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Molecular & Cellular Analysis Technologies
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High selectivity mutation detection, which frequently relies on PCR, often falls short by 1-2 orders of magnitude of the selectivity required to identify cancer cells at an early stage, to investigate mechanisms of tumorigenesis, to detect mutations in single cells or to reliably detect minimal residual disease. A major cause of this deficiency is that PCR poses a selectivity limit, typically 1 mutant in 105- 106 wild type alleles, since all DNA polymerases invariably generate errors during DNA synthesis which can be misinterpreted as mutations (false positives). We have now developed hairpin-PCR, a novel method that allows elimination or major reduction (>100-fold) of PCR errors in a sequence of interest, and can supply existing mutation detection technologies with the necessary 'selectivity leap'. By converting a DNA sequence to a hairpin and performing PCR in a hairpin structure, true mutations can be separated from polymerase-generated misincorporations, thereby providing practically error-free DNA. This project will develop and optimize the technology to PCR amplify sequences from genomic DNA, eliminate error-containing sequences, and demonstrate its application for highly improved mutation detection using technologies previously limited by PCR errors. In the R21 phase we shall demonstrate amplification of hairpins up to 500 bp long directly from human genomic DNA, and we shall optimize isolation of hairpins containing genuine mutations (homoduplex hairpins) from hairpins containing polymerase errors (heteroduplex hairpins), using dHPLC separation. In the R33 phase the technology will be further developed to (a) reduce PCR errors by at least 1000 times, (b) demonstrate that hairpin PCR allows current mutation detection methods a major selectivity leap relative to their current limit, and (c) amplify large portions of the human genome in an error-free manner, for multi-gene mutation screening. By providing error-free amplified DNA for analysis, the present hairpin PCR will allow a major boost to almost every existing genotypic selection method and enable studies and diagnostic tests that were not possible with previous technology. The new technology will also improve the accuracy of microsatellite analysis and will have additional applications in molecular beacons and real time PCR, and in DNA cloning or protein functional analysis by in vitro translation.