SITE SPECIFIC CHEMICAL METHODS FOR CLEAVING GENOMIC DNA

点击翻译按钮获取中文摘要

Physical mapping of chromosomes would be facilitated by methods of breaking large DNA into manageable fragments or cutting uniquely at genetic markers of interest. Our long-term objective is to provide chemical methods for efficient cleavage at single sites in megabase and gigabase DNA (human chromosomes). Oligonucleotide-directed triple helix formation is a versatile method for the sequence specific recognition of 15 base pairs of DNA. Due to the length of the recognition site, in a formal sense, this is one million times more sequence specific than that available with most restriction enzymes. It is important to determine whether the full potential of this cleavage specificity can be realized. Our program combines synthetic organic chemistry, biophysical methods, and molecular biology to learn about the relationship of structure to function in the area of creating reagents for recognition and modification of DNA biopolymers. Specific aims are (1) test the high yield double strand cleavage reactions at 5'-(pur)/m.(pyr)/n-3' target sequences in megabase DNA utilizing a new class of pyrimidine oligonucleotides equipped with the N-bromoacetamide at the 3' end, (2) test purine N-bromoacetamide- oligonucleotides for high yield double strand cleavage of single sites in megabase DNA and examine cleavage of DNA at an internal GC base pair utilizing a novel designed base (nebularine-N-bromoacetamide), (3) study sequence composition effects, modified bases, and mismatches at a single site in DNA by cooperatively binding oligonucleotides, (4) study the thermodynamics of oligonucleotides containing the novel base D/3 and test their use for targeting trinucleotide repeats such as (CAG)n, (5) characterize the intermediates in the joint molecule formed in a RecA- mediated strand exchange using affinity cleavage methods on a RecA nucleoprotein filament.