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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

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Pcr based genotyping methods

PCR-Based Genotyping Methods

An introduction to PCR-RFLP/CAPS, and dCAPS


Common pcr based genotyping methods for snp analysis
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
Genotyping – PCR-RFLP / CAPS

[T/A] SNP

EcoRVsite: GATATC

GATATC

T/T

GATATC

GAAATC

A/A

GAAATC

GATATC

T/A

GAAATC


Genotyping pcr rflp caps1
Genotyping – PCR-RFLP / CAPS

[T/A] SNP

EcoRVsite: GATATC

GATATC

T/T

PCR primers

GATATC

GAAATC

A/A

GAAATC

L T/T A/A T/A

200

GATATC

T/A

GAAATC

100

150 bp

50 bp

50

200 bp



Genotyping dcaps
Genotyping - dCAPS site?

  • 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 dCAPS example site?

  • 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 example1
Dog SNP dCAPS example site?

  • 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 example2
Dog SNP dCAPS example site?

  • 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 example3
Dog SNP dCAPS example site?

  • 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 overlapor mutate the SNP!


Dog snp dcaps example4
Dog SNP dCAPS example site?

  • 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