ERROR-FREE DNA AMPLIFICATION FOR MUTATION DETECTION

描述（由申请人提供）： 经常依赖于PCR的高选择性突变检测通常比在早期阶段鉴定癌细胞、研究肿瘤发生机制、检测单细胞中的突变或可靠地检测最小残留疾病所需的选择性低福尔斯1-2个数量级。这种缺陷的主要原因是PCR造成选择性限制，通常在105- 106个野生型等位基因中有1个突变体，因为所有DNA聚合酶在DNA合成期间总是产生错误，这可能被误解为突变（假阳性）。 我们现在已经开发了发夹PCR，这是一种新的方法，可以消除或大幅减少（>100倍）感兴趣的序列中的PCR错误，并可以为现有的突变检测技术提供必要的“选择性飞跃”。通过将DNA序列转化为发夹并在发夹结构中进行PCR，可以将真正的突变与聚合酶产生的错配分离，从而提供几乎无错误的DNA。该项目将开发和优化从基因组DNA中PCR扩增序列的技术，消除含有错误的序列，并展示其在使用以前受PCR错误限制的技术进行高度改进的突变检测中的应用。在R21阶段，我们将证明直接从人类基因组DNA扩增长达500 bp的发夹，我们将使用dHPLC分离优化含有真正突变的发夹（同源双链发夹）与含有聚合酶错误的发夹（异源双链发夹）的分离。在R33阶段，将进一步开发该技术，以（a）将PCR错误减少至少1000倍，（B）证明发夹PCR允许当前突变检测方法相对于其当前限制的主要选择性飞跃，以及（c）以无错误的方式扩增大部分人类基因组，用于多基因突变筛查。通过提供无错误的扩增DNA用于分析，本发明的发夹PCR将允许对几乎每一种现有的基因型选择方法进行重大推动，并使先前技术不可能进行的研究和诊断测试成为可能。这项新技术还将提高微卫星分析的准确性，并将在分子信标和真实的时间聚合酶链反应以及通过体外翻译进行DNA克隆或蛋白质功能分析方面有更多的应用。

DESCRIPTION (provided by applicant): 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.