wherein the second primer binding sequence is formed by ligation of the first or second hairpin adapter to the first product fragment.

19. The method of claim 1, wherein the first primer binding sequence is in one of the two single-stranded hairpin end regions in the template.

20. The method of claim 1, wherein the nucleic acid template and/or the first circular progeny nucleic acid is not subject to being cut by the first endonuclease.

21. The method of claim 1, wherein the target region comprises at least five tandem copies of a mononucleotide, dinucleotide, trinucleotide, tetranucleotide, or pentanucleotide repeat sequence.

6. A method of amplifying a nucleic acid target region, the method comprising:

a) providing a nucleic acid template, which template is a circular nucleic acid having a double-stranded central region and two single-stranded hairpin end regions;

wherein the double-stranded central region comprises a first polynucleotide sequence and a second polynucleotide sequence complementary to the first polynucleotide sequence, which first and second polynucleotide sequences collectively comprise the target region and a recognition site for a first site-specific endonuclease; and

wherein the circular nucleic acid comprises a first primer binding sequence;

b) binding a first primer to the first primer binding sequence;

c) performing polymerase-mediated template-directed primer extension of the first primer with a polymerase comprising strand displacement activity, thereby producing a first nucleic acid product comprising at least two copies of the first polynucleotide sequence and the second polynucleotide sequence, at least one copy of each of which is not base paired to the template;

d) cutting the first product with the first endonuclease and releasing at least one first product hairpin having at least one single-stranded hairpin end region, a double-stranded region that comprises the target region, a free 5′

terminus, and a free 3′

terminus; and

e) circularizing the at least one first product hairpin, thereby producing at least one first circular progeny nucleic acid.

7. The method of claim 6, wherein the recognition site for the first endonuclease is proximal to the first primer binding sequence in the template.

8. The method of claim 6, wherein the first endonuclease is a restriction endonuclease, and wherein circularizing the at least one first product hairpin comprises ligating a first hairpin adapter to the first product hairpin.

9. The method of claim 8, wherein the first circular progeny nucleic acid comprises a second primer binding sequence and a recognition site for a second restriction endonuclease, the method comprising:

without denaturing the first circular progeny nucleic acid, binding a second primer to the second primer binding sequence;

performing polymerase-mediated template-directed primer extension of the second primer with a polymerase comprising strand displacement activity, thereby producing a second nucleic acid product comprising at least two copies of the complement of the first copy nucleic acid, at least one copy of which is not base paired to the first circular progeny nucleic acid;

digesting the second product with the second restriction endonuclease and releasing at least one second product hairpin having one single-stranded hairpin end region and a double-stranded region that comprises the target region; and

ligating a second hairpin adapter to the second product hairpin, thereby producing at least one second circular progeny nucleic acid.

10. The method of claim 9, wherein the first hairpin adapter comprises the second primer binding sequence.

11. The method of claim 9, wherein the second primer binding sequence is formed by ligation of the first hairpin adapter to the first product hairpin.

12. The method of claim 9, wherein the primer extension, digesting, and ligating steps to produce the first circular progeny nucleic acid and the primer extension, digesting, and ligating steps to produce the second circular progeny nucleic acid are performed in a single reaction mixture.

13. The method of claim 8, wherein the first hairpin adapter has a single-stranded overhang that is complementary to a single-stranded overhang on the first product hairpin.

14. The method of claim 8, wherein the cleavage site for the first restriction endonuclease is within the target region.

15. The method of claim 6, wherein the first endonuclease is a nicking endonuclease, and wherein circularizing the at least one first product hairpin comprises ligating the 5′

terminus of the first product hairpin intramolecularly to the 3′

terminus.