Preparation of leading and lagging strand templates, p-t junctions and miniforks. The DNA fragments in the 100 base pair (bp) ladder are 100, 200, 300, 400, 500/517, 600, 700, 800, 900, and 1000 pbs. (a) The agarose gels stained by ethidium bromide (EtBr) show the products of the PCR obtained with the four plasmids (p-Empty, p-5′GTT16, p-5′CTG17, and p-5′CAG23) and the oligonucleotide couple specific of the leading (“PCR leading”; left) or the lagging (“PCR lagging”; right) strand. The name of the plasmids used for the PCR is indicated at the top of the gels. (b) The agarose gels stained by EtBr show the products of the T7 exonuclease digestion. The PCR products obtained with p-5′CTG17 (CTG17) and p-5′CAG23 (CAG23) and the oligonucleotide couple specific of the leading (“PCR leading”; left) or the lagging (“PCR lagging”; right) strand were treated (+) or not (−) by T7 exonuclease. After treatment with T7 exonuclease, the appearance of a DNA band with a slower electrophoretic migration and a weaker intensity (indicated by a backward arrow) than the ds DNA is indicative of ss DNA production. (c) The ss leading strand templates containing either 17 repeats of CTG (CTG17) or 23 repeats of (CAG23) are mixed with increasing amounts of radiolabelled p821 (p821*) to generate the p-t junctions. Species are resolved on a native gel. Free p821 migrates faster than the p-t junctions. (d) The p-t junctions containing 17 repeats of CTG (CTG17) or 23 repeats of (CAG23) on their leading strand are mixed with increasing amounts of ss lagging strand template to assemble the miniforks. Species are resolved on a native gel. The miniforks migrate more slowly than the p-t junctions.