Other

Dna Polymerase Technology, Inc.

Takara Shuzo Co., Inc.

Takara Bio Inc.

Dna Poleymerase Technology, Inc.

Barnes, Wayne M.

Wayne Morris Barnes

Milko B Kermekchiev

Zhian Zhang

Katherine R Rowlyk

Provided are mutant polymerases having DNA polymerase activity and reverse transcriptase activity or strand displacement activity, along with target nucleic acid amplification methods employing such mutant polymerases.

Mutant polymerases and uses thereof

Provided herein is a method for homologous end cloning using a 5'-exonuclease. One aspect provides a method of generating a recombinant DNA molecule comprising 5&#x2032; exonuclease digestion of DNA molecules, exonuclease inactivation, annealing of 3&#x2032; overhangs to form a annealed complex. Also provided is a method a method of generating a recombinant DNA molecule comprising 5&#x2032; exonuclease digestion of DNA molecules, exonuclease inactivation, annealing of 3&#x2032; overhangs to form a annealed complex, and extension of 3&#x2032; ends to fill gaps corresponding to exonuclease removal to form a substantially non-gapped annealed complex. Also provided is a one reaction method in which no further additions need be made to the reaction mixture and the reaction mixture is only manipulated thereafter as to incubation time and incubation temperature. Complexes formed according to methods described herein can be transformed into a host cell without further in vitro processing.

In vitro recombination methods using 5' exonuclease

The present invention provides an improved prokaryotic cell expression system employing a tightly controlled host strain construct that controls uninduced, leaky expression of proteins while still auto-inducing well. Various aspects of the present invention address and overcome the problem of uninduced basal expression by providing a host strain that comprises a T7 polymerase gene, and mutants thereof, inserted between lac Z and lac Y of the lac operon (a 'ZRY' construct), downstream of an otherwise wild-type lac operon control region.

T7 expression system

A method of obtaining DNA amplification of a nucleic acid target from a volume of whole blood comprising performing DNA amplification in a PCR assay mixture with a blood-resistant polymerase.

Use of whole blood in PCR reactions

A formulation and kit of thermostable or other DNA polymerases comprising at least one thermostable or other DNA polymerase which lacks 3'-exonuclease activity, and at least one thermostable DNA polymerase exhibiting 3&#x2032;-exonuclease activity. 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.

DNA polymerases with enhanced length of primer extension

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&#x2032;-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.

The present invention provides methods of linking nucleic acids without the use of restriction enzymes or any joining enzyme such as ligase. More specifically, the present invention provides methods for cloning or rearranging a double-stranded target DNA which is a PCR product into a double-stranded vector DNA which is a PCR product, said DNAs being amplified using primers that contain at least one ribonucleotide, preferably at or near the respective 3' ends of the primers, such that RNA-specific cleavage, preferably by RNAse A, will allow release of the primers to create long matching 3&#x2032;-sticky ends.

Ribocloning: recombinant DNA construction using primers with ribo bases

A formulation and kit of thermostable or other DNA polymerases comprising at least one thermostable or other DNA polymerase which lacks 3&prime;-exonuclease activity, and at least one thermostable DNA polymerase exhibiting 3&prime;-exonuclease activity. 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.

DNA polymersases with enhanced length of primer extension

The present invention provides a method of performing hot start PCR reactions. The method is based on sequestration of magnesium ions in the form of a precipitate which renders a DNA polymerase inactive until the appropriate time in the PCR reaction when a certain temperature is reached and the magnesium ions are released from the precipitate. Also provided are kits comprising reagents and instructions for amplifying a target nucleic acid by the magnesium precipitate hot start.

Magnesium precipitate hot start method for PCR

A method of inhibiting pyrophosphorolysis during DNA chain length elongation is provided. The method comprises including both Mn.sup.++ and Mg.sup.++ in chain-extension and chain-termination reaction mixtures such as those used in DNA sequencing. This method is useful in stabilizing dideoxy-ribonucleoside triphosphate-terminated DNA chains and improving the quality of DNA sequence data obtained via the use of DNA polymerases that do not discriminate against the incorporation of dideoxyribonucleoside triphosphates and other chain terminating agents. Also provided are a reaction mixture and kit for DNA sequencing employing this method.

Suppression of pyrophosphorolysis in DNA sequencing and in other

A vector which includes nucleic acid which encodes a DNA polymerase having an identical amino acid sequence to that of the DNA polymerase of Thermus aquaticus termed Taq DNA polymerase, except that it lacks the N-terminal 235 amino acids of Taq DNA polymerase.

Growing community of inventors

University City, MO, United States of America

Wayne Morris Barnes