Plasmid, direct sequencing

The exception to limited technical coverage concerns the trend toward direct sequencing of plasmids and polymerase chain reaction (PCR) products. This is a reflection of the increasing need for ease and rapidity in sequence determination. However, many published protocols lack consistency. As discussed at greater length below, there are few or no (as yet) tried-and-true favorites. Thus, the techniques presented in this chapter comprise detailed protocols for only one fairly robust rapid isolation and... 

Figure 2 Comparison of cloning and expression methods. In the conventional strategy (left), dehydrogenase genes obtained by PCR amplification of the original source DNAs are cloned into overexpression plasmids and verified by sequencing. Those with the desired structure are individually transformed into suitable host strains and the proteins are obtained, either as crude extracts or as purified samples. In the proposed streamlined approach (right), full-length dehydrogenase genes obtained by chemical synthesis are used directly in coupled transcription/translation reactions to obtain the proteins of interest.

Figure 4.4. Schematic illustration of directional topoisomerase cloning of PCR products into the pUNI vector. The PCR product to be cloned has the sequence 5 -CACC appended at the 5 end to direct the orientation of cloning. The Vaccinia virus topoisomerase I enzyme forms a covalent adduct with the cloning vector to create a cloning competent plasmid construct. The loxP site is 5 to the insertion site. The vector and PCR product are designed to fuse the ORF in-frame with loxP.

Figure 2.3 Metagenomic cloning experiments. Isolation of genomic DNA directly from environments (soil, plants, mixed environments or thermal-vent worms are the examples Illustrated here) can recover DNA fragments which could encode for enzymes. The DNA fragments can be ligated to plasmids or DNA linkers, and then subjected to functional screening (expression cloning) and/or sequence analysis. Amplification by PCR can sometimes be used to yield libraries enriched with clones containing selected sequence motifs relating to families of enzymes...

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