原位聚合酶链反应:一项新兴技术的评价。

The Histochemical Journal Pub Date : 1995-09-01
I A Teo, S Shaunak
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引用次数: 0

摘要

原位聚合酶链反应(PCR)是一种新技术,有望大大提高我们在固定组织和细胞中检测目标核酸序列的能力。它在病毒性疾病的组织病理学诊断和基因表达研究方面具有巨大的应用潜力。然而,原位PCR在技术上是困难的,目标DNA的扩增只有30-300倍。在这篇文章中,我们介绍了用于扩增DNA和RNA靶标的PCR原位技术(RT-PCR原位)的概述。我们还确定了可能降低技术效率或可能导致假阳性结果的问题。它们包括(1)组蛋白与DNA交联或PCR扩增的抑制作用,(2)用于涂覆玻片的组织结合剂提取PCR试剂,(3)由于组蛋白与DNA的广泛交联,或由于热循环器的温度调节不正确,导致靶DNA变性和随后的DNA变性不佳,(4)Taq聚合酶对DNA链断裂的末端标记引起的假阳性结果。(5) PCR产物在细胞内外扩散导致假阳性结果。我们提出了一些已经被用来克服这些困难的方法,并提出了进一步改进这项技术的新研究途径。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Polymerase chain reaction in situ: an appraisal of an emerging technique.

Polymerase chain reaction (PCR) in situ is a new technique which promises to enhance considerably our ability to detect a few copies of target nucleic acid sequences in fixed tissues and cells. It has an enormous potential for application in diagnostic histopathology of viral diseases and in the study of gene expression. PCR in situ is, however, technically difficult, and amplification of the target DNA is only 30-300 fold. In this article we present an overview of PCR in situ techniques used to amplify both DNA and RNA targets (RT-PCR in situ). We also identify problems which can reduce the efficiency of the technique or which can give rise to false-positive results. They include (1) the inhibitory effects of cross-linking of histones to DNA or PCR amplification, (2) abstraction of PCR reagents by tissue-bonding agents which are used to coat glass slides, (3) poor denaturation of target DNA and subsequent DNA renaturation due to extensive cross-linking of histones to DNA, or because of incorrect temperature regulation of thermal cyclers, (4) false-positive results which arise from end-labelling of DNA strand breaks by Taq polymerase, and (5) diffusion of PCR products into and out of cells leading to false-positive results. We present some of the approaches that have been used to overcome some of these difficulties and suggest new avenues for investigation to improve this technique further.

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