High temperature reverse transcription using mutant DNA polymerases
First Claim
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1. A method for reverse transcribing an RNA, that comprises:
- (a) providing a reverse transcription reaction mixture comprising said RNA, a primer, a divalent cation, and a mutant thermoactive DNA polymerase, wherein said mutant DNA polymerase is characterized in that i) in its native form said DNA polymerase comprises an amino acid sequence that is SEQ ID NO;
1;
ii) the amino acid at position 4 of said amino acid sequence is mutated in comparison to said native sequence to an amino acid other than E, A, G, or P; and
(b) treating said reaction mixture at a temperature sufficient for said mutant DNA polymerase to initiate synthesis of an extension product of said primer to provide a cDNA molecule complementary to said RNA.
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Abstract
The present invention relates to improved reverse transcription methods using a modified thermostable DNA polymerases, particularly in a magnesium ion buffer. These methods are particularly useful in combined reverse-transcription/amplification reactions.
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Citations
52 Claims
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1. A method for reverse transcribing an RNA, that comprises:
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(a) providing a reverse transcription reaction mixture comprising said RNA, a primer, a divalent cation, and a mutant thermoactive DNA polymerase, wherein said mutant DNA polymerase is characterized in that i) in its native form said DNA polymerase comprises an amino acid sequence that is SEQ ID NO;
1;
ii) the amino acid at position 4 of said amino acid sequence is mutated in comparison to said native sequence to an amino acid other than E, A, G, or P; and
(b) treating said reaction mixture at a temperature sufficient for said mutant DNA polymerase to initiate synthesis of an extension product of said primer to provide a cDNA molecule complementary to said RNA. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 25, 26)
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13. A method for reverse transcribing an RNA, that comprises:
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(a) providing a reverse transcription reaction mixture comprising said RNA, a primer, Mg+2, and a mutant thermoactive DNA polymerase, wherein said mutant DNA polymerase is characterized in that i) in its native form said DNA polymerase comprises an amino acid sequence that is SEQ ID NO;
1;
ii) the amino acid at position 4 of said amino acid sequence is mutated in comparison to said native sequence to an amino acid other than E, A, G, or P; and
(b) treating said reaction mixture at a temperature sufficient for said mutant DNA polymerase to initiate synthesis of an extension product of said primer to provide a cDNA molecule complementary to said RNA. - View Dependent Claims (14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 27, 28)
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29. A method for amplifying an RNA using a single-enzyme reverse transcription/amplification reaction, that comprises:
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(a) providing an amplification reaction mixture comprising said RNA, a pair of primers, a divalent cation, and a mutant thermostable DNA polymerase, wherein said mutant DNA polymerase is characterized in that i) in its native form said DNA polymerase comprises an amino acid sequence that is SEQ ID NO;
1;
ii) the amino acid at position 4 of said amino acid sequence is mutated in comparison to said native sequence to an amino acid other than E, A, G, or P; and
(b) treating said reaction mixture at a temperature sufficient for said mutant DNA polymerase to initiate synthesis of an extension product of said primer to provide a cDNA molecule complementary to said RNA;
(c) treating said reaction mixture at an appropriate temperature for said mutant DNA polymerase to initiate synthesis of an extension product of said second primer to provide a double-stranded cDNA molecule; and
(d) amplifying said double-stranded cDNA molecule of step (c) by a polymerase chain reaction. - View Dependent Claims (30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40)
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41. A method for amplifying an RNA using a single-enzyme reverse transcription/amplification reaction, that comprises:
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(a) providing an amplification reaction mixture comprising said RNA, a pair of primers, Mg+2, and a mutant thermostable DNA polymerase, wherein said mutant DNA polymerase is characterized in that i) in its native form said DNA polymerase comprises an amino acid sequence that is SEQ ID NO;
1;
ii) the amino acid at position 4 of said amino acid sequence is mutated in comparison to said native sequence to an amino acid other than E, A, G, or P; and
(b) treating said reaction mixture at a temperature sufficient for said mutant DNA polymerase to initiate synthesis of an extension product of said primer to provide a cDNA molecule complementary to said RNA;
(c) treating said reaction mixture at an appropriate temperature for said mutant DNA polymerase to initiate synthesis of an extension product of said second primer to provide a double-stranded cDNA molecule; and
(d) amplifying said double-stranded cDNA molecule of step (c) by a polymerase chain reaction. - View Dependent Claims (42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52)
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Specification