Agilent Technologies, Inc.

Stratagene

Stratagene California

Holly Hogrefe

Connie Jo Hansen

Joseph A Sorge

Bahram Arezi

Michael Borns

Jeffrey Fox

Janice Marie Cline

Michelle H Cayouette

Weidong Wu

Joel Myerson

Jeffrey Robert Sampson

Robert A Ach

Brian Jon Peter

Jeffrey Carl Braman

Zoltan Timar

Gothami Padmabandu

Reinhold Mueller

Peter James Sheffield

Nancy McKinney

Lydia Wu

Nick Roelofs

Michael Muhich

Weimei Xing

Nancy Mckinney

Jennifer Lapira

Mutant polymerases are provided that have improved ability to incorporate modified nucleotides, including 3'-OH unblocked reversible terminators. The mutant polymerases may be used in a variety of applications, such as for polynucleotide sequencing, primer extension reactions, and template-independent enzymatic oligonucleotide synthesis.

Polymerase mutants and use with 3'-OH unblocked reversible terminators

Methods and compositions are provided for amplifying a pool of oligonucleotides, such as dual guide oligonucleotide constructs comprising sequences encoding a first guide RNA segment and a sequence encoding a second guide RNA segment. An amplification mixture is formed comprising the pool of oligonucleotides, an amplification enzyme, deoxyribonucleotide triphosphates, and primers. The amplification mixture is thermocycled a sufficient number of times and under conditions to produce a library of oligonucleotide constructs. The present methods and compositions provide dual guide libraries, including libraries that are essentially free of scrambled library members.

Amplifying oligonucleotides and producing libraries of dual guide constructs

Provided herein, among other things, are a variety of methods for transposase-mediated tagging and amplification of short DNA fragments, e.g., between about 150 bp and 1.5 Kb in length. In some aspects, the method includes tagging the DNA fragments with a first primer sequence using barcoded transposases followed by a primer extension reaction to introduce a second primer sequence, e.g., using random or gene-specific primers. Kits for performing this method are also provided.

Transposase-random priming DNA sample preparation

This disclosure provides a method comprising: a) clamping the top and bottom strands of a double stranded DNA molecule to produce a duplex in which the top and bottom strands are linked; b) denaturing the duplex to produce a denatured product; and c) renaturing the denatured product in the presence of a labeled oligonucleotide that is complementary to a sequence of nucleotides in the double stranded DNA molecule, thereby producing a D-loop-containing product. Kits for performing the method and products made by the method are also provided.

Target enrichment and labeling for multi-kilobase DNA

The invention relates to the generation and characterization of stable MMLV reverse transcriptase mutants. The invention also discloses methods of using stable MMLV reverse transcriptase mutants.

Mutant reverse transcriptase and methods of use

The invention provides mutants of DNA polymerases having an enhanced resistance to inhibitors of DNA polymerase activity. The mutant polymerases are well suited for PCR amplification of targets in samples that contain inhibitors of wild-type polymerases.

Thermostable type-A DNA polymerase mutant with increased resistance to inhibitors in blood

The present invention provides mutants of DNA polymerases having an increased rate of incorporation of nucleotides into nucleic acids undergoing polymerization and having an enhanced resistance to inhibitors of DNA polymerase activity. The mutant polymerases are well suited for fast PCR applications, for PCR amplification of targets in samples that contain inhibitors of wild-type polymerases, and for fast PCR amplification of samples containing DNA polymerase inhibitors. In exemplary embodiments, the mutants are mutants of Taq DNA polymerase.

Thermostable type-A DNA polymerase mutants with increased polymerization rate and resistance to inhibitors

The subject invention relates to compositions comprising an enzyme mixture which comprises a first enzyme and a second enzyme, where the first enzyme comprises a DNA polymerization activity and the second enzyme comprises an 5'-3&#x2032; exonuclease activity and a reduced DNA polymerization activity. The invention also relates to the above compositions in kit format and methods for high fidelity DNA synthesis using the subject compositions of the invention.

High fidelity DNA polymerase compositions and uses thereof

The invention relates to compositions and methods utilizing split polymerase enzymes composed of at least two discrete polypeptides that stably associate to form a single polymerase. The invention further relates to nucleic acid constructs for expressing the split polymerases of the invention, and methods for using the split polymerases of the invention. The enzymes of the invention are useful in many applications calling for the detectable labeling of nucleic acids and are particularly useful in quantitative PCR (QPCR) and DNA sequencing applications.

Compositions and methods using split polymerases

The invention features a novel isolated Family B DNA polymerase, apolymerase JDF-3, and mutant recombinant forms thereof. Mutant polymerases of the invention are deficient in 3' to 5&#x2032; exonuclease activity and/or exhibit reduced discrimination against non-conventional nucleotides relative to the wild-type form of the polymerase.

Compositions and methods utilizing DNA polymerases

The present invention provides compositions and improved methods for multi-site directed mutagenesis and DNA shuffling. The present compositions and methods provide increased mutation frequency and increased number of transformants which allow one to sequence only a few clones in order to identify the correct mutants and to obtain the desired mutant by screening large number of transformants in a short time. Moreover, the inclusion of FEN-1, PEF and optimized buffer and cycling conditions provided in the present invention should also facilitate random mutagenized library construction and the mutagenesis of large or difficult templates.

Multi-site mutagenesis

The invention relates to the generation and characterization of Archaeal DNA polymerase mutants with deficient 3'-5&#x2032; exonuclease activity and reduced base analog detection activity. The invention further provides for Archaeal DNA polymerase mutants with deficient 3&#x2032;-5&#x2032; exonuclease activity and reduced base analog detection activity containing additional mutations that modulate other DNA polymerase activities including DNA polymerization or reverse transcriptase activity. The invention also discloses methods and applications of DNA polymerases with deficient 3&#x2032;-5&#x2032; exonuclease activity and reduced base analog detection activity.

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San Diego, CA, United States of America

Holly Hogrefe