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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.
Combined with the right computational tools, genomic data can uncover unknown pathways to cellular processes. Because few researchers have the resources to develop their own bioinformatics software, companies have stepped in to meet this need. Laura Bonetta reports.
