pcr based genotyping methods
Download
Skip this Video
Download Presentation
PCR-Based Genotyping Methods

Loading in 2 Seconds...

play fullscreen
1 / 11

PCR-Based Genotyping Methods - PowerPoint PPT Presentation


  • 98 Views
  • Uploaded on

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

loader
I am the owner, or an agent authorized to act on behalf of the owner, of the copyrighted work described.
capcha
Download Presentation

PowerPoint Slideshow about ' PCR-Based Genotyping Methods' - tatiana-stanley


An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.


- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -
Presentation Transcript
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
  • 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
  • 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
  • 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
  • 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
  • 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
  • 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

ad