通过滚环扩增检测单个细胞中的DNA点突变和mRNA表达水平。
Detection of DNA point mutations and mRNA expression levels by rolling circle amplification in individual cells.
作者信息
Christian A T, Pattee M S, Attix C M, Reed B E, Sorensen K J, Tucker J D
机构信息
Biology and Biotechnology Research Program, Lawrence Livermore National Laboratory, Livermore, CA 94551, USA.
出版信息
Proc Natl Acad Sci U S A. 2001 Dec 4;98(25):14238-43. doi: 10.1073/pnas.251383598. Epub 2001 Nov 27.
Abstract
Rolling circle amplification has been useful for detecting point mutations in isolated nucleic acids, but its application in cytological preparations has been problematic. By pretreating cells with a combination of restriction enzymes and exonucleases, we demonstrate that rolling circle amplification in situ can detect gene copy number and single base mutations in fixed cells with efficiencies up to 90%. It can also detect and quantify transcribed RNA in individual cells, making it a versatile tool for cell-based assays.
摘要
滚环扩增技术在检测分离核酸中的点突变方面很有用，但它在细胞学制剂中的应用一直存在问题。通过用限制性内切酶和外切核酸酶组合对细胞进行预处理，我们证明原位滚环扩增能够以高达90%的效率检测固定细胞中的基因拷贝数和单碱基突变。它还能检测和定量单个细胞中转录的RNA，使其成为基于细胞的检测的一种通用工具。
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