SUBSTANTIALLY PURE REVERSE TRANSCRIPTASES AND METHODS OF PRODUCTION THEREOF
The present invention provides substantially pure reverse transcriptases, which are preferably substantially free from contamination with nucleic acids. The invention also provides methods for the production of these enzymes, and kits comprising these enzymes which may be used in synthesizing, amplifying or sequencing nucleic acid molecules, including through the use of the polymerase chain reaction, particularly RT-PCR.
1 . A method of isolating or purifying a reverse transcriptase, said method comprising permeabilizing a cellular source of reverse transcriptase, subjecting said permeabilized cellular source of reverse transcriptase to filtration, and isolating said reverse transcriptase.
2 . The method of claim 1 , wherein said reverse transcriptase is substantially free of nucleic acids.
3 . The method of claim 1 , wherein said cellular source is a bacterial cell or a recombinant bacterial cell.
4 . The method of claim 3 , wherein said permeabilization forms spheroplasts and/or protoplasts.
5 . The method of claim 4 , wherein said filtration comprises microfiltration and/or ultrafiltration.
6 . The method of claim 1 , wherein said permeabilization is accomplished by contacting said cellular source with an aqueous solution comprising at least one component selected from the group consisting of a chaeotropic agent and/or a nonionic detergent.
7 . The method of claim 6 , wherein said nonionic detergent is selected from the group consisting of Triton X-100 and sodium deoxycholic acid.
8 . The method of claim 1 , wherein said isolation step comprises column chromatography.
9 . The method of claim 1 , wherein said method is conducted under conditions favoring the partitioning of nucleic acids, from said reverse transcriptase.
10 . The method of claim 9 , wherein said conditions comprise microfiltration of spheroplast or protoplasts in the presence of ammonium sulfate.
11 . The method of claim 1 , wherein said reverse transcriptase is M-MLV RT or M-MLV RT substantially reduced in RNase H activity.
12 . A reverse transcriptase made according to the method of claim 1 .
13 . The reverse transcriptase of claim 12 , wherein said reverse transcriptase is selected from the group consisting of M-MLV RT or M-MLV RT substantially reduced in RNase H activity.
14 . A kit for synthesizing a nucleic acid molecule comprising a reverse transcriptase made according to the method of claim 1 .
15 . The kit of claim 14 , wherein said kit further comprises at least one component selected from the group consisting of one or more DNA polymerases, one or more nucleotides, one or more buffers, and one or more primers.
16 . The kit of claim 15 , wherein said kit may be used for RT PCR.