Figure 2

Generation of shRNA constructs using YIU. (A) Intermediate products produced during the YIU procedure. Lanes 2 and 3. The Y oligonucleotide, designed with a single 3' T overhang and therefore unable to self-ligate. Lane 2, without ligase. Lane 3, with ligase. Note that the forked shape of the Y oligonucleotide causes abnormal mobility on PAGE. The ligated and MmeI-digested YI molecules are indicated by an arrowhead in lane 4; the digested YI molecules shift in migration when ligated to the U oligonucleotide (arrowhead, lane 5). Note that the YIU molecules are a minor fraction of the product molecules because the Y and U oligonucleotides were added at a large molar excess over cDNAs and that the ligation was efficient, as almost all of the U oligonucleotides have been converted to dimers. (B) PCR amplification of YIU ligation products. The template used for PCR in lane 2 was the PAGE-purified DNA band corresponding to the one labeled with arrowhead in lane 5 of Panel A. The template for lane 3 was the whole YIU ligation mixture shown in lane 5 of Panel A. The calculated size of YIU double-stranded DNA is 160 bp. (C) Conversion of the X molecule into double-stranded DNA by a "PCR+1" protocol. PCR amplification of YIU with 20 cycles results in an extra band (the X molecule) seen in the 200–300 bp region on agarose gels (lane 2). However, when the PCR products are diluted with fresh PCR reaction mix (1 × buffer, dNTPs, primers and enzyme) and subjected to one additional PCR cycle, the high molecular weight band is lost (lane 3). (D) Scheme of the generation of X molecules. Vent polymerase, through its strand-displacement activity, can open hairpin structures to generate fully double-stranded products. As the PCR progresses, however, YIU double-stranded DNA molecules accumulate and the concentration of primers decreases. When the templates are heat denatured and cooled, two heterogeneous molecules (shown in red or purple) can form an X-shaped (cruciform) heterodimer via the common complementary regions (shown in green). (E) Restriction digestion of double-stranded DNA of YIU. The YIU double-stranded DNA generated by PCR (the "uncut" lane) is digested with AflII or MlyI individually, or in combination to generate suitable inserts for cloning.