SNP genotyping by heteroduplex analysis.
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.
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, paniego.norma@inta.gob.ar.
Norma
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Paniego
Corina
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Lia
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D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D019075:Electrophoresis, Capillary; D004720:Endonucleases / Q000378:metabolism; D005838:Genotype; D060005:Genotyping Techniques / Q000379:methods*; D006368:Helianthus / Q000235:genetics; D020180:Heteroduplex Analysis / Q000379:methods*; D008969:Molecular Sequence Data; D009691:Nucleic Acid Denaturation; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*
D004720:Endonucleases