The Targeted Selected Cloning technique is an extremely simple mechanism for cloning specific genomic or cDNA fragments using only a single oligonucleotide primer in a single thermal replication cycle. Uniprime Corporation wishes to develop this method for commercial applications. The steps for Targeted Selected Cloning of a sequence of interest are summarized below: l - Prepare double-stranded phagemid DNA; 2 - Cut the double-stranded target DNA at one or more unique restriction sites; 3 - Ligate the phagemid DNA to genomic or cDNA cut with restriction enzymes yielding compatible ends to the restricted phagemid DNA; 4 - Transform the resulting library into a suitable strain of E. coli; 5 - Infect with helper phage to recover the library as a single-stranded form; 6 - Anneal a primer specific for a sequence of interest; 7 - Replicate the annealed templates by extending from the primer to form a double-stranded DNA; 8 - Selectively degrade single-stranded DNAs and transform the remaining double-stranded DNA into E. coli. The goals of this proposal are to: l - Determine optimum conditions to elongate primed single-stranded templates; 2 - Determine methods which select for desired duplexes and against transformation by single-stranded templates; 3 - Develop protocols for cloning specific fragments from genomic (or cDNA) restriction fragments.
