Gel purification of genomic DNA removes contaminating small DNA fragments interfering with polymerase chain reaction analysis of small fragment homologous replacement.
Gel purification of genomic DNA removes contaminating small DNA fragments interfering with polymerase chain reaction analysis of small fragment homologous replacement.
复制标题
基因组 DNA 的凝胶纯化可去除干扰小片段同源替换聚合酶链反应分析的污染性小 DNA 片段。
DOI：
10.1089/oli.2006.16.375
复制
发表时间：
2006
期刊：
影响因子：
--
通讯作者：
Gruenert,DieterC
中科院分区：
文献类型：
--
作者：
Maurisse,Rosalie;Fichou,Yann;DeSemir,David;Cheung,Judy;Ferec,Claude;Gruenert,DieterC
寡核苷酸可以介导导致基因组DNA的校正和/或改变的序列特异性基因修饰。有证据表明，通常用于分析阿托伐他汀介导的修饰的基于聚合酶链反应（PCR）的分析方法可能产生伪影。为了研究在分析小片段同源置换（SHFR）介导的修饰时可以产生和消除PCR伪影的条件，将ΔF508突变（CFBE 41 o-）纯合细胞与含有野生型CFTR（wt-CFTR）序列的小DNA片段（SDF）混合。野生型等位基因特异性PCR（wtAS-PCR）后，如果基因组DNA没有凝胶纯化，可能会产生一个假象。在没有凝胶纯化的情况下，产生伪影所需的SDF/细胞的量取决于所使用的AS引物对。当凝胶纯化基因组DNA时，无论SDF与细胞混合还是转染到细胞中，用任何wtAS-PCR引物都不能检测到伪影。此外，用DNA酶处理细胞mRNA足以消除逆转录酶-聚合酶链反应（RT-PCR）分析中的潜在伪影。因此，当通过PCR分析SFHR介导的修饰时，凝胶纯化基因组DNA和DNase处理mRNA是关键的。
Oligonucleotides can mediate sequence-specific gene modification that results in the correction and/or alteration of genomic DNA. There is evidence to suggest that the polymerase chain reaction (PCR)-based analytical methods usually used to analyze oligonucleotide-mediated modification can generate artifacts. To investigate the conditions under which a PCR artifact can be generated and eliminated when analyzing small fragment homologous replacement (SHFR)-mediated modification, cells homozygous for the ΔF508 mutation (CFBE41o-) were mixed with small DNA fragments (SDFs) containing the wild-type CFTR (wt-CFTR) sequence. An artifact could be generated after wild-type allele-specific PCR (wtAS-PCR) if the genomic DNA was not gel purified. Without gel purification, the amount of SDF/cell required to generate the artifact was dependent to the AS primer pairs used. When the genomic DNA was gel purified, no artifact could be detected with any of the wtAS-PCR primers whether the SDF was mixed with the cells or transfected into the cells. Furthermore, treatment of cellular mRNA with DNase was sufficient to eliminate potential artifacts in the reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. Thus, it is critical to gel purify genomic DNA and DNase treat mRNA when analyzing SFHR-mediated modification by PCR.