Compositions and methods for reverse transcriptase-polymerase chain reaction (RT-PCR)
View Patent ↗The present invention is directed to compositions and methods useful for the amplification of nucleic acid molecules by reverse transcriptase-polymerase chain reaction (RT-PCR). Specifically, the invention provides compositions and methods for the amplification of nucleic acid molecules in a simplified one- or two-step RT-PCR procedure using combinations of reverse transcriptase and thermostable DNA polymerase enzymes in conjunction with sulfur-containing molecules or acetate-containing molecules (or combinations of such sulfur-containing molecules and acetate-containing molecules), and optionally bovine serum albumin. The invention thus facilitates the rapid and efficient amplification of nucleic acid molecules and the detection and quantitation of RNA molecules. The invention also is useful in the rapid production and amplification of cDNAs which may be used for a variety of industrial, medical and forensic purposes.
1. A method for one-step reverse transcriptase-polymerase chain reaction (RT-PCR), said method comprising:
(a) forming a mixture by combining an RNA template with a composition comprising a Moloney murine leukemia virus (M-MLV) reverse transcriptase and one or more DNA polymerases, and bovine serum albumin (BSA), wherein the unit ratio of said M-MLV to said one or more DNA polymerases is greater than 3:2 and wherein said BSA is at a concentration of about 2 to 20 μg/mL;
(b) incubating the mixture under conditions sufficient to amplify a DNA molecule complementary to all or a portion of said RNA template in which said BSA is capable of relieving inhibition of said RT-PCR by said M-MLV.
2. The method of claim 1 , wherein said M-MLV is substantially reduced in RNase H activity.
3. The method of claim 1 , wherein said one or more DNA polymerases is selected from the group consisting of Tne, Tma, Taq, Pfu, Tth, Pwo, Ttl, and mutants, variants and derivatives thereof.
4. The method of claim 1 , wherein said mixture further comprises one or more sulfur-containing molecules.
5. The method of claim 1 or 4 , wherein said mixture further comprises one or more acetate-containing molecules.
6. The method of claim 1 or 4 , wherein said mixture further comprises one or more potassium-containing molecules.