Ligase-assisted nucleic acid circularization and amplification

Patent metadata

Field	Value
Publication number	US-9938568-B2
Application number	US-201514933275-A
Country	US
Kind code	B2
Filing date	Nov 5, 2015
Priority date	Jul 26, 2013
Publication date	Apr 10, 2018
Grant date	Apr 10, 2018

Abstract

Provided herein are methods for generation and amplification of a single-stranded DNA circle in a single reaction vessel from a linear DNA without any intervening purification steps. The single-stranded DNA circle is generated via a template-independent single-stranded DNA ligation. Whole-genome amplification of linear chromosomal DNA in a single tube using ligation-assisted DNA amplification is also provided.

First claim

The invention claimed is:

1. A method for nucleic acid amplification, the method comprising:
(a) providing a linear chromosomal DNA;
(b) incubating the linear chromosomal DNA with a ligase that is capable of template-independent, intra-molecular ligation of a single-stranded DNA sequence to generate a single-stranded DNA circle; and
(c) amplifying the single-stranded DNA circle via rolling circle amplification using a random primer mixture to form an amplified DNA product,
wherein the random primer mixture comprises oligonucleotide sequences comprising at least one nucleotide analogue, and
wherein all the steps of the method are performed in a single reaction vessel without any intervening isolation or purification steps.

2. The method of claim 1 , wherein the at least one nucleotide analogue comprises a 2-amino-deoxyadenosine.

3. The method of claim 1 , wherein the at least one nucleotide analogue comprises a 2-thio-deoxythymidine.

4. The method of claim 1 , wherein the random primer mixture comprises selective binding complementary oligonucleotides.

5. The method of claim 4 , wherein each member of the selective binding complementary oligonucleotides comprises at least one nucleotide comprising a 2-amino-deoxyadenosine or at least one nucleotide comprising a 2-thio-deoxythymidine.

6. The method of claim 1 , wherein the random primer mixture comprises oligonucleotide sequences comprising a phosphorothioate modified nucleotide, a LNA nucleotide, a nucleotide comprising a 2-amino-deoxyadenosine, a nucleotide comprising a 2-thio-deoxythymidine, or combinations thereof.

7. The method of claim 1 , wherein the random primer mixture is a hexamer comprising oligonucleotide sequences having a general structure +N+N(at N)(at N)(at N)*N.

8. The method of claim 1 , wherein each individual oligonucleotide sequence in the random primer mixture comprises at least one nucleotide analogue.

9. The method of claim 1 , wherein the concentration of the random primer mixture is higher than the concentration of the single-stranded DNA circle to promote multiple random-primed rolling circle amplification.

10. The method of claim 1 , wherein the linear chromosomal DNA is selected from the group consisting of a cell-free circulating DNA, a DNA isolated from a formalin fixed paraffin-embedded sample, a forensic DNA sample that has been exposed to environmental conditions, an ancient DNA sample, and combinations thereof.

11. The method of claim 1 , wherein the linear chromosomal DNA is a fragmented DNA.

12. The method of claim 1 , further comprising denaturing the linear chromosomal DNA to a single-stranded DNA prior to step (b), if the linear chromosomal DNA is in double-stranded form.

13. The method of claim 1 , wherein the ligase is selected from the group consisting of a TS2126 RNA ligase, a T4 RNA ligase, a T4 DNA ligase, a T3 DNA ligase, an E. Coli DNA ligase, and combinations thereof.

14. The method of claim 13 , wherein the ligase is a pre-adenylated ligase.

15. The method of claim 14 , wherein the pre-adenylated ligase is a pre-adenylated TS2126 RNA ligase.

16. The method of claim 1 , wherein the generation of the single-stranded DNA circle is performed in the absence of adenosine triphosphate or deoxyadenosine triphosphate.

17. The method of claim 1 , wherein steps (a) to (c) are performed in a sequential manner in the single reaction vessel.

18. The method of claim 1 , wherein all the steps of the method are performed in HEPES buffer.

19. The method of claim 1 , further comprising treating the linear chromosomal DNA with a polynucleotide kinase in the presence of a phosphate donor to generate a ligatable DNA sequence having a phosphate group at a 5′ terminal end and a hydroxyl group at a 3′ terminal end prior to incubating the linear chromosomal DNA with the ligase.

20. The method of claim 19 , wherein the linear chromosomal DNA is treated with the polynucleotide kinase in the presence of a phosphate donor other than adenosine triphosphate or deoxyadenosine triphosphate.

21. The method of claim 1 , further comprising sequencing the amplified DNA product.

22. The method of claim 1 , further comprising fragmenting the amplified DNA product to generate a genomic DNA library.

23. The method of claim 22 , further comprising employing the genomic DNA library for hybridization-based capture of a target DNA sequence.

24. The method of claim 23 , further comprising sequencing the captured target DNA sequence.

25. The method of claim 24 , wherein the target DNA sequence is an exome sequence.

26. The method of claim 12 , further comprising treating the single stranded DNA circles prior to step (c) to modify any damaged nucleobases.

27. The method of claim 26 , wherein the treatment is performed by incubating the ligation mixture of step (b) with a uracil DNA glycosylase, a formamidopyrimidine-DNA glycosylase, or combinations thereof.

28. The method of claim 1 , wherein the amplification is a whole genome amplification.

29. The method of claim 1 , wherein the rolling circle amplification is performed using a decontaminated DNA polymerase.

Assignees

• Gen Electric

Inventors

• Heller Ryan Charles
• Kvam Erik Leeming
• Nelson John Richard

Classifications

• C12Q1/6844 Primary

  Nucleic acid amplification reactions · CPC title

• C12Q1/6869

  Methods for sequencing · CPC title

• C12Q2527/137

  Concentration of a component of medium · CPC title

• C12Q2521/501

  Ligase · CPC title

• C12Q2531/125

  Rolling circle · CPC title