Compositions and methods for reverse transcription of nucleic acid molecules

Abstract

The present invention is generally related to compositions and methods for the reverse transcription of nucleic acid molecules, especially messenger RNA molecules. Specifically, the invention relates to compositions comprising mixtures of polypeptides having reverse transcriptase (RT) activity, and to methods of producing, amplifying or sequencing nucleic acid molecules (particularly cDNA molecules) using these compositions or polypeptides, particularly at temperatures above about 55° C. The invention also relates to nucleic acid molecules produced by these methods, to vectors and host cells comprising these nucleic acid molecules, and to the use of such nucleic acid molecules to produce desired polypeptides. The invention also relates to methods for producing Rous Sarcoma Virus (RSV) and Avian Myeloblastosis Virus (AMV) RTs or other Avian Sarcoma-Leukosis Virus (ASLV) RTs (α and/or β subunits thereof), to isolated nucleic acid molecules encoding such RSV RT, AMV RT or other ASLV RT subunits, to vectors and host cells comprising these isolated nucleic acid molecules and to RSV RT, AMV RT and other ASLV RT subunits produced by these methods. The invention further relates to nucleic acid molecules encoding recombinant heterodimeric RT holoenzymes, particularly heterodimeric RSV RTs, AMV RTs or other ASLV RTs (which may be αβ RTs, ββ RTs, or α RTs), vectors (particularly baculovirus vectors) and host cells (particularly insect and yeast cells) comprising these nucleic acid molecules, methods for producing these heterodimeric RTs and heterodimeric RTs produced by these methods. The invention also relates to kits comprising the compositions, polypeptides, or RSV RTs, AMV RTs or other ASLV RTs of the invention.

1 . A composition for use in reverse transcription of a nucleic acid molecule, said composition comprising two or more polypeptides having reverse transcriptase activity. 
     
     
         2 . The composition of  claim 1 , wherein said polypeptides are obtained from different sources. 
     
     
         3 . The composition of  claim 1 , wherein the transcription pause site of each of said polypeptides is different from that of each of the other polypeptides in said composition. 
     
     
         4 . The composition of  claim 1 , wherein said polypeptides are reduced or substantially reduced in RNase H activity. 
     
     
         5 .- 9 . (canceled) 
     
     
         10 . A method for reverse transcription of one or more nucleic acid molecules comprising
 (a) mixing one or more nucleic acid templates with two or more polypeptides having reverse transcriptase activity; and   (b) incubating said mixture under conditions sufficient to make one or more first nucleic acid molecules complementary to all or a portion of said one or more templates.   
     
     
         11 .- 21 . (canceled) 
     
     
         22 . A kit for use in reverse transcription, amplification or sequencing of a nucleic acid molecule, said kit comprising two or more polypeptides having reverse transcriptase activity. 
     
     
         23 .- 115 . (canceled) 
     
     
         116 . The composition of  claim 1 , wherein at least one of said two or more polypeptides having reverse transcriptase activity is selected from the group consisting of retroviral reverse transcriptase, retrotransposon reverse transcriptase, hepatitis B reverse transcriptase, cauliflower mosaic virus reverse transcriptase, bacterial reverse transcriptase, Tth DNA polymerase, Taq DNA polymerase, Tne DNA polymerase, Tma DNA polymerase and mutants, fragments, variants, or derivatives thereof. 
     
     
         117 . The method of  claim 10 , wherein at least one of said two or more polypeptides having reverse transcriptase activity is selected from the group consisting of retroviral reverse transcriptase, retrotransposon reverse transcriptase, hepatitis B reverse transcriptase, cauliflower mosaic virus reverse transcriptase, bacterial reverse transcriptase, Tth DNA polymerase, Taq DNA polymerase, Tne DNA polymerase, Tma DNA polymerase and mutants, fragments, variants, or derivatives thereof. 
     
     
         118 . The kit of  claim 22 , wherein at least one of said two or more polypeptides having reverse transcriptase activity is selected from the group consisting of retroviral reverse transcriptase, retrotransposon reverse transcriptase, hepatitis B reverse transcriptase, cauliflower mosaic virus reverse transcriptase, bacterial reverse transcriptase, Tth DNA polymerase, Taq DNA polymerase, Tne DNA polymerase, Tma DNA polymerase and mutants, fragments, variants, or derivatives thereof.