DNA polymerases with enhanced length of primer extension
AssigneeTakara Bio Inc.
InventorWayne M. Barnes
Life of this patent
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Abstract
A formulation of thermostable or other DNA polymerases comprising (a) at least one thermostable or other DNA polymerase which lacks 3′-exonuclease activity, and (b) at least one thermostable DNA polymerase exhibiting 3′-exonuclease activity, wherein the ratio of (a) to (b) is greater than 1 to 1. Also provided is an improved method for enzymatic extension of DNA strands, especially while, but not limited to, amplifying nucleic acid sequences by polymerase chain reaction wherein the above formulation is made and used to catalyze primer extension, are also provided.
Claims16
A method for amplifying a nucleic acid, comprising:
- preparing a composition comprising a DNA polymerase comprising at least one thermostable DNA polymerase which lacks 3′-exonuclease activity, at least one thermostable DNA polymerase which exhibits 3′-exonuclease activity, and a nucleic acid comprising a sequence to be amplified, wherein the ratio of DNA polymerase activity of the at least one thermostable DNA polymerase which lack 3′-exonuclease activity to DNA polymerase activity of the at least one thermostable DNA polymerase which exhibits 3′-exonuclease activity from about 100:1 up to about 600:1;
- and
- subjecting the composition to conditions effective for amplifying the nucleic acid sequence,
- wherein the nucleic acid sequence to be amplified is a nucleic acid sequence 6 kb or more in length.
A method for amplifying a nucleic acid, comprising:
- preparing a composition comprising a DNA polymerase comprising at least one thermostable DNA polymerase which lacks 3′-exonuclease activity, at least one thermostable DNA polymerase which exhibits 3′-exonuclease activity, and a nucleic acid comprising a sequence to be amplified, wherein the ratio of DNA polymerase activity of the at least one thermostable DNA polymerase which lack 3′-exonuclease activity to DNA polymerase activity of the at least one thermostable DNA polymerase which exhibits 3′-exonuclease activity exceeds 1 to 1;
- and wherein the nucleic acid sequence to be amplified is a nucleic acid sequence 6 kb or more in length wherein the conditions effective for amplifying the nucleic acid sequence comprise conditions effective for denaturing the nucleic acid sequence, and wherein the denaturing has a duration of less than 20 seconds.
A method of amplifying a polynucleotide sequence, said method comprising:
- the steps of mixing a composition with a synthesis primer and a synthesis template, said composition comprising:
- (a) a first DNA polymerase, wherein said first polymerase possesses 3′-5′ exonuclease activity selected from the group consisting of Archaebacterial DNA polymerases and
- (b) a second DNA polymerase, wherein said second polymerase lacks 3′-5′ exonuclease activity selected from the group consisting of thermostable DNA polymerases lacking 3′-5′ exonuclease activity;
- wherein the ratio of DNA polymerase activity of the first DNA polymerase to the DNA polymerase activity of the second DNA polymerase is from about 1:100 up to about 1:600.
Dependencies are inferred from the claim text.