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

GATATC

[T/A] SNPEcoRV site: GATATC

GAAATC

GATATC

GATATC

GAAATCGAAATC

T/T

A/A

T/A

Genotyping – PCR-RFLP / CAPS

200 bp

GATATC

[T/A] SNPEcoRV site: GATATC

GAAATC

GATATC

GATATC

GAAATC

GAAATC

T/T

A/A

T/A

PCR primers

150 bp50 bp

200

100

50

L T/T A/A T/A

5

How do you genotype a SNP that does not make a restriction site?

6

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’

Important Note: The primer does not overlap or mutate the SNP!

Dog SNP dCAPS example

1. PCR with dCAPs primers2. Digest products with BamHI3. Run on gelExpected 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