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 - 115 . (canceled)  
     
     
         116 . A recombinant, heterodimeric αβ Avian Myeloblastosis Virus (AMV) reverse transcriptase, wherein said AMV reverse transcriptase has a polymerase specific activity of at least about 30,000 units per milligram.  
     
     
         117 . The AMV reverse transcriptase of  claim 116 , wherein said AMV reverse transcriptase has a polymerase specific activity of about 30,000 units per milligram to about 150,000 units per milligram.  
     
     
         118 . The AMV reverse transcriptase of  claim 116 , wherein said AMV reverse transcriptase has a polymerase specific activity of about 40,000 units per milligram to about 150,000 units per milligram.  
     
     
         119 . The AMV reverse transcriptase of  claim 116 , wherein said AMV reverse transcriptase has a polymerase specific activity of about 50,000 units per milligram to about 150,000 units per milligram.  
     
     
         120 . The AMV reverse transcriptase of  claim 116 , wherein said AMV reverse transcriptase has a polymerase specific activity of about 75,000 units per milligram to about 150,000 units per milligram.  
     
     
         121 . The AMV reverse transcriptase of  claim 116 , wherein said AMV reverse transcriptase is present in a kit or composition at working concentrations at a concentration of about 100 to about 5,000 units per milliliter.  
     
     
         122 . A composition comprising a recombinant, heterodimeric up AMV reverse transcriptase, wherein said AMV reverse transcriptase has a polymerase specific activity of at least about 30,000 units per milligram.  
     
     
         123 . The composition of  claim 122 , wherein said AMV reverse transcriptase has a polymerase specific activity of about 30,000 units per milligram to about 150,000 units per milligram.  
     
     
         124 . The composition of  claim 122 , wherein said AMV reverse transcriptase has a polymerase specific activity of about 40,000 units per milligram to about 150,000 units per milligram.  
     
     
         125 . The composition of  claim 122 , wherein said AMV reverse transcriptase has a polymerase specific activity of about 50,000 units per milligram to about 150,000 units per milligram.  
     
     
         126 . The composition of  claim 122 , wherein said AMV reverse transcriptase has a polymerase specific activity of about 75,000 units per milligram to about 150,000 units per milligram.  
     
     
         127 . The composition of  claim 122 , wherein said AMV reverse transcriptase is present in said composition at working concentrations a concentration of about 100 to about 5,000 units per milliliter.  
     
     
         128 . A kit comprising a heterodimeric up AMV reverse transcriptase, wherein said AMV reverse transcriptase has a specific activity of at least about 30,000 units per milligram.  
     
     
         129 . The kit of  claim 128 , wherein said AMV reverse transcriptase has a polymerase specific activity of about 30,000 units per milligram to about 150,000 units per milligram.  
     
     
         130 . The kit of  claim 128 , wherein said AMV reverse transcriptase has a polymerase specific activity of about 40,000 units per milligram to about 150,000 units per milligram.  
     
     
         131 . The kit of  claim 128 , wherein said AMV reverse transcriptase has a polymerase specific activity of about 50,000 units per milligram to about 150,000 units per milligram.  
     
     
         132 . The kit of  claim 128 , wherein said AMV reverse transcriptase has a polymerase specific activity of about 75,000 units per milligram to about 150,000 units per milligram.  
     
     
         133 . The kit of  claim 128 , wherein said AMV reverse transcriptase is present in said kit at working concentrations a concentration of about 100 to about 5,000 units per milliliter.  
     
     
         134 . The kit of  claim 128 , said kit further comprising one or more components selected from the group consisting of one or more nucleotides selected from the group consisting of ATP, CTTP, GTP, and TTP, one or more DNA polymerases, a suitable buffer, one or more primers and or one or more terminating agents.  
     
     
         135 . The kit of  claim 128 , wherein said terminating agent is a dideoxynucleotide.  
     
     
         136 . The kit of  claim 128 , wherein two or more of the components of said kit are present as a mixture or are present as separate components.  
     
     
         137 . A recombinant, heterodimeric up AMV reverse transcriptase produced by a method comprising: 
 (a) obtaining a host cell comprising one or more nucleic acid sequences encoding at least one AMV reverse transcriptase subunit; and    (b) culturing said host cell under conditions sufficient to produce said AMV reverse transcriptase; and    (c) isolating or purifying said reverse transcriptase thereby obtaining said reverse transcriptase wherein said AMV reverse transcriptase has a polymerase specific activity of at least about 30,000 units per milligram.    
     
     
         138 . The AMV reverse transcriptase of  claim 137 , wherein said AMV reverse transcriptase has a polymerase specific activity of about 30,000 units per milligram to about 150,000 units per milligram.  
     
     
         139 . The AMV reverse transcriptase of  claim 137 , wherein said AMV reverse transcriptase has a polymerase specific activity of about 40,000 units per milligram to about 150,000 units per milligram.  
     
     
         140 . The AMV reverse transcriptase of  claim 137 , wherein said AMV reverse transcriptase has a polymerase specific activity of about 50,000 units per milligram to about 150,000 units per milligram.  
     
     
         141 . The AMV reverse transcriptase of  claim 137 , wherein said AMV reverse transcriptase has a polymerase specific activity of about 75,000 units per milligram to about 150,000 units per milligram.  
     
     
         142 . The AMV reverse transcriptase of  claim 137 , wherein said AMV reverse transcriptase is present in a kit or composition at working concentrations a concentration of about 100 to about 5,000 units per milliliter.