Isothermal amplification of DNA
AssigneePresident and Fellows of Harvard College
InventorsStanley Tabor, Charles C. Richardson
Life of this patent
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Abstract
A method of amplifying a template DNA molecule comprising incubating the template DNA molecule in a reaction mixture comprising a DNA polymerase and at least one accessory protein at a constant temperature to produce amplified product, wherein production of amplified product does not require exogenously-added oligonucleotide primers and the template DNA molecule does not have have terminal protein covalently bound to either 5′ end.
Claims34
A method of amplifying a template DNA molecule, said method comprising incubating said template DNA molecule in an in vitro reaction mixture comprising:
- wild-type T7 DNA polymerase,
- a T7 DNA polymerase modified to have reduced 3′ to 5′ exonuclease activity,
- the 63-kDa form of a gene 4 protein from bacteriophage T7, and
- a single-stranded DNA binding protein from Escherichia coli at a constant temperature to produce amplified product,
- wherein the amplification reaction is conducted in the absence of exogenously added oligonucleotide primers, and
- wherein the amount of the amplified product is at least 10-fold greater than the amount of template DNA put into the mixture.
A method of amplifying a template DNA molecule, said method comprising incubating said template DNA molecule in an in vitro reaction mixture comprising:
- wild-type T7 DNA polymerase,
- Δ28 T7 DNA polymerase,
- the 63-kDa form of a gene 4 protein from bacteriophage T7,
- a single-stranded DNA binding protein from Escherichia coli, and
- one or more components selected from the group consisting of nucleoside diphosphokinase, T7 single-stranded DNA binding protein, T7 gene 6 exonuclease, T7 DNA ligase, and a combination of creatine kinase and phosphocreatine, at a constant temperature to produce amplified product,
- wherein the amplification reaction is conducted in the absence of exogenously added oligonucleotide primers, and
- wherein the amount of the amplified product is at least 10-fold greater than the amount of template DNA put into the mixture.
Dependencies are inferred from the claim text.