Exact Sciences Corporation

Exact Sciences Development Company, LLC

Other

Qiagen North American Holdings, Inc

Pel-freez

Michael J Domanico

Graham P Lidgard

Hatim T Allawi

Hongzhi Zou

James P Light, Ii

Keith Kopitzke

Rebecca Oldham-Haltom

Janelle J Bruinsma

Hemanth D Shenoi

William G Weisburg

John Zeis

Keith D Fourrier

Scott E Castanon

Brian Aizenstein

Zubin Gagrat

Oliver Hunt

Tobias Charles Zutz

Ilse A Tyler

Daniel J Simpson

Jacquelyn T Hennek

Kathleen S Harings

Keith Yaeger

Robert E Novy

Lars-Erik Peters

Timothy E Higgins

Thomas Kolzau

Bridget Z Gagrat

Kevin T Conroy

Matt Myers

Kristian Keane

Lisa R Braun

Sven Buelow

Keith W Yaeger

Warren Kroeker

James P Light

Scott Castanon

James P Ii Light

Ii James P Light

Provided herein is technology relating to isolating nucleic acids. In particular, the technology relates to methods and kits for extracting nucleic acids from problematic samples such as stool.

Isolation of nucleic acids

A cleavage-based real-time PCR assay method is provided. In general terms, the assay method includes subjecting a reaction mixture comprising a) PCR reagents for amplifying a nucleic acid target, and b) flap cleavage reagents for performing a flap cleavage assay on the amplified nucleic acid target to two sets of thermocycling conditions. No additional reagents are added to the reaction between said first and second sets of cycles and, in each cycle of the second set of cycles, cleavage of a flap probe is measured.

Real time cleavage assay

Provided herein is technology relating to the amplification-based detection of nucleic acids and particularly, but not exclusively, to methods and compositions for minimizing variability in the activity between different samples or manufacturing lots of DNA polymerases, such as Taq DNA polymerase.

Normalization of polymerase activity

A method for detecting a methylated genomic locus is provided. In certain embodiments, the method comprises: a) treating a nucleic acid sample that contains both unmethylated and methylated copies of a genomic locus with an agent that modifies cytosine to uracil to produce a treated nucleic acid; b) amplifying a product from the treated nucleic acid using a first primer and a second primer, wherein the first primer hybridizes to a site in the locus that contain methylcytosines and the amplifying preferentially amplifies the methylated copies of the genomic locus, to produce an amplified sample; and c) detecting the presence of amplified methylated copies of the genomic locus in the amplified sample using a flap assay that employs an invasive oligonucleotide having a 3' terminal G or C nucleotide that corresponds to a site of methylation in the genomic locus.

Methylation assay

Sample collector device

Provided herein is reagent mixture comprising multiplexed amplification reagents and flap assay reagents for detecting, in a single reaction, mutant copies of the KRAS gene that contain any of the 34A, 34C, 34T, 35A, 35C, 35T or 38A point mutations. Methods that employ the reagent mix and kits for performing the same are also provided.

Multiplexed KRAS mutation detection assay

Provided herein is technology relating to collecting and preparing samples. For example, the technology relates particularly, but not exclusively, to devices, systems, and kits that allow a subject to collect and prepare a sample for analysis.

Sample collection device

A stool resuspension solution comprising a hemoglobin stabilization reagent is provided. In some embodiments, the hemoglobin stabilization reagent may be an osmolyte, a polyvalent cation, a sugar or polysaccharide and, optionally, a polyvalent cation, a protoporphyrin, or an HRP stabilization component and, optionally, a polyvalent cation. A method of stabilizing hemoglobin in a stool sample in the solution is also provided, as well as a sample collection device containing the solution.

Method for stabilizing hemoglobin and reagents for performing the same

A method of sample analysis is provided. In certain embodiments, the method involves: a) amplifying a product from a sample that comprises both wild type copies of a genomic locus and mutant copies of the genomic locus that have a point mutation relative to said wild type copies of the genomic locus, to produce an amplified sample, where: i. the amplifying is done using a first primer and a second primer; and ii. the first primer comprises a 3′ terminal nucleotide that base pairs with the point mutation and also comprises a nucleotide sequence that is fully complementary to a sequence in the locus with the exception of a single base mismatch within 6 bases of the 3′ terminal nucleotide; and b) detecting the presence of said product in said amplified sample using a flap assay that employs an invasive oligonucleotide. A kit for performing the method is also provided.

Mutation detection assay

Provided herein is technology related to the chemical modification and purification of DNA. Specifically, the technology provides methods for performing a bisulfite conversion reaction on small amounts of single-stranded, fragmented DNA and performing the subsequent desulfonation and purification steps on magnetic beads.

Modification of DNA on magnetic beads

The present invention provides synthetic DNA strands that find use as controls or in nucleic acid testing methods. In particular, provided herein are synthetic DNA strands of known composition for use as control molecules in stool DNA testing, e.g., of mutations and/or methylation of DNA isolated from stool samples.

Synthetic nucleic acid control molecules

Growing community of inventors

Middleton, WI, United States of America

Michael J Domanico