Reverse Transcriptases and Related Methods
Reverse transcriptases that may have an improvement in one or more properties are provided. For example, the present reverse transcriptases are believed to be more efficient than other reverse transcriptases, particularly in the presence of substances that typically inhibit reverse transcriptases. Also provided are related compositions, kits, and methods for using the reverse transcriptases.
1 . A reverse transcriptase comprising:
an amino acid sequence having at least 90% identity with SEQ ID NO:1; or
an amino acid sequence having at least 90% identity with SEQ ID NO:2.
2 . The reverse transcriptase of claim 1 , wherein the amino acid sequence has at least 95% sequence identity with SEQ ID NO:1.
3 . The reverse transcriptase of claim 1 , wherein the amino acid sequence has at least 95% sequence identity with SEQ ID NO:2.
4 . The reverse transcriptase of claim 1 , comprising:
an amino acid sequence having at least 90% identity with SEQ ID NO:1, wherein the amino acid sequence has one or more of the following amino acid substitutions:
an R at position 190,
an R at position 445,
a K at position 509, and
a K at position 588,
wherein the positions correspond to positions in SEQ ID NO:1.
5 . The reverse transcriptase of claim 4 , wherein the amino acid sequence contains:
an R at position 190,
an R at position 445,
a K at position 509, and
a K at position 588,
wherein the positions correspond to positions in SEQ ID NO:1.
6 . The reverse transcriptase of claim 1 , comprising:
an amino acid sequence having at least 90% identity with SEQ ID NO:2, wherein the amino acid sequence has one or more of following amino acid substitutions:
an R at position 190,
an R at position 444,
a K at position 507, and
a K at position 586,
wherein the positions correspond to positions in SEQ ID NO:2.
7 . The reverse transcriptase of claim 6 , wherein the amino acid sequence contains
an R at position 190,
an R at position 444,
a K at position 507, and
a K at position 586,
wherein the positions correspond to positions in SEQ ID NO:2.
8 . The reverse transcriptase of claim 1 , wherein the reverse transcriptase is a fusion protein comprising: (i) a polymerase domain comprising the amino acid sequence; and (ii) an exogenous amino acid sequence.
9 . A reverse transcriptase fusion protein comprising:
(i) a polymerase domain having reverse transcriptase activity, comprising an amino acid sequence that is a portion of:
an amino acid sequence having at least 90% identity with SEQ ID NO:1; and
an amino acid sequence having at least 90% identity with SEQ ID NO:2; and
(ii) an exogenous amino acid sequence.
10 . The reverse transcriptase of claim 8 , wherein the exogenous amino acid sequence comprises a purification tag.
11 . A composition comprising a reverse transcriptase of claim 1 .
12 . The composition of claim 11 , further comprising one or more of: dNTPs, a buffering agent, an oligonucleotide, an aptamer, a template switching oligonucleotide, a DNA polymerase, an RNA template and a DNA template.
13 . The composition of claim 11 , wherein reverse transcriptase is associated with a solid support.
14 . The composition of claim 11 , wherein the composition is in a form selected from lyophilized, dried, and in solution.
15 . A kit comprising:
(i) a reverse transcriptase of claim 1 ; and
(ii) a component selected from a buffering agent, a cationic salt, dNTPs, a primer, an aptamer, a detergent, a template switching oligonucleotide, and nucleic acid binding protein.
16 . The kit of claim 15 , wherein the reverse transcriptase is in a form selected from lyophilized, dried, and in solution.
17 . The kit of claim 15 , wherein one or more components is associated with a solid support.
18 . A kit comprising:
(i) a reverse transcriptase fusion protein of claim 9 ; and
(ii) a component selected from a buffering agent, a cationic salt, dNTPs, a primer, an aptamer, a detergent, a template switching oligonucleotide, and nucleic acid binding protein.
19 . A method comprising:
incubating a reaction mixture comprising
(i) a reverse transcriptase of claim 1 ,
(ii) a primer,
(ii) a target nucleic acid, and
(iii) dNTPs,
under conditions suitable for the reverse transcriptase to copy the target nucleic acid to generate copied DNA.
20 . The method of claim 19 , wherein the incubating is done at a temperature of between 55° C.-65° C.
21 . The method of claim 19 , wherein the primer is selected from an oligo(dT) primer, a random primer, a target-specific primer.
22 . The method of claim 19 , wherein the target nucleic acid is RNA.
23 . The method of claim 19 , wherein the target nucleic acid is DNA.
24 . The method of claim 18 , wherein the target nucleic acid is an RNA, and further comprising incubating the generated copied DNA with an enzyme to generate second copied DNA.
25 . The method of claim 19 , further comprising quantifying the amount of the copies of the target nucleic acid.
26 . The method of claim 22 , wherein the RNA template is of a size of at least about 1 kb, and full length copied DNA is generated within a time period selected from 10 minutes, 9 minutes, 8 minutes, less than 10 minutes, less than 9 minutes, and less than 8 minutes.
27 . A method comprising:
incubating a reaction mixture comprising
(i) a reverse transcriptase fusion protein of claim 9 ,
(ii) a primer,
(ii) a target nucleic acid, and
(iii) dNTPs,
under conditions suitable for the reverse transcriptase to copy the target nucleic acid to generate copied DNA.