Multiplex Assays with Fluorescent Microbead Readout: A Powerful Tool for Mutation Detection

[摘要] After the completion of the Human Genome Project, the goal for biomedical research is to apply genomic approaches for the improvement of human health (1). The study of human DNA variation promises to have a great impact on understanding how genetic factors contribute to human disease, conferring susceptibility or resistance; it is also expected to help uncover the reasons that individuals respond differentially to therapeutics.Technologies for mutation detection can be classified into methods for the detection of unknown mutations and methods for the detection of known mutations (2). Methods for the detection of unknown mutations (3)(4)(5)(6)(7)(8), also known as screening or scanning methods, are used during the discovery phase. Once a new mutation associated with a genetic disease has been characterized and validated, it is usually detected over and over in many DNA samples by specific, cost-efficient, and easy-to-use methods (9)(10)(11)(12)(13), also known as “diagnostic methods” (14).Typically, genotyping assays for known mutations start with sample preparation followed by amplification of the target DNA sequence containing the mutation to be tested. This latter step is most commonly accomplished by use of the PCR, but other amplification strategies are available (15)(16)(17). Subsequently, the presence or absence of the mutation is determined by allele-specific biochemical reactions. The selected allele-specific method largely determines the specificity, accuracy, and reproducibility of the genotyping assay. The discriminating power of DNA ligases (13)(15) and DNA polymerases (10)(11)(12), as well as the difference in thermodynamics between matched and mismatched DNA duplexes (9), has been extensively exploited for mutation detection. The final step of a genotyping approach is the detection of the signal resulting from the allele-specific assay and …

[学科分类] 过敏症与临床免疫学