217x Filetype PPTX File size 0.09 MB Source: legacy.genetics-gsa.org
Common PCR-based Genotyping Methods for SNP Analysis SNPs can have up to 4 alleles (A/C/G/T), but two alleles are most common. These methods can only positively detect one allele. • PCR-RFLP / CAPS - Polymerase Chain Reaction - Restriction Fragment Length Polymorphisms - Also called Cleaved Amplified Polymorphic Sequences (CAPS) - A Single Nucleotide Polymorphism (SNP) where one allele creates (or removes) a naturally occurring restriction site. Amplifying the sequence surrounding these SNPs from individuals, cutting the products with a restriction enzyme and resolving on a gel will reveal which alleles an individual carries. • dCAPS - derived Cleaved Amplified Polymorphic Sequences - For SNPs that do not create a natural restriction site. Uses mismatches in one PCR primer to create or remove a restriction site for one allele. Genotyping – PCR-RFLP / CAPS [T/A] SNP EcoRV site: GATATC GATATC T/T GATATC GAAATC A/A GAAATC GATATC T/A GAAATC Genotyping – PCR-RFLP / CAPS [T/A] SNP EcoRV site: GATATC GATATC T/T GATATC PCR primers GAAATC A/A GAAATC L T/T A/A T/A GATATC 200 T/A GAAATC 100 50 bp 150 bp 50 200 bp How do you genotype a SNP that does not make a restriction site? 5 Genotyping - dCAPS • Derived CAPS uses a mismatched PCR primer to create or remove a restriction site based on the genotype of a SNP. • Advantages: - Can be used when the SNP does not create a natural CAPS/RFLP marker. - Can be used to change a natural CAPS marker from a site using an expensive or rare enzyme to a cheap or common enzyme. • Disadvantages: - Mismatches in primer lowers PCR specificity. - Laborious compared to hybridization with gene chip methods for SNP detection. - Finding the right enzyme. Can use web site: http://helix.wustl.edu/dcaps/dcaps.html to find dCAPS primers for SNPs. 6
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