Automated purification of dye terminator sequencing reactions: an approach to high-throughput capillary electrophoresis sequencing of large templates

Gernon, Amy; Woldu, Ermias; Godlevski, M M; Wilson, Willie; Gilmore, Rodney C.; Grant, Delores J.; Chatterjee, Pranam; Kephart, Dan

doi:10.1016/s1535-5535(03)00006-6

Cited by 2 publications

(1 citation statement)

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“…The DNA of 20 clones from each deletion series was sequenced directly using a transposon-end primer (19) and Big Dye Terminator chemistry on an ABI-3100 AVANT genetic analyzer. The primer extended products were purified using Magnesil (Promega Corporation) according to the manufacturer's procedures as described previously (27). New primers used to sequence the newly created ends of deletions from the mutant loxP side of insert DNA already trimmed from the wild-type loxP-end are listed below:…”

Section: End-sequencing Of Pac Deletion Clonesmentioning

confidence: 99%

Mutually exclusive recombination of wild-type and mutant loxP sites in vivo facilitates transposon-mediated deletions from both ends of genomic DNA in PACs

Chatterjee

Shakes²,

Srivastava³

et al. 2004

Nucleic Acids Research

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Recombination of wild-type and mutant loxP sites mediated by wild-type Cre protein was analyzed in vivo using a sensitive phage P1 transduction assay. Contrary to some earlier reports, recombination between loxP sites was found to be highly specific: a loxP site recombined in vivo only with another of identical sequence, with no crossover recombination either between a wild-type and mutant site; or between two different mutant sites tested. Mutant loxP sites of identical sequence recombined as efficiently as wild-type. The highly specific and efficient recombination of mutant loxP sites in vivo helped in developing a procedure to progressively truncate DNA from either end of large genomic inserts in P1-derived artificial chromosomes (PACs) using transposons that carry either a wild-type or mutant loxP sequence. PAC libraries of human DNA were constructed with inserts flanked by a wild-type and one of the two mutant loxP sites, and deletions from both ends generated in clones using newly constructed wild-type and mutant loxP transposons. Analysis of the results provides new insight into the very large co-integrates formed during P1 transduction of plasmids with loxP sites: a model with tri- and possibly multimeric co-integrates comprising the PAC plasmid, phage DNA, and transposon plasmid(s) as intermediates in the cell appears best to fit the data. The ability to truncate a large piece of DNA from both ends is likely to facilitate functionally mapping gene boundaries more efficiently, and make available precisely trimmed genes in their chromosomal contexts for therapeutic applications.

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Section: End-sequencing Of Pac Deletion Clonesmentioning

confidence: 99%