SNP Genotyping by Heteroduplex Analysis

PANIEGO NORMA; CORINA FUSARI; LIA VERONICA; ANDREA PUEBLA

Plant Genotyping: Methods and Protocols

Sringer. Humana Press

Año: 2015;

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 to SNP detection and haplotype analysis of candidate genes in association studies. The protocols involve enzymatic mismatch cleavage with endonuclease CEL 1 from celery associated to fragment separation using capillary electrophoresis (CEL1 cleavage), and differential retention of the homo/heteroduplex DNA molecules under partial denaturing conditions on ion pair reversed-phase liquid chromatography (dHPLC), respectively. 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 SNP 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.