Capture methodologies for circulating cell free DNA
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 targeted region between two unknown sequences;
reducing said sample of said fgDNA to single stranded fgDNA;
adding oligonucleotide patches to said single stranded fgDNA, each of said oligonucleotide patches comprising:
a nucleotide sequence complementary to an end of said targeted region;
a universal primer sequence; and
a protecting group on at least one end;
one of said oligonucleotide patches binding to each of said ends of said targeted region creating double stranded DNA at both said ends of said targeted region and leaving single stranded DNA flanking regions outside said targeted region, said single stranded DNA flanking regions being of unknown length and sequence;
adding enzymes to said single stranded fgDNA, the enzymes 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 targeted region with said double stranded DNA at both said 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.