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Dideoxyfingerprinting for Mutation Detection

2025-03-27 生物化学 加入收藏
Screening for mutations of the thousands of the sequence products provided by hu

Screening for mutations of the thousands of the sequence products provided by human genome analysis has proven to be a daunting task. The gold standard for identifying sequence alterations is direct sequencing. However, this method is labor- intensive and the least costeffective. Since the mid-1980s, the need for rapid, high-throughput, accurate, and economical mutation analysis systems has lead to the development of several technologies, as an alternative to analysis by direct sequencing, which allowed detection of single mutations in long stretches of DNA (200–600 bp). These techniques include restriction endonuclease digestion of polymerase chain reaction (PCR) products (PCR-RFLP), denaturing gradient gel electrophoresis (DGGE), single-strand conformation polymorphism (SSCP), dideoxyfingerprinting (ddF), and heteroduplex mobility assay (HMA). Most of these methods utilize PCR for amplification of a region of the DNA, a physical or chemical treatment of amplified DNA (by restriction digestion or denaturation), separation of the amplicons by gel electrophoresis (by denaturing or nondenaturing), and visualization of the separated sequence strands (by autoradiography or fluorescence-based detection). Most recent modifications in some of these techniques allow the simultaneous separation and detection of DNA fragments with the use of sophisticated equipment such as high-performance liquid chromatography (HPLC) and capillary electrophoresis.

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