Ligase-assisted nucleic acid circularization and amplification
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.
1. A method for nucleic acid amplification, the method comprising:
(a) ligating a linear chromosomal DNA to generate a single-stranded DNA circle; and
(b) 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 2-amino-deoxyadenosine and 2-thio-deoxythymidine nucleotide analogs, 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 random primer mixture comprises selective binding complementary oligonucleotides.
3. The method of claim 1 , wherein the random primer mixture further comprises a phosphorothioate modified nucleotide, a LNA nucleotide, or combinations thereof.
4. 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.
5. The method of claim 1 , wherein the linear chromosomal DNA is a fragmented DNA.
6. The method of claim 1 , further comprising denaturing the linear chromosomal DNA to a single-stranded DNA prior to step (a), if the linear chromosomal DNA is in double-stranded form.
7. 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.
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 TS2 126 RNA ligase.
10. 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.
11. The method of claim 1 , wherein steps (a) to (b) are performed in a sequential manner in the single reaction vessel.
12. 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.
13. The method of claim 12 , 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.
14. The method of claim 1 , further comprising sequencing the amplified DNA product.
15. The method of claim 6 , further comprising treating the single stranded DNA circles prior to step (b) to modify any damaged nucleobases.
16. The method of claim 1 , wherein the amplification is a whole genome amplification.