Paniego Norma, Fusari Corina, Lia Verónica, Puebla Andrea
Instituto de Biotecnología, Centro de Investigación en Ciencias Veterinarias y Agronómicas (CICVyA), Instituto Nacional de Tecnología Agropecuaria (INTA), Nicolas Repeto y Los Reseros, 1686 Hurlingham, Buenos Aires, Argentina,
Methods Mol Biol. 2015;1245:141-50. doi: 10.1007/978-1-4939-1966-6_10.
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.
基于异源双链的基因分型方法已被证明在中低通量单核苷酸多态性（SNP）测定方面技术上有效且经济高效。在本章中，我们描述了两种成功应用于关联研究中候选基因SNP检测和单倍型分析的方案。这些方案包括：（1）使用芹菜中的核酸内切酶CEL1进行酶促错配切割，并通过毛细管电泳进行片段分离（CEL1切割）；（2）在部分变性条件下，离子对反相液相色谱（dHPLC）对同源/异源双链DNA分子的差异保留。这两种方法是互补的，因为对于每个目标区域识别多个SNP，dHPLC比CEL1切割更通用，而后者对于SNP较少或小插入/缺失多态性的序列很容易优化。此外，当使用毛细管电泳进行片段分辨率分析时，CEL1切割是定位突变位置的有力方法。
