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
T/A
GAAATC
Genotyping – PCR-RFLP / CAPS
[T/A] SNP
EcoRV site: GATATC
GATATC
T/T
GATATC
PCR primers
GAAATC
A/A
GAAATC
L
200
GATATC
T/A
GAAATC
100
150 bp
50 bp
200 bp
50
T/T A/A T/A
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
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’
Important Note: The primer does
not overlap or mutate the SNP!
DS1 Locus:
5’-GCCTTGTCCTAAATGTAGTGGATC-3’
5’-GCCTTGTCCTAAATGTAGTCGATA[C/T]N(116) TCTTCTCCCCTTCTGGGTTTAAA-3’
3’-AGAAGAGGGGAAGACCCAAA-5’
Dog SNP dCAPS example
1. PCR with dCAPs primers
2. Digest products with BamHI
3. 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
Note: The 20 bp product will run off gel,
since we run gel long enough to resolve
between 163 and 143 bp
L
200
150
100
C/C T/T C/T