Method for amplification of DNA

A method for detecting the presence of a target nucleic acid sequence on a sample nucleic acid strand from an individual comprising the steps of:

• treating the sample, together or sequentially with appropriate nucleoside triphosphates, an agent for polymerization of the nucleoside triphosphates, a diagnostic primer and an amplification primer under hybridizing conditions;
• wherein the nucleotide sequence of said diagnostic primer comprises (1) a priming region at its 3′-end which is substantially complementary to the target nucleic acid sequence, and (2) a probe region located 5′ to said priming region which is substantially complementary to a reference nucleic acid sequence which is 3′ to the target nucleic acid sequence on the sample nucleic acid strand wherein when said reference nucleic acid sequence is contiguous with said target nucleic acid sequence on the sample nucleic acid strand then the priming region and probe region on the diagnostic probe are separated by a spacer region of nucleic acid,
• whereby for selected hybridization and extension conditions an extension product of the diagnostic primer is synthesized when the priming region is substantially complementary to the target nucleic acid sequence and when the probe region is substantially complementary to the reference nucleic acid sequence,
• but wherein for said selected hybridization and extension conditions no extension product of the diagnostic primer is synthesized when either the priming region or the probe region are not substantially complementary to the target and reference nucleic acid sequences respectively;
• any extension product of the diagnostic primer formed being capable of serving as a template for synthesis of an extension product of said amplification primer after separation from its complement;
• amplifying any extension product;
• and
• detecting the presence or absence of the target polynucleic acid sequence from the presence of absence of amplification product obtained as above.