Pyrophosphorolysis activated polymerization (PAP) offers a novel approach for retrieving multiple types of information from nucleic acids. The exceptional specificity of PAP derives from an inactive dideoxy terminated oligonucleotide (P*). P* is activated by pyrophosphorolysis of the 3? terminal nucleotide, followed by extension of the activated oligonucleotide by DNA polymerization. The initial studies established proof of principle and recently the efficiency of PAP was improved greatly with genetically engineered polymerases that have a high affinity for dideoxy nucleotides. Herein, we propose to demonstrate that PAP is a platform technology. Three novel methods will be developed on the platform: i) allele-specific detection of a single nucleotide change in the presence of 10expl6-10exp9 normal alleles (PAP-A): ii) microarray-based resequencing (PAP-R) and iii) analysis of in vivo chromatin structure (LM-PAP). These methods have many applications including: i) ultra sensitive detection of minimal residual disease or measurement of mutation load (PAP-A); ii) molecular epidemiological analysis of sequence variants that predispose to complex disease or rapid molecular diagnosis (PAP-R); and iii) analysis of chromatin structure as a function of imprinting, X-inactivation and gene expression or quantitation of the level of methylation (LM-PAP).
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