High Throughput Cloning of Open Reading Frames

HIGH-THROUGHPUT CLONING OF OPEN READING FRAMES 

As discussed above, alternative recombinant DNA techniques are necessary to efficiently generate genome-scale clone sets. One alternative exploits the ability of the Vaccinia virus DNA topoisomerase I to both cleave and rejoin DNA strands with high sequence specificity (Shuman, 1992a Shuman, 1992b). In the reaction, the enzyme recognizes the sequence 5 -CCCTT and cleaves at the final T whereby a covalent adduct is formed between the 3 phosphate of the cleaved strand and a tyrosine residue in the enzyme (Fig. 4.1). The covalent complex can combine with a heterologous acceptor DNA that has a 5 hydroxyl tail complementary to the sequence on the covalent adduct to create a recombinant molecule (Shuman, 1994). 

An alternative to repeated cloning of PCR products is a recombination-based approach developed by Liu et al. (1998) to permit the cloning of a PCR product into a plasmid and the rapid conversion of the plasmid to a number of different expression systems without the necessity of cloning the PCR product multiple, independent times. The method, termed the univector plasmid-fusion system (UPS), involves the insertion of the PCR product into a particular type of plasmid, called the univector, which can then be placed under the control of a variety of promoters or fused in-frame to various tag sequences. The system is based upon plasmid fusion using the Cre-lox site-specific recombination system of bacteriophage PI (Sternberg et al., 1981). The Cre enzyme is a site-specific recombinase that catalyzes recombination between two 34 base pair (bp) loxP sequences and is involved in the resolution of dimers formed during replication of the 

The topoisomerase and Cre-fox-based cloning systems described above may not be ideal for all experimental situations. However, alternative 

The reaction differs from excision of the X chromosome because the Entry Clone contains two attL sites and the destination vector contains two attR sites (Hartley et al., 2000). The att sites are mutated to ensure recombination only occurs between attLl and attRl and between attL2 and att.R2. The recombination reaction proceeds through a cointegrate molecule that is resolved to create a destination vector containing the gene of interest with the desired promoter and tag sequences (Fig. 4.6). 

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