Method for hypermutagenesis of nucleotides
AssigneeInstitut Pasteur
InventorsSimon Wain-Hobson, Miguel Angel Martinez, Valerie Pezo
Life of this patent
Estimating the term…
Abstract
The present invention features a method for introducing hypermutations into a target DNA or RNA sequence of interest, characterized in that said method comprises the steps of: (a) transcribing a RNA into DNA in a reaction mixture comprising a reverse transcriptase, varying biased concentrations of deoxynucleoside triphosphates to produce hypermutations and an oligonucleotide primer that is partially complementary to the 3' end of said RNA; and (b) recovering said DNA sequences.
Claims45
A method for introducing hypermutations into a target nucleotide sequence, wherein said method comprises:
- reverse transcribing an RNA sequence in a reaction mixture comprising a reverse transcriptase, dATP, dTTP, dCTP, and dGTP with varying biased concentrations of said deoxynucleoside triphosphates, and an oligonucleotide primer that is partially complementary to the 3' end of said RNA sequence, to thereby produce a hypermutated DNA sequence.
A method for introducing hypermutations into a target nucleotide sequence, wherein said method comprises:
- (a) providing a vector containing a nucleotide sequence in double-stranded form;
- (b) transcribing the nucleotide sequence into an RNA sequence in vitro;
- reverse transcribing said RNA sequence in a reaction mixture comprising a reverse transcriptase, dATP, dTTP. dCTP, and dGTP with varying biased concentrations of said deoxynucleoside triphosphates, and an oligonucleotide primer that is partially complementary to the 3' end of said RNA sequence, to thereby produce a hypermutated DNA sequence.
A process for introducing hypermutations into a target nucleotide sequence, wherein said process comprises:
- (a) providing a vector containing a nucleotide sequence and a T7 or a T3 promoter sequence;
- (b) transcribing the nucleotide sequence into an RNA sequence by using T7 or T3 RNA polymerase;
- (c) reverse transcribing said RNA sequence in a reaction mixture comprising a reverse transcriptase, dATP, dTTP, dCTP, and dGTP with varying biased concentrations of said deoxynucleoside triphosphates, and an oligonucleotide primer that is partially complementary to the 3' end of said target RNA sequence, to thereby produce a hypermutated DNA sequence.
A method for introducing hypermutations into a target nucleotide sequence, wherein the method comprises:
- (A) providing a reaction mixture consisting essentially of
- (1) a RNA template to be hypermutated;
- (2) reverse transcriptase for reverse transcribing the RNA template to form cDNA, wherein the RNA template and reverse transcriptase are in a molar ratio of about 30:1 to about 50:1;
- (3) an oligonucleotide primer for initiating cDNA synthesis along the template, wherein the oligonucleotide primer is present in a molar excess relative to the RNA template;
- and
- (4) a mixture of deoxynucleoside triphosphates comprising dATP, dTTP, dGTP, dCTP, wherein molar concentrations of said deoxynucleoside triphosphates are biased such that
- (a) the concentration of dCTP is low relative to the concentration of dTTP;
- or
- (b) the concentration of dATP is low relative to the concentration of dGTP;
- or
- (c) the sum of the concentrations of dCTP and dATP is low relative to the sum of the concentrations of dTTP and dGTP;
- and
- (B) reverse transcribing said RNA template to form a hypermutated nucleotide sequence having G.fwdarw.A mutations, U.fwdarw.C mutations, or mixed G.fwdarw.A and U.fwdarw.C mutations in which about 2% to about 99% of the nucleotides in the RNA template are mutated.
Dependencies are inferred from the claim text.