RAPID DETECTION OF GENETIC MUTATIONS USING THE CHEMILUMINESCENT HYBRIDIZATION PROTECTION ASSAY (HPA) - OVERVIEW AND COMPARISON WITH OTHER METHODS

The detection of genetic mutations is of paramount importance for the study, diagnosis, and treatment of human genetic disease. Methods of d etection generally fall into one of two categories: those to scan for unknown mutations and those to detect known mutations. This review foc uses on methods for the detection of known mutations. The hybridizatio n protection assay (HPA) is described in detail. The HPA method utiliz es short oligonucleotide probes covalently labeled with a highly chemi luminescent acridinium ester (AE). The assay format is completely homo geneous, requiring no physical separation steps, and can rapidly and s ensitively detect all single-base mismatches as well as multiple misma tches, insertions, deletions, and genetic translocations. When very lo w copy number targets are assayed, HPA is coupled with transcription-m ediated amplification (TMA), an isothermal method that amplifies DNA o r RNA targets. Other methods that are described for the detection of k nown mutations include hybridization with sequence-specific oligonucle otides, hybridization to oligonucleotide arrays, allele-specific ampli fication, ligase-mediated detection, primer extension, and restriction fragment analysis. The advantages and limitations of each of these me thods are discussed. Methods to scan for unknown mutations are briefly described.