Modified reverse transcriptases
The present disclosure provides methods and systems for amplifying and analyzing nucleic acid samples. The present disclosure provides methods for preparing cDNA and/or DNA molecules and cDNA and/or DNA libraries using modified reverse transcriptases.
1. A non-naturally occurring enzyme, comprising (i) a finger domain derived from an R2 retrotransposon; (ii) a thumb domain derived from an R2 retrotransposon; and (iii) an endonuclease domain derived from an R2 retrotransposon, and an amino acid sequence with at least 95% identity to SEQ ID NO: 20.
2. The non-naturally occurring enzyme of claim 1 , further comprising a palm domain derived from an R2 retrotransposon.
3. The non-naturally occurring enzyme of claim 1 , further comprising a fusion-tag molecule.
4. The non-naturally occurring enzyme of claim 3 , wherein the fusion-tag molecule stabilizes the non-naturally occurring enzyme.
5. The non-naturally occurring enzyme of claim 3 , wherein the fusion-tag molecule is selected from the group consisting of: Fh8, MBP, NusA, Trx, SUMO, GST, SET, GB1, ZZ, HaloTag, SNUT, Skp, T7PK, EspA, Mocr, Ecotin, CaBP, ArsC, IF2-domain I, an IF2-domain I derived tag, RpoA, SlyD, Tsf, RpoS, PotD, Crr, msyB, yjgD, rpoD, and His6.
6. The non-naturally occurring enzyme of claim 3 , wherein the fusion-tag molecule is selected from the group consisting of: His-tag, His6-tag, Calmodulin-tag, CBP, CYD (covalent yet dissociable NorpD peptide), Strep II, FLAG-tag, HA-tag, Myc-tag, S-tag, SBP-tag, Softag-1, Softag-3, V5-tag, Xpress-tag, Isopeptag, SpyTag, B, HPC (heavy chain of protein C) peptide tags, GST, MBP, biotin, biotin carboxyl carrier protein, glutathione-S-transferase-tag, green fluorescent protein-tag, maltose binding protein-tag, Nus-tag, Strep-tag, and thioredoxin-tag.
7. The non-naturally occurring enzyme of claim 1 , wherein at least one of the finger domain, the thumb domain, or the endonuclease domain is derived from an arthropod.
8. The non-naturally occurring enzyme of claim 1 , wherein at least one of the finger domain, the thumb domain, or the endonuclease domain is derived from a silkmoth.
9. The non-naturally occurring enzyme of claim 1 , wherein at least one of the finger domain, the thumb domain, or the endonuclease domain is derived from a vertebrate or an echinoderm.
10. The non-naturally occurring enzyme of claim 1 , wherein at least one of the finger domain, the thumb domain, or the endonuclease domain is derived from a flatworm or a hydra.
11. The non-naturally occurring enzyme of claim 1 , wherein the enzyme comprises a mutagenized motif-1 sequence.
12. The non-naturally occurring enzyme of claim 11 , wherein the mutagenized motif-1 sequence has an improved jumping activity as compared to a wild-type sequence.
13. The non-naturally occurring enzyme of claim 1 , wherein the enzyme comprises a mutagenized motif 0 sequence.
14. The non-naturally occurring enzyme of claim 13 , wherein the mutagenized motif 0 sequence has an improved jumping activity as compared to a wild-type sequence.
15. The non-naturally occurring enzyme of claim 1 , wherein the enzyme comprises a mutagenized thumb sequence.
16. The non-naturally occurring enzyme of claim 15 , wherein the mutagenized thumb sequence has an improved single-stranded priming efficiency.
17. The non-naturally occurring enzyme of claim 15 , wherein the mutagenized thumb sequence has an improved processivity.
18. The non-naturally occurring enzyme of claim 1 , wherein the enzyme has a processivity of 20 nucleotides or longer.
19. The non-naturally occurring enzyme of claim 1 , wherein the enzyme comprises SEQ ID NO: 20.