Capture methodologies for circulating cell free dna

Abstract

A nucleic acid patch method for amplifying target nucleic acid sequences in circulating free DNA or residual DNA samples where the defining ends of the target nucleic acid sequences are unknown.

1 . A method for the capture and amplification of a targeted region of fragmented deoxyribonucleic acid (fgDNA), the method comprising:
 providing a sample of an fgDNA having a minimal targeted region between two unknown sequences;   reducing said sample of said fgDNA to single stranded fgDNA;   adding oligonucleotide patches to said single stranded fgDNA, said oligonucleotide patches comprising:
 a nucleotide sequence that is a reverse compliment to an end sequence that defines the minimal targeted region; 
 a universal primer sequence; and 
 a protecting group on at least one end; 
   said oligonucleotide patches binding to said end sequence to create:
 double stranded DNA at said end sequence; 
 a single stranded DNA flanking region outside said minimal targeted region, said single stranded DNA flanking region being of unknown length and sequence; and 
 a single stranded DNA gap region between said universal primer sequence and said end sequence; 
   filling in said single stranded DNA gap region with polymerase to make double stranded DNA at said gap region;   adding enzymes to said single stranded fgDNA, said enzymes cleaving said single stranded DNA flanking regions from said double stranded DNA, resulting in said single stranded fgDNA being reduced to a portion of said minimal targeted region with said double stranded DNA at both ends, said protecting group preventing said oligonucleotide patch from degradation by said enzymes;   ligating a complementary universal primer to each of said ends of said targeted region;   performing polymerase chain reaction; and   sequencing the polymerase chain reaction amplicons of said targeted region.   
     
     
         2 . The method of  claim 1 , wherein a multiplicity of oligonucleotide patch pairs are added to the reaction concurrently. 
     
     
         3 . A system for the capture and amplification of a targeted region of fragmented deoxyribonucleic acid (fgDNA), the system comprising:
 a sample comprising:
 single stranded fgDNA reduced from double stranded fgDNA, said single stranded fgDNA having a minimal targeted region between two unknown sequences; and 
   a plurality of oligonucleotide patches, each of which comprises:
 a nucleotide sequence that is a reverse compliment to an end sequence that defines the minimal targeted region; 
 a universal primer sequence; and 
 a protecting group on at least one end; 
   said oligonucleotide patches binding to said end sequence to create:
 double stranded DNA at said end sequence; 
 a single stranded DNA flanking region outside said minimal targeted region, said single stranded DNA flanking region being of unknown length and sequence; and 
 a single stranded DNA gap region between said universal primer sequence and said end sequence; 
   enzymes for cleaving said single stranded DNA flanking regions from said double stranded DNA at both said ends of said single stranded fgDNA, resulting in said single stranded fgDNA being reduced to said targeted region with said double stranded DNA at both said ends said protecting group preventing said oligonucleotide patch from degradation by said enzymes; and   a complementary universal primer having a complementary sequence to said universal primer sequences of said oligonucleotide patches bound to each of said ends of said targeted region.   
     
     
         4 . The system of  claim 3 , wherein all said plurality of oligonucleotide patches are bound to said ends.