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The DNA marketplace is changing: the technology is maturing, whole human genome arrays, as well as those of model organisms, are beginning to make it onto the market, and researchers have more choices of commercial platform than ever before. Diane Gershon takes a closer look at what's on offer.
The 'megaprimer' method of site-directed mutagenesis uses three oligonucleotide primers and two rounds of polymerase chain reaction (PCR)1. One oligonucleotides is mutagenic; the others are forward and reverse primers that lie upstream and downstream from the binding site for the mutagenic oligonucleotide. The mutagenic primer and the nearer of the external primers are used in the first PCR to generate and amplify a mutated fragment of DNA. This amplified fragment—the megaprimer—is used in the second PCR in conjunction with the remaining external primer to amplify a longer region of the template DNA. This protocol is based on a method that uses forward and reverse external primers with significantly different melting temperatures (Tm)2.
With the sequencing of the human genome, millions of single-nucleotide polymorphisms, or SNPs, have been discovered and can be used as markers to identify genes contributing to common human diseases. Two large sets of SNPs have now been organized in panels for high-throughput genotyping.