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?
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       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.
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