Abstract
Heteroduplex-based genotyping methods have proven to be technologically effective and economically efficient for low- to medium-range throughput single-nucleotide polymorphism (SNP) determination. In this chapter we describe two protocols that were successfully applied for SNP detection and haplotype analysis of candidate genes in association studies. The protocols involve (1) enzymatic mismatch cleavage with endonuclease CEL1 from celery, associated with fragment separation using capillary electrophoresis (CEL1 cleavage), and (2) differential retention of the homo/heteroduplex DNA molecules under partial denaturing conditions on ion pair reversed-phase liquid chromatography (dHPLC). Both methods are complementary since dHPLC is more versatile than CEL1 cleavage for identifying multiple SNP per target region, and the latter is easily optimized for sequences with fewer SNPs or small insertion/deletion polymorphisms. Besides, CEL1 cleavage is a powerful method to localize the position of the mutation when fragment resolution is done using capillary electrophoresis.
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Acknowledgments
We gratefully thank Lic Alberto Maligne for dHPLC technical assistance and Verónica Nishinakamasu and Pablo Vera for fluorescence capillary electrophoresis technical support. This research was supported by ANPCyT/FONCYT, PID 2007 00073, INTA-PRR AEBIO 245001 and 245005, INTA-PE AEBIO 24554711 and 241351. Drs. V.L. and N.P. are career members of the Consejo Nacional deInvestigaciones Científicas y Técnicas (CONICET).
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Paniego, N., Fusari, C., Lia, V., Puebla, A. (2015). SNP Genotyping by Heteroduplex Analysis. In: Batley, J. (eds) Plant Genotyping. Methods in Molecular Biology, vol 1245. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1966-6_10
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DOI: https://doi.org/10.1007/978-1-4939-1966-6_10
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