Rapid, nonradioactive detection of mutations in the human genome by allele-specific amplification.

A simple, rapid, nonradioactive method has been developed to facilitate the direct detection of point mutations that cause genetic disease. The method operates on the basis of the specific amplification of a target allele by the polymerase chain reaction with extension primers designed such that their 3' end is placed at the mutation site. When this base is complementary to that of the specific allele, the DNA segment is amplified; when it is not complementary, the polymerase chain reaction cannot proceed. When alpha 1-antitrypsin (alpha 1AT) deficiency was used as a model, the technique of allele-specific amplification was capable of selective detection of five different mutations that cause the alpha 1AT deficiency state, including three different naturally occurring single-base substitution mutations (alleles Z, S, and Nullbellingham), an insertion mutation (Nullmattawa), and a deletion mutation (Nullgranite falls). Double-blind evaluation of 47 samples of genomic DNA demonstrated 100% accuracy of the method. The technique of allele-specific amplification is rapid, simple, and does not require the existence of a convenient restriction endonuclease site or the use of radioactive materials, and thus should have broad applicability for the detection of known genetic diseases in a highly sensitive and specific fashion.