Research Project
3499150
In this project we will develop kits, services, and reagents useful in the processes of generating and employing amplified DNA mini-libraries (i.e., amplified products) from microdissected human chromosome fragments. The focus of Phase I studies will be development of standard protocols for generating such mini-libraries. Two amplification strategies are proposed based on their use of recognition sites distributed throughout the human genome at about 250 bp intervals (insuring representative coverage of genomic sequences in the min- library). The strategies are based on DNA sequence recognition by Mbo I cleavage and by oligonucleotide hybridization to the most common 6 bp sequences in the human genome. DNA products obtained from both strategies are PCR amplified with a single adaptor oligonucleotide. The products of the amplification reactions will be analyzed for the amount and size of DNA, degree of genome coverage, hybridization specificity, retrievability upon reamplification, ease of use and reproducibility. Based on amplification strategies developed in Phase I, we will ultimately make available to the human genom research community standardized reagents that can be used: a) for obtaining region-specific clones (i.e., genomic clones for contig assembly/map closure, and/or cDNA clones to identify novel genes), b) as starting material for isolation of region-specific STSs, and c) as hybridization/amplification targets for subchromosome localization.
