Compositions and methods for reverse transcriptase-polymerase chain reaction (RT-PCR)
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 composition suitable for carrying out a one-step reverse transcriptase-polymerase chain reaction (RT-PCR) comprising a Moloney murine leukemic virus (M-MLV) reverse transcriptase (RT), one or more DNA polymerases, a non-naturally occurring buffering agent, and bovine serum albumin, wherein said one or more DNA polymerases is selected from the group consisting of Tne, Tma, Taq, Pfu, Tth, Tfi, Pwo, and Tfl; or wherein said one or more DNA polymerases comprises a first DNA polymerase having 3′ exonuclease activity and a second DNA polymerase having substantially reduced 3′ exonuclease activity; and wherein the unit ratio of said RT to said DNA polymerase(s) is greater than about 3:2; and wherein said bovine serum albumin is at a concentration of about 2 to 20 μg/ml.
2. The composition of claim 1 , wherein said bovine serum albumin is at a concentration which relieves RT inhibition of nucleic acid amplification.
3. The composition of claim 1 , further comprising one or more sulfur-containing molecules, one or more acetate-containing molecules, and/or one or more potassium-containing molecules.
4. The composition of claim 3 , wherein said one or more acetate-containing molecules is at a concentration of about 1 mM to about 500 mM.
5. The composition of claim 3 , wherein said one or more sulfur-containing molecules is at a concentration of at least 18 mM sulphur.
6. The composition of claim 1 , wherein
(a) said DNA polymerase having 3′ exonuclease activity is selected from the group consisting of Pfu, Pwo, Tne, Tma, Kod, and mutants, variants and derivatives thereof; and/or
(b) said DNA polymerase having substantially reduced 3′ exonuclease activity is selected from the group consisting of Taq, Tfl, Tth, and mutants, variants and derivatives thereof.
7. The composition of claim 3 , wherein a source of said one or more sulfur-containing molecules is selected from:
(a) a sulfur-containing buffer; or
(b) a sulfur-containing salt.
8. The composition of claim 3 , wherein a source of said one or more acetate-containing molecules is selected from:
(a) an acetate-containing salt; or
(b) an acetate-containing buffer.
9. The composition of claim 1 , further comprising one or more nucleotides which are deoxyribonucleoside triphosphates or derivatives thereof, or dideoxyribonucleoside triphosphates or derivatives thereof.
10. The composition of claim 1 , wherein said reverse transcriptase is substantially reduced in RNase H activity.
11. The composition of claim 1 , wherein said reverse transcriptase is a mutant Moloney murine leukemic virus (M-MLV) reverse transcriptase.
12. The composition of claim 1 , further comprising one or more oligonucleotide primers.