Template switch-based methods for producing a product nucleic acid
View Patent ↗Provided are methods of producing a product nucleic acid. The methods include combining a template deoxyribonucleic acid (DNA), a polymerase, a template switch oligonucleotide, and dNTPs into a reaction mixture. The components are combined into the reaction mixture under conditions sufficient to produce a product nucleic acid that includes the template DNA and the template switch oligonucleotide each hybridized to adjacent regions of a single product nucleic acid that includes a region polymerized from the dNTPs by the polymerase. Aspects of the invention further include compositions and kits.
1. A method comprising:
combining:
a template deoxyribonucleic acid (DNA);
a retroviral reverse transcriptase;
a template switch oligonucleotide;
a first DNA primer comprising a 3′ domain that hybridizes to the template DNA; and
dNTPs;
into a reaction mixture under conditions sufficient to produce a product nucleic acid, wherein the product nucleic acid comprises the template DNA and the template switch oligonucleotide each hybridized to adjacent regions of a single hybrid nucleic acid comprising a region polymerized from the dNTPs by the retroviral reverse transcriptase, wherein the region polymerized from the dNTPs comprises a domain that is complementary to and hybridized to the 5′ end of the template DNA.
2. The method according to claim 1 , wherein the template switch oligonucleotide comprises a 3′ hybridization domain and a 5′ adapter sequence.
3. The method according to claim 2 , wherein the 3′ hybridization domain comprises a homo-trinucleotide.
4. The method according to claim 2 , wherein the 3′ hybridization domain comprises a hetero-trinucleotide.
5. The method according to claim 4 , wherein the hetero-trinucleotide comprises a nucleotide comprising cytosine and a nucleotide comprising guanine.
6. The method according to claim 1 , wherein the template switch oligonucleotide comprises a modification that prevents the retroviral reverse transcriptase from switching from the template switch oligonucleotide to a different template nucleic acid after synthesizing the complement of the template switch oligonucleotide.
7. The method according to claim 1 , wherein the method further comprises contacting a 3′ region of the single hybrid nucleic acid with a second strand primer configured to bind thereto under hybridization conditions.
8. The method according to claim 1 , wherein the method further comprises subjecting the single hybrid nucleic acid to nucleic acid amplification conditions.
9. The method according to claim 1 , wherein the template DNA is single stranded.
10. The method according to claim 1 , wherein the template DNA is double-stranded.
11. The method according to claim 10 , wherein the double-stranded template DNA comprises a 3′ overhang.
12. The method according to claim 11 , wherein the method further comprises producing the template DNA from a precursor double-stranded DNA.
13. The method according to claim 12 , wherein the method comprises contacting the precursor double-stranded DNA with a terminal transferase under conditions sufficient to produce the template DNA.
14. The method according to claim 12 , wherein the method comprises contacting the precursor double-stranded DNA with a restriction enzyme to produce the template DNA.
15. The method according to claim 2 , wherein the first DNA primer comprises a 5′ adapter sequence.