PCR-Based Genotyping Methods An introduction to PCR-RFLP/CAPS, and dCAPS

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 To find SNPs, one can use RFLP – as long as the SNP modifies a naturally occurring restriction site. This is showing an A/T SNP where the T allele has a natural EcoRV site, and the A does not. There are 3 potential Genotypes, T/T, A/A, and T/A. The T/T will have two EcoRV spots at this locus, the A/A will have none, and the A/T will have one. 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/T will have the smaller products: 50 and 150. A/A will have only large products: 200. And the heterozygote will have both the small and large products: 50, 150 and 200 bp bands. T/A GAAATC 100 50 bp 150 bp 50 200 bp

How do you genotype a SNP that does not make a restriction site? Pose the question to the class and see if they can come up with a method. If you have covered site directed mutagenesis, lead them to consider combining the RFLP technique with the mutagenic primers of site directed mutagenesis.

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.

Dog SNP dCAPS example Dog SNP #1 (DS1) is polymorphism of C/T on chromosome 10 in the dog genome. The C allele is associated with small size and weight (< 30 lb). We will create a dCAPS primer set that is a positive assay for the C allele using BamHI enzyme (C will be cut with BamHI). BamHI site: 5’-GGATCC-3’ 3’-CCTAGG-3’ DS1 Locus: 5’-GCCTTGTCCTAAATGTAGTCGATA[C/T]N(119) TCTTCTCCCCTTCTGGGTTTAAA-3’

Dog SNP dCAPS example The reverse primer will be a standard reverse primer with no mismatches about 100 bp downstream of SNP. In this case the best primer site was 123 bp downstream of the SNP. BamHI site: 5’-GGATCC-3’ 3’-CCTAGG-3’ DS1 Locus: 5’-GCCTTGTCCTAAATGTAGTCGATA[C/T]N(119) TCTTCTCCCCTTCTGGGTTTAAA-3’ 3’-AGAAGAGGGGAAGACCCAAA-5’

Dog SNP dCAPS example The Forward primer will be mismatched to create the BamHI site with the C allele but not the T allele. We will need to mutate two sites: the first and fifth site in the recognition sequence. This will create a GGATCC site with the C allele and a GGATCT site with the T allele (only the C will be cut by BamHI) BamHI site: 5’-GGATCC-3’ 3’-CCTAGG-3’ DS1 Locus: 5’-GCCTTGTCCTAAATGTAGTCGATA[C/T]N(119) TCTTCTCCCCTTCTGGGTTTAAA-3’ 3’-AGAAGAGGGGAAGACCCAAA-5’

Dog SNP dCAPS example The Forward primer will be mismatched to create the BamHI site with the C allele but not the T allele. We will need to mutate two sites: the first and fifth site in the recognition sequence. This will create a GGATCC site with the C allele and a GGATCT site with the T allele (only the C will be cut by BamHI) BamHI site: 5’-GGATCC-3’ 3’-CCTAGG-3’ DS1 Locus: 5’-GCCTTGTCCTAAATGTAGTGGATC-3’ 5’-GCCTTGTCCTAAATGTAGTCGATA[C/T]N(116) TCTTCTCCCCTTCTGGGTTTAAA-3’ 3’-AGAAGAGGGGAAGACCCAAA-5’ PCR will result in 163 bp product. Cutting with BamHI will only cut off the length of the Forward primer ~ 20 bp. In contrast to RFLP where you can design the primer further away from the SNP, here we are limited to the length of the primer. This is why we usually prefer longer primers, which will also increase specificity. Important Note: The primer does not overlap or mutate the SNP!

Dog SNP dCAPS example PCR with dCAPs primers Digest products with BamHI Run on gel Expected results for three genotypes: Homozygous C/C – 143, 20 bp Homozygous T/T – 163 bp Heterozygous C/T – 163, 143, 20 bp L C/C T/T C/T 200 150 100 Note: The 20 bp product will run off gel, since we run gel long enough to resolve between 163 and 143 bp