Integrated Dna Technologies, Inc.

Joseph R Dobosy

Mark Aaron Behlke

Scott Rose

Joseph Alan Walder

Garrett Richard Rettig

Caifu Chen

Richard Owczarzy

Susan M Rupp

Shawn Allen

Jeffrey A Manthey

Kristin Beltz

Adam Clore

Jessica Lister

Sarah Jacobi

John Froehlig, Jr

Katherine Perschbacher

Steven A Henck

Jessica Woodley

Bernice Thommandru

Beimeng Sun

Scott D Rose

Described herein are compositions and methods for improving homology directed repair (HDR) efficiency and reducing homology-independent integration following introduction of double strand breaks with engineered nucleases. Additionally, modifications to double stranded DNA donors to improve the donor potency and efficiency of homology directed repair following introduction of double stranded breaks with programmable nucleases.

Modified double-stranded donor templates

The present invention pertains to hybrid RNase H2 proteins that include fragments of amino acid sequences from(P.a.),(T.kod), andorganisms, as well as methods of using the same to improve mismatch discrimination and activity in a high-fidelity DNA polymerase buffer.

RNASE H2 mutants that reduce primer dimers and off-target amplification in RHPCR-based amplicon sequencing with high-fidelity DNA polymerases

Described herein are compositions and methods for the production and quantification of barcoded or unique molecular identifier (UMI)-labeled substrates. In one aspect, the substrate is a bead comprising a template oligonucleotide that is elongated by successive extension reactions to provide a bead with an oligonucleotide comprising a plurality of barcodes and conserved anchor regions. Methods are also described for quantifying the amount of template oligonucleotide loaded onto the substrate and the products of the extension reaction after each round and after the final extension.

Methods for production and quantification of unique molecular identifier-labeled beads

Methods for detecting on-target and predicted off-target genome editing events by providing a multiplex PCR reaction mixture with an on-target oligonucleotide primer and one or more off-target oligonucleotide primers and then hybridizing the on-target oligonucleotide primer and the one or more off-target oligonucleotide primers to target nucleic acid sequences, followed by cleaving blocking groups from the primers and extending the primers.

Method for detecting on-target and predicted off-target genome editing events

This invention relates to mutant DNA polymerases having an enhanced template discrimination activity compared with the corresponding unmodified DNA polymerase counterparts, wherein the amino acid sequence of the mutant DNA polymerase includes at least one substitution at residue positions structurally and functionally homologous or orthologous positions 783 or 784 of an unmodified Taq DNA polymerase.

DNA polymerase mutants having enhanced template discrimination activity

The invention is directed to methods and kits for performing an RNase H2-mediated cleavage reaction on a sample in an emulsion.

RNase H mutants in an emulsion

The invention can be used to provide a more efficient and less error-prone method of detecting variants in DNA, such as SNPs and indels. The invention also provides a method for performing inexpensive multiplex assays. The invention also provides methods for detection of DNA sequences altered after cleavage by a targetable endonuclease, such as the CRISPR Cas9 protein from the bacterium

Methods for variant detection

The invention describes composition and methods of use for novel hairpin blocked-cleavable primers. In one embodiment unblocking occurs through action of RNase H2. The method improves the specificity of PCR and reduces primer dimer events, enabling higher level multiplex reactions. Additionally, the invention protects RNA-containing primers from attack by single-strand RNases.

Cleavable hairpin primers

Methods and compositions are provided for improving specificity during amplification of a target DNA sequence. The methods and compositions rely upon the use of an RNase H enzyme, a polymerase, and RNase H enzyme-sensitive, blocked-cleavable oligonucleotide primers in the amplification reactions, wherein the reaction mixtures include either an optimized final concentration of a divalent metal salt comprising 2.0 mM or less of free Mgcation and/or an optimized final concentration of a non-ionic detergent comprising at least about 0.001% polyethylene glycol hexadecyl ether.

RNase H-based assays utilizing modified RNA monomers

The present invention provides methods of cleaving a nucleic acid strand to initiate, assist, monitor or perform biological assays.

The present invention pertains to novel oligonucleotide compounds for use in various biological assays, such as nucleic acid amplification, ligation and sequencing reactions. The novel oligonucleotides comprise a ribonucleic acid domain and a blocking group at or near the 3' end of the oligonucleotide. These compounds offer an added level of specificity previously unseen. Methods for performing nucleic acid amplification, ligation and sequencing are also provided. Additionally, kits containing the oligonucleotides are also disclosed herein.

Growing community of inventors

Coralville, IA, United States of America

Joseph R Dobosy