Novel polymerase compositions and uses thereof

Abstract

The subject invention provides novel compositions containing a mixture of (a) an enzyme that possesses substantial 3′-5′ exonuclease activity (b) a DNA polymerase with less 3′-5′ exonuclease activity than the enzyme with substantial 3′-5′ exonuclease activity. Preferably, the DNA polymerase for inclusion in the compositions are DNA polymerases that substantially lack 3′-5′ exonuclease activity. A preferred embodiment of the invention is a composition comprising the Taq DNA polymerase (isolated from Thermus aquaticus) and the Pfu DNA polymerase (isolated from Pyrococcus furiosus). Another aspect of the invention is to provide methods for synthesizing polynucleotides, typically DNA, using compositions comprising an enzyme that possesses substantial 3′-5′ exonuclease activity and a DNA polymerase with less 3′-5′ exonuclease activity than the enzymes possessing substantial 3′-5′ exonuclease activity, preferably a DNA polymerase that substantially lacks 3′-5′ exonuclease activity. Another aspect of the invention involves the use the subject method of polynucleotide synthesis to carry out the synthesis step in a polymerase chain reaction experiment. Yet another aspect of the invention is to provide kits for the synthesis of polynucleotides, wherein the kits comprise an enzyme that possesses substantial 3′-5′ exonuclease activity and a DNA polymerase with less 3′-5′ exonuclease activity than the enzyme possessing substantial 3′-5′ exonuclease activity.

Description

RELATED APPLICATIONS [0001] This application is a continuation-in-part of co-pending U.S. patent application Ser. No. 08 / 164,290 which was filed Dec. 8, 1993.FIELD OF THE INVENTION [0002] The present invention is related to the field of molecular biology, and more particularly, to polynucleotide synthesis. BACKGROUND OF THE INVENTION [0003] DNA polymerases catalyze the synthesis of DNA and can be found in all cells as well as being encoded in numerous viruses. Although all DNA polymerases possess 5′-3′ DNA polymerase activity, DNA polymerases differ from one another in numerous other properties. For example, some enzymatic activities that are possessed by some DNA polymerases, but absent in other DNA polymerases include: double stranded DNA 5′-3′ exonuclease activity, single-stranded DNA 3′-5′ exonuclease activity, double-stranded 3′-5′ DNA exonuclease activity, RNase H activity, reverse transcriptase activity, and the like. Additionally, different DNA polymerases may have different...

AI Technical Summary

Benefits of technology

The invention provides new mixtures of enzymes and DNA polymerases with different levels of activity for synthesizing polynucleotides, such as DNA. These mixtures can be used in polymerase chain reaction experiments and can help improve the accuracy and efficiency of the reaction. The mixtures contain an enzyme with substantial 3′-5′ exonuclease activity and a DNA polymerase with less 3′-5′ exonuclease activity than the first enzyme. The use of these mixtures can help prevent errors and improve the accuracy of the polynucleotide synthesis process.

Problems solved by technology

This proofreading activity allows Pfu to extend from mismatched primer:templates by first removing the mismatched base(s) followed by polymerization and results in an error rate of 1.6×10−6 mutations per nucleotide per cycle in PCR reactions.

This presents a problem when the exact template sequence is not known such as when the nucleotide sequence of the template is derived from amino acid sequence due to the redundancy of the amino acid code and when designing primers for templates of families of genes which are heterogeneous.

Taq DNA polymerase is unable to correct nucleotide misincorporations made during polymerization due to its lack of 3′ to 5′ exonuclease activity.

In general, this would result in the inability of Taq to extend from a newly polymerized strand annealed to a template when an incorrect nucleotide has been inserted.