FIG. 1.

Constructs and ligation conditions. (A) An intact U2 repeat unit and the five fragments used to construct artificial tandem arrays. Key restriction sites are shown (Bg, BglII; K, KpnI; Ea, EagI; A, AseI; N, NdeI; Hc, HincII; Sm, SmaI; D, DraI; RI, EcoRI; Bm, BamHI; Sf, SfuI; F, FokI). The 5′ and 3′ ends of each construct are BglII and BamHI, respectively. Sites in parentheses were destroyed by ligation. Open arrow, U2 snRNA gene; asterisk, U87C mutation; open circle and square, DSE and PSE, respectively; hatched rectangle, d(CT)_n · d(GA)_n or CT microsatellite, where n ≈ 75; shaded rectangle, truncated L1 repeat. LTR, long terminal repeat. (B) Representative ligation reactions. The mU2 and mU2+CT constructs were subjected to the two-step ligation as described in Materials and Methods, and the products were separated by agarose FIGE under conditions that resolve both large and small fragments. FIGE gels were then blotted and probed with the labeled mU2+CT construct. Either 1.0 (left) or 0.02 (right) μg of each ligation reaction was loaded per lane. As an internal control, 100 ng of the mU2 fragment was loaded to the right of the mU2 lane in each of these panels. The residual monomer and dimer are indicated; these species account for <30% of the input fragments compared to DNA mass markers (data not shown). Size markers are shown in kilobases (center).