Generation and amplification of nucleic acids from ribonucleic acids
First Claim
1. In a method for generating a nucleic acid from an RNA template comprising the steps:
- a) adding said RNA template to a reaction mixture, said reaction mixture comprising at least one reverse transcriptase and at least one nucleic acid polymerase, or derivatives thereof; and
b) incubating said reaction mixture under conditions sufficient to allow polymerization of a nucleic acid molecule complementary to a portion of said RNA template, wherein the improvement comprises a reaction mixture further comprising a homopolymeric oligonucleotide at least 50 bases in length.
1 Assignment
0 Petitions
Accused Products
Abstract
Novel compositions and methods useful for the generation of nucleic acids from a ribonucleic acid template and further nucleic acid replication are disclosed. It is shown that the generation and amplification of nucleic acids by methods that utilize two or more different polymerases, such as reverse transcriptase-polymerase chain reaction (RT-PCR), are dramatically more sensitive and efficient in the presence of a homopolymeric nucleic acid. Homopolymeric nucleic acids have been found to reduce or negate the inhibitory effect reverse transcriptases have on DNA polymerase activity. It is demonstrated that this inhibition-relieving effect of homopolymeric nucleic acids is general in nature; independent of the chemical species of homopolymer used, or the chemical composition of the polymerization reaction mixture.
-
Citations
45 Claims
-
1. In a method for generating a nucleic acid from an RNA template comprising the steps:
-
a) adding said RNA template to a reaction mixture, said reaction mixture comprising at least one reverse transcriptase and at least one nucleic acid polymerase, or derivatives thereof; and
b) incubating said reaction mixture under conditions sufficient to allow polymerization of a nucleic acid molecule complementary to a portion of said RNA template, wherein the improvement comprises a reaction mixture further comprising a homopolymeric oligonucleotide at least 50 bases in length. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11)
-
-
12. In a method for generating a nucleic acid from an RNA template comprising the steps:
-
a) adding said RNA template to a reaction mixture, said reaction mixture comprising at least one reverse transcriptase, at least one nucleic acid polymerase, and one or more ribonucleotide triphosphate or derivatives thereof; and
b) incubating said reaction mixture under conditions sufficient to allow polymerization of a nucleic acid molecule complementary to a portion of said RNA template, wherein the improvement comprises a reaction mixture further comprising a homopolymeric oligonucleotide at least 50 bases in length. - View Dependent Claims (13, 14, 15, 16, 17, 18, 19)
-
-
20. In a composition suitable for reverse transcription of RNA, comprising at least one member of the group consisting of:
- a reverse transcriptase, a DNA polymerase, one or more oligonucleotide primers, any one or more nucleotides or derivatives thereof, a buffering agent, a salt, and additives useful in RT-PCR, wherein the improvement comprises a homopolymeric oligonucleotide at least 50 bases in length.
- View Dependent Claims (21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45)
Specification