Detection of nucleic acid sequence differences using coupled ligase detection and polymerase chain reactions

A method comprising:

• providing a sample potentially containing one or more target nucleotide sequences;
• providing one or more primary oligonucleotide primer sets, each set characterized by (a) a first oligonucleotide primer, having a target-specific portion and a 5′ upstream secondary primer-specific portion, and (b) a second oligonucleotide primer, having a target-specific portion and a 5′ upstream secondary primer-specific portion;
• subjecting a primary polymerase chain reaction mixture, comprising the sample, the one or more primary oligonucleotide primer sets, and a polymerase, to two or more polymerase chain reaction cycles to form primary extension products;
• providing one or a plurality of secondary oligonucleotide primer sets, each set characterized by (a) a first secondary primer containing the same sequence as the 5′ upstream portion of a first primary oligonucleotide primer, and (b) a second secondary primer containing the same sequence as the 5′ upstream portion of a second primary oligonucleotide primer from the same primary oligonucleotide primer set as the first primary oligonucleotide primer contained by the first secondary primer;
• and
• subjecting a secondary polymerase chain reaction mixture, comprising the primary extension products, the one or plurality of secondary oligonucleotide primer sets, and the polymerase to two or more polymerase chain reaction cycles to form secondary extension products complementary to the primary extension products.