 PV92 teaching experiment

The DNA Learning Center's Detection of an Alu Insertion Polymorphism by PCR by DNA Learning Center, Cold Spring Harbor Laboratory supplies a concrete length-based example. It collects mouthwash biological cells, concentrates them with a microcentrifuge, heats them by Chelex extraction and transfers the DNA-containing supernatant. Its $25\mu \mathrm{L}$ reaction combines $2.5\mu \mathrm{L}$ of that preparation with primer mix and a reagent bead in a thermal cycler. Thirty cycles use $94$, $68$ and $72^{\circ }\mathrm{C}$, each for thirty seconds. An electrophoresis chamber separates products by agarose gel electrophoresis in a 2 percent gel; a UV transilluminator reveals stained DNA.^[ref]

Its PCR primers flank an insertion site at the PV92 locus. The student protocol predicts a 715-base pair product with the Alu element and a 415-base pair product without it. Two bands support a heterozygous genotype; one corresponding band supports a homozygous genotype only if amplification and detection of both alleles were adequate. These are the protocol's expected readouts, not newly collected observations.^[ref]

The instructor manual describes extra bands from nonspecific priming and a small diffuse primer dimer band. A missing target band therefore cannot on its own distinguish failed extraction, inhibited copying, poor binding or lack of the target. Nor does detection of the Alu element at this one genetic locus establish a complete genome, a unique personal identity or a clinical conclusion. The PCR controls help separate these possibilities; DNA sequencing can add sequence evidence to the size comparison.^[ref]