Methods and compositions for preventing concatemerization during template-switching
View Patent ↗Compositions and methods for performing a template-switching reaction are provided that may include reducing or eliminating concatemerization of the template-switching oligonucleotide (TSO). In some embodiments, the composition may comprise: a reverse transcriptase; a TSO that includes a recognition sequence for a site-specific double strand nucleic acid cleaving enzyme, wherein the TSO has at its 3′ end at least one nucleotide capable of hybridizing to at least one or more non-templated nucleotides added to a templated cDNA strand by the reverse transcriptase; and a site-specific double strand nucleic acid cleaving enzyme that cleaves the TSO at the recognition sequence.
1. A method for making cDNA comprising:
(a) obtaining a reaction mix comprising:
a reverse transcriptase;
an RNA template;
a cDNA synthesis primer;
an oligonucleotide for template switching, the oligonucleotide comprising a 3′ end having at least one nucleotide that can hybridize to one or more non-templated nucleotides added to a templated cDNA strand by the reverse transcriptase; and
polyethylene glycol having an average molecular weight in the range of about 3350 to about 8000; and
(b) incubating the reaction mix so as to produce cDNA molecules that comprise, at the 3′ end of the cDNA, an added sequence comprising the complement of a sequence at the 3′ end of the template switching oligonucleotide.
2. The method of claim 1 , wherein (b) further comprises incubating the reaction mix with a site-specific double stranded nucleic acid cleaving enzyme for preventing concatamers of the double stranded form of the template-switching oligonucleotide at the 3′ end of the cDNA molecules.
3. The method of claim 1 , wherein step (b) comprises incubating the reaction mix at a temperature of 35° C. to 55° C. for a period of at least 30 minutes.
4. The method of claim 2 , further comprising, after step (b), inactivating the reverse transcriptase and the site-specific nucleic acid cleaving enzyme by incubating the reaction mix at a temperature of at least 65° C. for a period of at least 10 minutes.
5. The method of claim 1 , further comprising: (c) amplifying the cDNA molecules to produce amplification products.
6. The method of claim 5 , wherein (c) is performed using a first primer and a second primer, wherein the first primer hybridizes to the added sequence at the 3′ end of the cDNA and the second primer hybridizes to the complement of a sequence in the cDNA synthesis primer; and wherein the amplification is performed by PCR.
7. A method according to claim 1 , further comprising obtaining an enhanced yield of amplified cDNA in the presence of the crowding agent compared to in the absence of the crowding agent.
8. The method of claim 7 , wherein the method further comprises sequencing the amplification products.
9. The method of claim 1 , wherein the RNA template is from a clinical sample.
10. A composition comprising:
(a) a reverse transcriptase;
(b) an oligonucleotide for template switching, wherein the oligonucleotide comprises a 3′ end having at least one nucleotide that can hybridize to one or more non-templated nucleotides added to a templated cDNA strand by the reverse transcriptase; and
(c) polyethylene glycol having an average molecular weight in the range of about 3350 to about 8000.
11. The composition according to claim 10 , wherein the template-switching oligonucleotide comprises a recognition sequence for a site-specific double stranded strand nucleic acid cleaving enzyme; and wherein the reaction mix further comprises a site-specific double strand nucleic acid cleaving enzyme that recognizes the recognition sequence in the template-switching oligonucleotide.
12. The composition of claim 10 , further comprising an RNA template.
13. The composition of claim 11 , wherein the RNA template is total RNA or polyA±RNA.
14. The composition of claim 11 , wherein the RNA template comprises a 5′ modified GMP cap.
15. The composition of claim 13 , wherein the 5′ modified GMP cap is a 7-methylguanosine or an affinity tag-labeled GMP cap.
16. The composition of claim 10 , further comprising a cDNA synthesis primer which is an oligo(dT) primer, a random primer, or a transcript-specific primer.
17. The composition of claim 12 , further comprising a cDNA synthesis primer having a 5′ tail that does not hybridize to the RNA template.
18. The composition of claim 10 , wherein the template-switching oligonucleotide is a DNA-RNA chimera.
19. The composition of claim 10 , wherein the template-switching oligonucleotide comprises an amplification primer sequence, and optionally a molecular barcode sequence.
20. The composition of claim 10 , wherein the template-switching oligonucleotide comprises three riboguanine residues at its 3′ end.
21. The composition of claim 10 , wherein the reverse transcriptase is Moloney murine leukemia virus (M-MLV), M-MLV reverse transcriptase lacking RNaseH activity, human T-cell leukemia virus type I (HTLV-I) reverse transcriptase, bovine leukemia virus (BLV) reverse transcriptase, Rous sarcoma virus (RSV) reverse transcriptase, or human immunodeficiency virus (HIV) reverse transcriptase, or a variant thereof.
22. The composition of claim 11 , wherein the site-specific double strand nucleic acid cleaving enzyme is selected from a restriction endonuclease, an N-glycosylase, a lyase (N-glycosylase-lyase, an endonuclease, a cas9 ortholog and an Argonaut ortholog.
23. The composition of claim 11 , wherein the restriction endonuclease is active at a temperature in the range of 40° C. to 55° C.
24. The composition of claim 22 , wherein the restriction endonuclease is capable of inactivation by incubation at a temperature of at least 65° C. for at least 10 minutes.