Comparison of germ line minisatellite mutation detection at the CEB1 locus by Southern blotting and PCR amplification.
Comparison of germ line minisatellite mutation detection at the CEB1 locus by Southern blotting and PCR amplification.
复制标题
通过 Southern blotting 和 PCR 扩增对 CEB1 基因座种系小卫星突变进行比较。
DOI：
10.1093/mutage/geq011
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发表时间：
2010
期刊：
影响因子：
2.7
通讯作者：
BoiceJr,JohnD
中科院分区：
文献类型：
--
作者：
Taylor,Malcolm;Cieslak,Marcin;Rees,GwenS;Oojageer,Anthony;Leith,Cheryl;Bristow,Claire;Tawn,EJanet;Winther,JeanetteF;BoiceJr,JohnD
在暴露于诱变剂的父母的后代中鉴定新生小卫星突变提供了电离辐射和遗传毒性化学品诱导的生殖系遗传事件的潜在敏感措施。生殖系小卫星突变（GMM）通常通过将未扩增的大小分级的基因组DNA与小卫星探针杂交来检测。然而，这消耗相对大量的DNA，需要几个步骤，并且可能缺乏灵敏度。我们已经开发了一种基于聚合酶链反应（PCR）的GMM检测，我们应用于超变小卫星，CEB 1。在这里，我们比较了这种检测的灵敏度和特异性与传统的Southern杂交方法使用的DNA从10个配偶对，每对的父母是一个幸存者的癌症在儿童时期，和他们的20个后代。我们报告说，这两种方法具有相似的特异性，但PCR方法使用的DNA少250倍，具有更少的步骤，更好地检测GMM与单重复，提供了等位基因大小的具体指导方针。PCR-GMM方法更容易应用于后代DNA样本量有限的家庭。
Identification ofde novominisatellite mutations in the offspring of parents exposed to mutagenic agents offers a potentially sensitive measure of germ line genetic events induced by ionizing radiation and genotoxic chemicals. Germ line minisatellite mutations (GMM) are usually detected by hybridizing Southern blots of unamplified size-fractionated genomic DNA with minisatellite probes. However, this consumes a relatively large amount of DNA, requires several steps and may lack sensitivity. We have developed a polymerase chain reaction (PCR)-based GMM assay, which we applied to the hypermutable minisatellite, CEB1. Here, we compare the sensitivity and specificity of this assay with the conventional Southern hybridization method using DNA from 10 spouse pairs, one parent of each pair being a survivor of cancer in childhood, and their 20 offspring. We report that both methods have similar specificity but that the PCR method uses 250 times less DNA, has fewer steps and is better at detecting GMM with single repeats provided that specific guidelines for allele sizing are followed. The PCR GMM method is easier to apply to families where the amount of offspring DNA sample is limited.