Ligase-assisted nucleic acid circularization and amplification
View Patent ↗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 circulating nucleic acids extracted from blood is provided. Kits for performing the disclosed methods are also provided.
1. A method for generating a single-stranded DNA circle from a linear DNA, the method comprising:
(a) providing the linear DNA;
(b) incubating the linear 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; and
(c) incubating the ligatable DNA sequence with a ligase that is capable of template-independent, intra-molecular ligation of a single-stranded DNA sequence to generate the single-stranded DNA circle,
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 step (b) is performed using a phosphate donor other than adenosine triphosphate or deoxyadenosine triphosphate.
3. The method of claim 2 , wherein the phosphate donor is selected form the group consisting of guanosine triphosphate, cytidine triphosphate, uridine triphosphate, deoxythymidine triphosphate and combinations thereof.
4. The method of claim 3 , wherein the phosphate donor is guanosine triphosphate.
5. The method of claim 4 , wherein the linear DNA is incubated with the polynucleotide kinase in the presence of less than 200 μM of guanosine triphosphate.
6. The method of claim 5 , wherein the linear DNA is incubated with the polynucleotide kinase in the presence of up to 30 μM of guanosine triphosphate and up to 2.5 mM of manganese ion.
7. The method of claim 4 , further comprising denaturing the ligatable DNA sequence prior to step (c) if the ligatable DNA sequence is in double-stranded form.
8. The method of claim 7 , wherein the ligase is a pre-adenylated ligase.
9. The method of claim 8 , wherein the pre-adenylated ligase is a pre-adenylated TS2126 RNA ligase.
10. The method of claim 9 , wherein steps (a) to (c) are performed in a sequential manner in the single reaction vessel.
11. The method of claim 10 , wherein all the steps of the method are performed in the absence of adenosine triphosphate or deoxyadenosine triphosphate.
12. The method of claim 11 , wherein all the steps of the method are performed in HEPES buffer.
13. The method of claim 1 , wherein the linear DNA is selected from the group consisting of a circulating DNA, a DNA isolated from formalin fixed paraffin-embedded samples, a forensic DNA sample that have been exposed to environmental conditions, and an ancient DNA sample.
14. The method of claim 1 , further comprising amplifying the single-stranded DNA circle under isothermal conditions.
15. The method of claim 14 , wherein the single-stranded DNA circle is amplified via rolling circle amplification.
16. The method of claim 15 , wherein the rolling circle amplification is performed using a nuclease-resistant primer.
17. The method of claim 16 , wherein the nuclease-resistant primer comprises at least one modified nucleotide.
18. The method of claim 15 , wherein the single-stranded DNA circle is amplified using a random primer mixture.
19. The method of claim 15 , wherein the single-stranded DNA circle is amplified using a primer solution that consists essentially of a partially constrained primer mixture comprising a terminal mismatch primer-dimer structure.
20. A method of generating a single-stranded DNA circle from a linear DNA, the method comprising:
(a) providing the linear DNA;
(b) optionally incubating the linear DNA with a polynucleotide kinase in the presence of adenosine triphosphate to generate a ligatable DNA sequence having a phosphate group at a 5′ terminal end and a hydroxyl group at a 3′ terminal end;
(c) incubating the linear DNA sequence or the ligatable DNA sequence with an adenylating enzyme in the presence of adenosine triphosphate to generate a 5′ adenylated DNA sequence; and
(d) incubating the 5′ adenylated DNA sequence with a non-adenylated ligase, which is capable of template-independent intra-molecular ligation of a 5′ adenylated, single-stranded DNA sequence to generate the single-stranded DNA circle,
wherein all the steps of the method are performed in single reaction vessel without any intervening isolation or purification steps.
21. The method of claim 20 , further comprising denaturing the 5′ adenylated DNA sequence prior to step (d) if the 5′ adenylated DNA sequence is in double-stranded form.
22. The method of claim 21 , wherein the adenylating enzyme is an RNA ligase 1 derived from thermophilic archeabacteria, Methanobacterium thermoautotrophicum.
23. The method of claim 22 , wherein the non-adenylated ligase is a mutant or engineered ligase, which is adenosine-triphosphate-independent and is defective in self-adenlyation, de-adenylation and adenylate transfer.
24. The method of claim 23 , wherein the mutant ligase is a mutant of RNA ligase 1 derived from thermophilic archeabacteria, Methanobacterium thermoautotrophicum.
25. The method claim 22 , further comprising incubating the reaction mixture of step (c) comprising the 5′ adenylated DNA sequence with a phosphatase to eliminate the adenosine triphosphate from the reaction mixture.
26. The method of claim 25 , wherein the non-adenylated ligase is a TS2126 RNA ligase derived from thermus bacteriophage, TS2126.
27. The method of claim 26 , wherein steps (a) to (d) are performed in a sequential manner in the single reaction vessel.
28. The method of claim 20 , wherein the linear DNA is selected from the group consisting of a circulating DNA, a DNA isolated from formalin fixed paraffin-embedded samples, a forensic DNA sample that have been exposed to environmental conditions, and an ancient DNA sample.
29. The method of claim 20 , further comprising amplifying the single-stranded DNA circle under isothermal conditions.
30. The method of claim 29 , wherein the single-stranded DNA circle is amplified via rolling circle amplification.
31. The method of claim 30 , wherein the single-stranded DNA circle is amplified using a nuclease-resistant primer.
32. The method of claim 31 , wherein the nuclease-resistant primer comprises at least one modified nucleotide.
33. The method of claim 30 , wherein the single-stranded DNA circle is amplified using a random primer mixture.
34. The method of claim 30 , wherein the single-stranded DNA circle is amplified using a primer solution that consists essentially of a partially constrained primer mixture comprising a terminal mismatch primer-dimer structure.