Generation of cohesive ends on PCR products by UDG-mediated excision of dU, and application for cloning into restriction digest-linearized vectors.

Abstract

We have Investigated the use of dU excision by uracil N-glycosylase (UDG) to create cohesive ends on PCR fragments "mimicking" those generated by restriction enzymes. The feasibility of this approach for directional and nondirectional cloning using cohesive ends mimicking Sacl or Pstl ends is demonstrated by the subcloning of a 383 to 388-bp fragment of bovine basic flbroblast growth factor into restriction enzyme-linearized pTTT318U. UDG-mediated cohesive ends imperfectly matched to Pstl-generated vector ends gave reasonable cloning efficiency and accuracy, suggesting that the approach may be extended to mimicry of other restriction enzymes producing 3' overhangs. The rapid and specific excision of dU by UDG (within 30 rain at 37~ has several potential advantages over the use of restriction site-modified primers, including the avoidance of restriction cleavage at internal sites within the PCR product. Also, following ligation, the approach described may be used to prevent subsequent cleavage of the joined DNA segments by the restriction enzyme, that is, by not recreating the restriction enzyme recognition sequence at the junction, which may find application in gene engineering. By adapting the approach to use dU-containing linkers or "vectorettes," the approach may be used for cloning unknown sequences (e.g., by cDNA or genomic library construction) or for mimicking S' overhang cohesive ends on PCR fragments.

Air Pollution Particles Induce IL-6 Gene Expression in Human Airway Epithelial Cells via NF- κ B Activation

“…that some particulate air pollutants also trigger an inflammatory response in the lung (13,15). We have demonstrated that NF-B, a common transcriptional activator of several inflammatory genes (26,27), regulates IL-6 transcription in a lung epithelial cell line exposed to ROFA. IL-6 is a multifunctional cytokine activated early in the inflammatory response, chronically elevated in association with several environmentally induced respiratory diseases, and released in vitro by lung epithelial cells after ROFA exposure.…”

“…Transcriptional activation of specific inflammatory genes such as IL-1, IL-6, IL-8, and TNF is mediated by NF-B in various cell types under a wide range of conditions (26,27). In lung epithelial cells, nuclear translocation of NF-B has been stimulated with IL-1, TNF (28, 29), phorbol esters (28,30), and asbestos (31).…”

“…Point mutations in the NF-B binding motif were made using a modification of the PCR-uracil DNA-glycosylase (UDG) method (26,27). Briefly, PCR primers spanning the mutation site were synthesized which replaced relevant bases with point mutations and with deoxyuracil-CE phosphoramidite in place of the thymidine-CE phosphoramidite (Cruachem, Sterling, VA).…”

Inefficient excision of uracil from loop regions of DNA oligomers byE.coliuracil DNA glycosylase

“…Earlier we used single-stranded DNA oligomers of different sizes containing dUMP in varying positions and showed that E.coli UNG excises even those uracil residues located near the ends of the single-stranded DNA (9). Such studies are important not only in considering the details of the mechanism of enzyme action but also in increasing the usefulness of UNG in DNA research (10)(11)(12)(13)(14)(15)(16).…”

Uracil Excision for Assembly of Complex Pathways

“…As an example, uracil excision DNA assembly makes use of oligonucleotides where selected thymines are replaced by uracils. This is a non-mutagenic and PCRtolerated replacement, as the uracil is able to form base pairs with adenine nucleotides on the complementary strand [2][3][4]. Following PCR, the uracils are selectively removed by treatment with uracil DNA glycosidase, leaving a chemically unstable phosphoribose backbone.…”