The Targeted Selected Cloning technique is an extremely simple mechanism for cloning specific genomic or cDNA fragments by 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: I - 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 are to I - 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.Awardee's statement of the potential commercial applications of the research: Potential commercial applications exist in molecular biology research laboratories for using our reagent kits. Other applications include uses in medical diagnostic tests for genetic disease, cancer and infectious disease.National Institute of General Medical Sciences (NIGMS)
