Methods for selectively suppressing non-target sequences

Abstract

The invention generally relates to negative selection of nucleic acids. The invention provides methods and systems that remove unwanted segments of nucleic acid in a sample so that a target gene or region of interest may be analyzed without interference from the unwanted segments. A sample is obtained that includes single-stranded nucleic acid with one or more unwanted segments. Complementary nucleic acid is added to the single-stranded nucleic acid to create a double-stranded region that includes the unwanted segment. The double-stranded region is then digested, leaving single-stranded nucleic acid that includes the target gene or region of interest. This allows paralogs, pseudogenes, repetitive elements, and other segments of the genome that may be similar to the target gene or region of interest to be removed from the sample.

What is claimed is: 
     
         1 . A method of removing unwanted segments of a nucleic acid from a sample, the method comprising:
 obtaining a single-stranded nucleic acid that contains an unwanted segment;   adding complementary nucleic acid to create a double-stranded region that contains the unwanted segment; and   digesting the double-stranded region, thereby removing the unwanted segment from the nucleic acid.   
     
     
         2 . The method of  claim 1 , wherein adding the complementary nucleic acid comprises annealing an oligonucleotide to the unwanted segment, thereby creating the double-stranded region. 
     
     
         3 . The method of  claim 2 , wherein the annealing step comprises annealing a plurality of primers to a plurality of portions of the nucleic acid that flank an unwanted segment. 
     
     
         4 . The method of  claim 1 , wherein adding the complementary nucleic acid comprises:
 annealing an oligonucleotide to a portion of the single-stranded nucleic acid that flanks the unwanted segment; and   extending the annealed oligonucleotide to create the double-stranded region.   
     
     
         5 . The method of  claim 4 , wherein the annealing step comprises annealing a plurality of primers to a plurality of portions of the nucleic acid that flank an unwanted segment. 
     
     
         6 . The method of  claim 4 , wherein the extending step is conducted using a polymerase enzyme under conditions sufficient to cause extension of the primer in a template-dependent manner. 
     
     
         7 . The method of  claim 1 , wherein the digesting step comprising exposing the sample to an enzyme that preferentially digests double-stranded nucleic acid. 
     
     
         8 . The method of  claim 7 , wherein the enzyme is selected from double-stranded endonucleases, restriction endonucleases, and nicking enzymes. 
     
     
         9 . The method of  claim 8 , further comprising the step of deactivating the enzyme. 
     
     
         10 . The method of  claim 1 , wherein the nucleic acid is selected from DNA, RNA, and modified nucleic acids. 
     
     
         11 . The method of  claim 1 , further comprising the step of analyzing nucleic acid remaining after the digesting step. 
     
     
         12 . The method of  claim 1 , wherein the digesting step results in intact genomic DNA lacking one or more unwanted segment and that is compatible with a nucleic acid analysis assay. 
     
     
         13 . The method of  claim 12 , wherein the assay comprises molecular inversion probe capture. 
     
     
         14 . The method of  claim 13 , wherein the assay further comprises sequencing. 
     
     
         15 . The method of  claim 14 , wherein the sequencing is selected from Sanger sequencing and Next Generation Sequencing. 
     
     
         16 . The method of  claim 1 , wherein the unwanted segment is a paralog, a pseudogene, or non-paralogous repetitive element. 
     
     
         17 . The method of  claim 1 , further comprising the step of obtaining a sample from a subject and denaturing double-stranded DNA in the sample. 
     
     
         18 . The method of  claim 17 , wherein the denaturing step comprises exposing the sample to heat, a detergent, or a basic solution.