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通过聚合酶链式反应(PCR)进行的微卫星(SSR)扩增通常会产生多态性条带:这表明在延伸的或新生的DNA链中发生了复制滑移。

Microsatellite (SSR) amplification by PCR usually led to polymorphic bands: Evidence which shows replication slippage occurs in extend or nascent DNA strands.

作者信息

Hosseinzadeh-Colagar Abasalt, Haghighatnia Mohammad Javad, Amiri Zahra, Mohadjerani Maryam, Tafrihi Majid

机构信息

Department of Molecular and Cell Biology, Faculty of Basic Sciences, University of Mazandaran, Babolsar, Mazandaran, Iran.

出版信息

Mol Biol Res Commun. 2016 Sep;5(3):167-174.

Abstract

Microsatellites or simple sequence repeats (SSRs) are very effective molecular markers in population genetics, genome mapping, taxonomic study and other large-scale studies. Variation in number of tandem repeats within microsatellite refers to simple sequence length polymorphism (SSLP); but there are a few studies that are showed SSRs replication slippage may be occurred during amplification which are produced 'stutter products' differing in length from the main products. The purpose of this study is introducing a reliable method to realize SSRs replication slippage. At first, three unique primers designed to amplify SSRs loci in the great gerbil () by PCR. Crush and soak method used to isolate interesting DNA bands from polyacrylamide gel. PCR products analyzed using by sequencing methods. Our study has been shown that DNA polymerase slipped during microsatellite amplification which led to insertion or deletion of repeats in sense or antisense DNA strands. It is produced amplified fragments with various lengths in gel electrophoresis showed as 'stutter bands'. Thus, in population studies by SSRs markers recommend that replication slippage effects and stutter bands have been considered.

摘要

微卫星或简单序列重复(SSRs)是群体遗传学、基因组作图、分类学研究及其他大规模研究中非常有效的分子标记。微卫星内串联重复数目的变异指简单序列长度多态性(SSLP);但有一些研究表明,在扩增过程中可能会发生SSRs复制滑动,从而产生与主要产物长度不同的“结巴产物”。本研究的目的是引入一种可靠的方法来实现SSRs复制滑动。首先,设计了三种独特的引物,通过PCR扩增大沙鼠()中的SSRs位点。采用压碎浸泡法从聚丙烯酰胺凝胶中分离出感兴趣的DNA条带。使用测序方法分析PCR产物。我们的研究表明,DNA聚合酶在微卫星扩增过程中发生滑动,导致有义或反义DNA链中重复序列的插入或缺失。在凝胶电泳中产生了不同长度的扩增片段,表现为“结巴条带”。因此,在使用SSRs标记进行群体研究时,建议考虑复制滑动效应和结巴条带。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7f61/5219911/b31d8e1210e2/mbrc-5-167-g001.jpg

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