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Advances in Nucleic Acid Detection and Quantification


Mutation scanning using high-resolution melting
Claire F. Taylor1
Cancer Research UK Genome Variation Service, St James’s University Hospital, Leeds LS9 7TF, U.K.

Mutation scanning techniques are used to detect sequence variants without the need for prior knowledge of the identity or precise location of the variant, incontrast with genotyping techniques, which determine the status of a specific variant. High-resolution melting is a recently developed method that shows great potential as a mutation scanning technique. Sensitivity and specificity for mutation detection are extremely high and the technique also has advantages of cost and throughput. Practical considerations for successful mutation scanning byhigh-resolution melting are also discussed in this review.

Principle of HRM (high-resolution melting)
HRM is a simple, PCR-based method for detecting DNA sequence variation by measuring changes in the melting of a DNA duplex. Melting of double-stranded DNA molecules is influenced by several factors. Some of these, such as the length, GC content and sequence, are propertiesof the individual molecule [1]. Others are not: these include the ionic strength of the buffer solution, the DNA concentration and the presence of substances such as DMSO or betaine [2,3]. For HRM analysis, duplexes may be formed from the two strands of a PCR amplicon [4–6] or from an oligonucleotide probe and an amplicon strand [7–9]. Amplicon melting has been used for both genotyping andmutation scanning [5,6]. Probe melting is restricted to detecting variation within the probe-binding site. It has generally been used for genotyping applications [10], although there is scope for limited scanning because the presence of variants under the probe other than the target variant also perturbs probe melting [7]. A homozygous sequence variant usually changes the T m of the duplex. Where thereis an exchange between G:C and T:A base pairs, the change in T m is relatively large: approx. 0.8– 1.4◦ C [6]. If, however, the bases swap strands but the base-pair does not change, the change in T m is smaller, becoming undetectable if there is also nearest-neighbour symmetry [6]. A heterozygous sample contains four duplex species (Figure 1A) and its observed melting curve is a composite of thefour individual melting curves. The contribution from the relatively unstable heteroduplexes changes the shape of the heterozygous melting curve (Figure 1B) [5,6]. Duplex melting is generally monitored using intercalating dyes, although fluorescently labelled primers have also been used [4]. These dyes, which bind to double-stranded but not single-stranded DNA, fluoresce when bound but not afterrelease on duplex melting. Dyes such as LCGreen, able to saturate available binding sites at a concentration compatible with PCR, are necessary for successful HRM. As a consequence of
Key words: amplicon, genotyping, high-resolution melting (HRM), mutation detection, mutation scanning, sequence variant. Abbreviations used: HRM, high-resolution melting. 1 email taylor_claire_f@yahoo.comBiochemical Society Transactions

dye redistribution during melting, non-saturating dyes, such as SYBR Green, are biased against low-temperature melting species and do not detect heteroduplexes [5,11]. HRM is a simple method: after PCR, carried out in the presence of a suitable dye, the product is heated while the level of fluorescence is measured. As the temperature rises and the duplex passes throughits melting transition, dye is released and fluorescence intensity is reduced. Although several instruments capable of performing the fluorescence acquisition exist, they vary in performance, with those designed for HRM giving a more satisfactory outcome [6,11–14]. Low-resolution melting data and HRM data in a genotyping context are often viewed as a derivative plot (−dF/dT against T), whereas...
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