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A) Schematic depiction of primers designed to anneal to 20-nt fixed sequences flanking template random region and indicating addition of 5′ overhangs to lengthen PCR products. B) qPCR profiles for various primers (20-nt, no overhang; 27-nt, 7-nt overhang; 35 nt, 15-nt overhang) with a 20(N40)20 template. C) Moderate qPCR profile improvement for 27-nt primer pairs with (Tm, forward/Tm, reverse) indicated in °C.
