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picture1_Rflp Slideshare 66903 | Hultman And Mellgren 2014 Fetching Snps Supplemental 2   Dcaps Genotyping Slides


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File: Rflp Slideshare 66903 | Hultman And Mellgren 2014 Fetching Snps Supplemental 2 Dcaps Genotyping Slides
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 ...

icon picture PPTX Filetype Power Point PPTX | Posted on 28 Aug 2022 | 3 years ago
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      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      T/T   A/A    T/A
                         GATATC                                                        200
   T/A                   GAAATC
                                                                                       100
                   50 bp                           150 bp                                50
                                      200 bp
     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
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...Common pcr based genotyping methods for snp analysis snps can have up to alleles a c g t but two are most these only positively detect one allele rflp caps polymerase chain reaction restriction fragment length polymorphisms also called cleaved amplified polymorphic sequences single nucleotide polymorphism where creates or removes naturally occurring site amplifying the sequence surrounding from individuals cutting products with enzyme and resolving on gel will reveal which an individual carries dcaps derived that do not create natural uses mismatches in primer remove ecorv gatatc gaaatc primers l bp how you genotype does make mismatched of advantages be used when marker change using expensive rare cheap disadvantages lowers specificity laborious compared hybridization gene chip detection finding right use web http helix wustl edu html find...

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