Genetics Research, LLC

William Glover

Anthony P Shuber

Thomas C Meyers

Provided herein are methods of detecting nucleic acids. The nucleic acid of interest may be detected by using Cas endonuclease to degrade substantially all nucleic acid in a sample except for the nucleic acid of interest, leaving the nucleic acid of interest isolated and amenable to detection. In related methods, Cas endonuclease complexes are used to protect the nucleic acid of interest while unprotected nucleic acid is digested, e.g., by exonuclease, after which the isolated nucleic acid of interest is detected.

Detection of targeted sequence regions

The invention provided methods and devices for performing sequential, regulated multiplex reactions in a single tube without the addition or removal of contents from the tube.

Regulated multiplex reactions in a single tube

The invention provides methods of isolating a target nucleic acid in a sample. A primer is hybridized to the target. A polymerase and modified nucleotide resistant to nuclease degradation are used to extend the primer to create a modified polynucleotide. The sample is exposed to a nuclease, thereby isolating the modified polynucleotide. Optionally, the target nucleic acid may be further protected by binding a protein in a sequence specific manner to one end of the target nucleic acid to create a protected target nucleic acid resistant to nuclease degradation. Thus, after exposing the sample to a nuclease, the modified polynucleotide and protected target nucleic acid are isolated.

Isolation of target nucleic acids

The invention provides methods for determining the mutation burden of a tumor by assaying tumor DNA that is representative of genetic loci that are themselves representative of genetics of the tumor. The assayed tumor DNA may be itself agnostic as to loci, so long as it is representative of loci that are representative of tumor mutation burden. The invention provides for assays in which the tumor DNA being sequenced or tested can be something other than, and possibly less than, a full panel of oncogenes that is expected to stand for a tumor's mutational load.

Tumor mutation burden

The invention provides methods for detecting small mutations and structural alterations in DNA by using binding proteins to protect those features while digesting unprotected DNA in a sample. To detect small mutations, a protein that binds exclusively to the mutation of interest, and not to wild-type, is used. For structural alterations, binding proteins are used that flank a breakpoint of the alteration. After digestion of unbound, unprotected nucleic acid in the sample, the mutation- or breakpoint-containing segment remains as an isolated DNA fragment. The sample is then assayed to detect any fragment of DNA and the detection of the fragment indicates the presence of the mutation or breakpoint in the subject.

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Wakefield, MA, United States of America

William Glover