Nucleic acid amplification allele specific

A method of allele-specific amplification of a variant of a target sequence, wherein the amplification involves the polymerase chain reaction (PCR), the target sequence may exist as several variant sequences, the method comprising (a) hybridizing a first and second oligonucleotide to the target sequence; wherein the first oligonucleotide is at least partially complementary to the target sequence and the second oligonucleotide is at least partially complementary on their portion 3 'and 5' of the target sequence, and has at least one complementary selective nucleotide at only one variant of the target sequence, wherein said selective nucleotide is 10 positioned internally of the 3 'end, and wherein said second oligonucleotide comprises at least one nucleotide with a covalently modified base in the exocyclic amino group, wherein the exocyclic amino group is amino group at the 6 position of adenosine, in position 2 or guanosine in position 4 of cytidine; (B) providing conditions for hybridizing said first and second oligonucleotides to at least one variant of the target sequence, suitable for the oligonucleotide extension by a nucleic acid polymerase, wherein said polymerase is capable of extending said second oligonucleotide preferably when it said second oligonucleotide hybridizes to said variant of the target sequence which is complementary to said selective nucleotide, and substantially less when said second oligonucleotide hybridizes to said variant of the target sequence that is not complementary to said selective nucleotide; and (c) repeating the sequence of steps of annealing and extension multiple times.