Mutation Detection by Real-Time PCR

Real-time PCR is ideally suited for analysis of single nucleotide polymorphisms (SNPs) and has been increasingly used for this purpose since the advent of real-time PCR and as whole genome sequences have become available. It requires methods that are rapid, sensitive, specific and inexpensive, and several real-time methods have evolved which fulfil these requirements. Additionally real-time PCR is a technique that is readily amenable to automation and no post-PCR handling is required. Different formats have been applied including hybridisation probes with melting curve analysis, hydrolysis probes, molecular beacons and scorpion primers. SNP detection by real-time PCR has found applications in diagnosis of human disease, pharmacogenetics, clinical microbiology and drug development, and has replaced techniques such as sequencing, single strand conformation polymorphism and restriction enzyme digestion. 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SNPs are the commonest type of DNA sequence variations and can occur once every 100-300 bases. As the requirement for rapid, reliable, sensitive and inexpensive methods for SNP detection grow the number of techniques available also increases, each with inherent strengths and weaknesses. Most of these techniques can be divided into hybridisation-based or enzymebased methods and have been extensively reviewed (Syvanen, 2001; Kirk et al., 2002; Kwok, 2002). Real-time PCR, a hybridisation-based method, has become widely used for mutation detection. The systems are flexible with a number of different probe systems that can be used and there is additional flexibility in the design of the probes. Several formats have evolved including, hybridisation probes, hydrolysis probes, molecular beacons and scorpion primers. These methods are sensitive and specific, inexpensive, rapid (some assays can be performed in as little as 30 minutes) and they are both easy to perform and interpret the results. All of the available platforms are semi-automated and none require additional post-PCR handling for example, agarose gel analysis. Depending on the platform used real-time PCR is suitable for low to medium sample throughput. The greatest advantage of these systems is the quality of the data that is generated, known mutations are easily detected and there are possibilities with some of the systems to detect new mutations. Due to increased demand there is now a number of commercially available tests developed, especially for the Applied Biosystems Sequence Detection Systems (ABI 7700 and 7000) and the LightCycler.