Minisequencing, sometimes referred to as SNaPshot, involves allele-specific primer extension with fluorescent dye-labeled dideoxynucleotide triphosphates (ddNTPs) to help visualize the results. There are three primary steps in performing minisequencing: amplification, primer extension, and analysis (Figure 8.1). First, the region around each SNP locus is amplified using PCR. Amplicons can be pooled following singleplex PCR or simultaneously generated using multiplex PCR. The remaining dNTPs and primers following PCR are destroyed by simply adding two different enzymes to the initial reaction tube or well. Exonuclease (Exo) chews up the single-stranded primers while shrimp alkaline phosphatase (SAP) destroys the dNTP building blocks. These enzymes are often sold together as 'ExoSAP'. It is necessary to remove the primers and dNTPs so they do not interfere with the subsequent primer extension reaction.
Primer extension is performed by adding SNP extension primers, a mixture of the four possible ddNTPs each with a unique fluorescent dye label, and a polymerase to the ExoSAP-treated PCR products. The SNaPshot 'kit' from Applied Biosystems only supplies the fluorescently labeled ddNTPs, buffer, and polymerase making it generic to any primer set. The SNP extension primers are designed to anneal immediately adjacent to a SNP site so that the addition of a single ddNTP will interrogate the nucleotide present at the SNP site in the PCR product. The SNP extension reaction is heated and cooled, usually through 25 cycles on a thermal cycler, to permit a linear amplification of the fluorescent ddNTP addition to the SNP primer by the polymerase. If any dNTPs remain from the preceding PCR reaction, then extension can go beyond the single base. Likewise, the presence of remaining PCR primers could mean that
Protocol Steps for Allele-Specific Primer Extension SNP Assay
Genomic _^ (Multiplex)
Amplification DNA sample PCR
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