David M. Francis, The Ohio State University
Current methods used to assay DNA sequence variation (SNPs) include: conversion to size polymorphism (cleaved amplified polymorphism—CAP) followed by detection using agarose gel electrophoresis, polyacrylamide gel electrophoresis, or capillary electrophoresis. Techniques to directly assay sequence variation include allele-specific amplification, allele specific primer extension (ASPE), and real time PCR. This page focuses on converting SNPs to cleaved amplified polymorphism markers (CAP) detected as size polymorphisms.
Conversion to size polymorphism
One means of detecting a DNA polymorphism (SNP) is to convert it into a size polymorphism. This can be done through digestion with a restriction endonuclease (Fig. 1). Restriction endonucleases (RE) are proteins that recognize a specific sequence of DNA, and cleave at or near that sequence. These enzymes are part of the bacterial defense system, serving to protect it from external DNA such as that which might arise from viral infection. Thus, identifying differences between PCR amplified fragments from two different varieties can be visualized as a fragment difference using gel electrophoresis after digestion with an RE that cuts the amplicon from one variety but not the second variety.
Figure 1. Restriction endonuclease site maps of Red Setter (A) and VRT-32-1 (B). The recognition site for PshAI (GACNNNNGTC, where N can be any nucleotide) is created by the A/G mutation. PshAI will cut the VRT-32-1 sequence but not the Red Setter sequence. Figure credit: David Francis, The Ohio State University.
For example, the A/G SNP in the PSY1 gene from tomato can be detected between Red Setter and VRT-32-1 by digestion with PshA1 (Fig. 1). Amplification of PSY1 DNA from Red Setter followed by digestion with PshAI will result in a single band, whereas VRT-32-1 amplification and digestion would result in two bands when assayed using agarose gel electrophoresis, polyacrylamide gel electrophoresis, or capillary electrophoresis.
Development of this page was supported in part by the National Institute of Food and Agriculture (NIFA) Solanaceae Coordinated Agricultural Project, agreement 2009-85606-05673, administered by Michigan State University. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the United States Department of Agriculture.