Heteroduplex analysis

DNA and RNA with different sequences and mutations show different electrophoretic migration behavior. There are simple electrophoretic methods, which can be used to detect mutations and minimal differences within the sequences of the PCR product. Popular methods are single-strand conformational polymorphism and heteroduplex analysis. 

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Tang, J. and Unnasch, T.R. (1997) Heteroduplex analysis in medical entomology a rapid and sequence-based tool for population and phylogenetic studies. Parasitology Today 13, 271—274. 

Alternatively, the denatured single strands can be made to reanneal to form double-stranded helices. Complementary strands will hybridize to each other. However, if there are sequence differences between two strands, one from each allele, they remain unpaired in the heteroduplex and, as a result, form open loops that reduce migration in the electrophoretic gel. This is the basis of heteroduplex analysis, in which distinct electrophoretic patterns are seen for different alleles, similar to that seen in SSCP (01). 

Jones AC, Austin J, Hansen N, Hoogendoorn B, Oefner PJ, Cheadle JP, O Donovan MC (1999) Optimal temperature selection for mutation detection by denaturing HPLC and comparison to single-stranded conformation polymorphism and heteroduplex analysis. Clin Chem 45 1133-1140... 

HRM MWP Discovery or scanning Heteroduplex analysis, no variant identification... 

Tian, H.J., Landers, J.P., Hydroxyethylcellulose as an effective polymer network for DNA analysis in uncoated glass microchips optimization and application to mutation detection via heteroduplex analysis. Anal. Biochem. 2002,309,212-223. 

Heteroduplex analysis can also be used in combination with long (overnight) runs on Hydrolink sequencing gels to detect allelic variation.9 The main appeal of this approach, as for SSCP, is its simplicity, but the factors controlling the level of resolution of this technique are unknown. 

Landers and co-workers compared various surface derivatization agents in conjunction with coating polymers for DNA heteroduplex analysis of the breast cancer susceptibility gene (BRCA1) [29]. The most effective coating combina-... 

Figure 11 Fast mutation detection via heteroduplex analysis on a microfabricated electrophoretic chip. The heterozygous mutants were specified in the figure. The separation buffer was 2.5% HEC containing 10% glycerol in 1xTBE (pH = 8.6) for (a-d) and 4.5% HEC containing 10% glycerol and 15% urea in 1xTBE (pH =8.6) for (e-f). The microchannel on the chip was coated with PVP (Mr 1,000,000) and detection mediated by laser-induced fluorescence (em/ex 520 nm/488 nm). The PCR products were injected into the channel for 100 s at 333 V/cm (effective microchannel length of 5.5 cm), and the separation voltage was 573 V/cm. (Reprinted with permission from Ref. 69.)...

Tian H, Brody LC, Landers JP. Rapid detection of deletion, insertion, and substitution mutations via heteroduplex analysis using capillary- and microchip-based electrophoresis. Genome Res 2000 10 1403-1413. 

Tian H, Brody LC, Fan S, Huang Z, Landers JP. Capillary and microchip electrophoresis for rapid detection of known mutations by combining allele-specific DNA amplification with heteroduplex analysis. Clin Chem 2001 47 173-185. 

Tian H, Brody LC, Mao D, Landers JP. Effective capillary electrophoresis-based heteroduplex analysis through optimization of surface coating and polymer networks. Anal Chem 2000 72 5483-5492. 

Shi, R. et al. Temperature-mediated heteroduplex analysis for the detection of drug-resistant gene mutations in clinical isolates of Mycobacterium tuberculosis by denaturing HPLC, SURVEYOR nuclease. Microb. Infect. 2006, 8, 128-135. 

Tchernitchko D, Lamoril J, Puy H, Robreau AM, Bogard C, Rosipal R, et al. Evaluation of mutation screening by heteroduplex analysis in acute intermittent porphyria. Clin Chim Acta 1999 279 133-43. 

Hestekin CN, Jakupciak JP, Chiesi TN, Kan CW, O Connell CD, Barron AE (2006). An optimized microchip electrophoresis system for mutation detection by tandem SSCP and heteroduplex analysis for p53 exons 5-9. Electrophoresis, 27 3823-3835. 

Robinson MD, (2005). Manual SSCP and heteroduplex analysis gels. In Taylor GR, Day EMM (eds.), Guide to Mutation Detection. Wiley-Liss, Hoboken, NJ, pp. 201-209. 

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