Methods of enriching and determining target nucleotide sequences

A method of enriching a target nucleotide sequence having a locus of interest from a nucleic acid sample comprising a nucleic acid template comprising the target nucleotide sequence, the method comprising:

• (a) ligating a universal adaptor to the nucleic acid template to provide a ligated nucleic acid, wherein the universal adaptor is an oligonucleotide comprising a duplex portion at a first end and a non-duplex portion at a second end, and wherein the nucleic acid template is ligated to the universal adaptor via the first end;
• (b) dissociating the ligated nucleic acid into a first strand and a second strand, wherein the first strand comprises the target nucleotide sequence;
• (c) annealing an outside primer to the first strand of the ligated nucleic acid in the vicinity of the target nucleotide sequence;
• (d) extending the outside primer over the full length of the first strand of the ligated nucleic acid using a DNA polymerase to provide a nascent DNA linear amplification duplex;
• (e) dissociating the nascent DNA linear amplification duplex at a sufficiently high temperature into the first strand of the ligated nucleic acid and a single-stranded DNA linear amplification product;
• (f) repeating steps (c)-(e) for nineteen or more DNA linear amplification cycles;
• (g) contacting the single-stranded DNA linear amplification products with a DNA polymerase, a universal adaptor primer and an inside primer under a condition sufficient for PCR amplification of the target nucleotide sequence, wherein the universal adaptor primer anneals to a complementary sequence of the non-duplex portion of the universal adaptor in the single-stranded DNA linear amplification products, wherein the inside primer comprises a sequence at the 3′ end that specifically anneals to the target nucleotide sequence, and wherein the inside primer is nested with respect to the outside primer for the locus of interest;
• and
• (h) repeating step (g) for one or more cycles of PCR amplification to provide amplicons of the target nucleotide sequence, thereby enriching the target nucleotide sequence;
• wherein, the nucleic acid sample input is from 5 ng to 50 ng.