PCR in situ: aspects which reduce amplification and generate false-positive results.

The Histochemical Journal Pub Date : 1995-09-01
I A Teo, S Shaunak
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Abstract

PCR in situ promises the ability to amplify and detect very low levels of target nucleic acid in tissues. Despite considerable effort, the technique is still technically difficult and has not yet proved to be reliable or reproducible. We have now identified a number of factors which can contribute to the poor amplification of the target DNA and to the generation of false-positive signals. These factors include the effects of fixation, reagent abstraction, DNA degradation, DNA end-labelling and product diffusion. We present evidence to show that formaldehyde fixation cross-links histones to DNA and thus restricts the subsequent amplification of target sequences by PCR. End-labelling of DNA occurs when direct incorporation is used to detect amplified products and this gives rise to false-positive signals. Amplified products can also diffuse out of cells and into neighbouring cells which do not contain target sequences. They can undergo re-amplification within these cells giving rise to false-positive signals. We believe considerable caution should be exercised in the interpretation of results generated using PCR in situ.

原位PCR:减少扩增和产生假阳性结果的方面。
原位PCR保证了在组织中扩增和检测极低水平靶核酸的能力。尽管付出了相当大的努力,但这项技术在技术上仍然困难重重,而且尚未被证明是可靠的或可重复的。我们现在已经确定了一些可能导致目标DNA扩增不良和产生假阳性信号的因素。这些因素包括固定、试剂提取、DNA降解、DNA末端标记和产物扩散的影响。我们提出的证据表明,甲醛固定交联组蛋白DNA,从而限制了随后的扩增目标序列的PCR。当使用直接掺入检测扩增产物时,会发生DNA末端标记,这会产生假阳性信号。扩增产物也可以扩散出细胞并进入不含靶序列的邻近细胞。它们可以在这些细胞内进行再扩增,从而产生假阳性信号。我们认为,在解释原位PCR产生的结果时应相当谨慎。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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