Alu element

Chelex extraction

Microcentrifuge

PV92 locus

{c}

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 $25\,\mathrm{\mu L}$ reaction combines $2.5\,\mathrm{\mu L}$ 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>.<Detection of an Alu Insertion Polymorphism by PCR by DNA Learning Center, Cold Spring Harbor Laboratory>[pp. 2–3, Procedures I–III: extraction, 25 microlitre reaction, cycle profile, gel and UV readout]{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.<Detection of an Alu Insertion Polymorphism by PCR by DNA Learning Center, Cold Spring Harbor Laboratory>[p. 1, Introduction: primer-flanked insertion and stated 715/415 bp genotype patterns]{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.<Using an Alu Insertion Polymorphism to Study Human Populations by Dolan DNA Learning Center, Cold Spring Harbor Laboratory>[printed pp. 18 and 29, Results and Discussion and Amplify DNA by PCR: primer dimer and nonspecific priming]{ref}


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 Incoming links: PV92 teaching experiment