The methods used to verify the identity of the amplicon(s) produced in real-time PCR are also sufficiently powerful to detect small variations between sequences. Variations in sequence, including single nucleotide polymorphisms (SNPs) have been successfully identified in real-time PCR assays. One common approach to the detection of sequence variation is to compare melting curves. In general, the effect of base substitutions on the melting kinetics of PCR products is too small to be detected reliably (if at all). However, heteroduplexes of relatively long amplicons differing by a SNP can be distinguished from the homoduplexes on the basis of their melting curves.

The melting curves of short fluorescent probes can be used to distinguish between amplicons. This method is sensitive to SNPs, which usually cause a shift in the melting peak of several degrees. A common alternative to the melting curve approach is to use hydrolysis (TaqMan) probes. The efficiency of the 5'-3' endonuclease reaction is greatly impaired when a well-designed probe mismatches its target sequence by even a single base. The detection of mutations by real-time PCR is discussed by Lyon et al Mutation Detection and by Pont-Kingdon Molecular Haplotyping. Although the melting curve and hydrolysis probe methods for mutation analysis are widely used they are only able to detect sequences that represent a large proportion of the population. A quantitative real-time ARMS method can be used (see Lyon et al). ARMS assays are designed to detect the emergence of significant sequence mutants within a background that remains mainly of the parent type.

Bibliography:

1. Real-Time PCR: Current Technology and Applications
2. Real-Time PCR in Microbiology: From Diagnosis to Characterization
3. PCR Troubleshooting: The Essential Guide
4. PCR Books

Labels: molecular haplotyping, mutation detection, real-time pcr, SNP