FIG. 3.

PCR analysis of nascent strand length from the II/9A locus of Sciara. (A) PCR analysis of nascent strand length at locus II/9A from various developmental stages in Sciara: DNA amplification stage to form DNA puffs in the salivary gland polytene chromosomes (upper panel), preamplification stage salivary glands where endoreplication results in polytenization of the chromosomes (middle panel), and 9-h embryos where mitotic division in cycle 11 occurs (lower panel). Selected size fractions of nascent DNA were pooled and assayed by PCR. Photographs of ethidium bromide-stained agarose gels show the PCR products from nascent strands; the length of nascent DNA in each fraction is indicated. The fractions where a PCR product was obtained from the smallest nascent DNA are marked with an asterisk and were used to estimate the boundaries of the initiation zone (Fig. 2). The pale band seen with primer pair A for the 0.2- to 0.3-kb size fraction of nascent DNA from embryos most likely is due to contamination from unligated Okazaki fragments. The positions of primer sets A to E are shown on Fig. 2. The lanes marked M contain a 100-bp DNA size marker (Gibco-BRL/Life Technologies). (B) Lane 1 of each panel contains a 100-bp size marker (Gibco-BRL/Life Technologies). Lane 2 of each panel shows the efficiency of the indicated primer pair (A to E) in amplifying 20 ng of chromosomal DNA under the same experimental conditions used above in panel A. The size of the PCR product from each primer set is indicated.