Patent ID: 8501403

Claim:
A method for selective amplification from a sample of a nucleic acid molecule having a target sequence adjacent a 3′ terminus, in the presence of molecules comprising the target sequence not adjacent a 3′ terminus but embedded within the molecule, the method comprising (i) contacting the sample with a template oligonucleotide having (a) a 3′ region substantially complementary to the target sequence adjacent the 3′ terminus of a nucleic acid molecule; (b) a 5′ tail comprising a nucleic acid sequence such that a free 5′ tail is formed when the template oligonucleotide anneals to the target sequence located adjacent a 3′ terminus or embedded within a nucleic acid molecule, the 5′ tail providing a template for extension of the 3′ terminus of the target sequence incorporating sequence complementary to the 5′ tail of the template oligonucleotide resulting in the addition of sequence complementary to the 5′ tail to the target sequence; and (c) a modification in the 3′ region that delays 3′ extension of said template oligonucleotide; (ii) contacting the sample with a second oligonucleotide for priming in a reverse direction to the template oligonucleotide, and optionally a third oligonucleotide sharing nucleotide sequence with the 5′ tail of the template oligonucleotide; (iii) carrying out amplification of the sample wherein (a) annealing of the template oligonucleotide to target sequence adjacent a 3′ terminus is stabilised over annealing of said oligonucleotide to target sequence not adjacent to a 3′ terminus by copying of the 5′ tail of the template oligonucleotide by extension from the 3′ terminus of the target sequence in the presence of delayed 3′ extension of said oligonucleotide; and (b) wherein consequent stabilised annealing of the template oligonucleotide to target sequence adjacent to a 3′ terminus enhances efficiency of 3′ extension of the template oligonucleotide compared to extension of the template oligonucleotide annealed to target sequence not adjacent a 3′ terminus, and (c) wherein amplification occurs using the template oligonucleotide and/or the third oligonucleotide in combination with the second oligonucleotide, resulting in selective amplification of target sequence adjacent a 3′ terminus in the presence of nucleic acid molecules comprising target sequence embedded within the molecule.