Deletion screening at the hypoxanthine-guanine phosphoribosyltransferase locus in Chinese hamster cells using the polymerase chain reaction.
Deletion screening at the hypoxanthine-guanine phosphoribosyltransferase locus in Chinese hamster cells using the polymerase chain reaction.
复制标题
使用聚合酶链式反应对中国仓鼠细胞中次黄嘌呤-鸟嘌呤磷酸核糖基转移酶位点进行缺失筛选。
DOI：
10.1002/tcm.1770090306
复制
发表时间：
1989
期刊：
影响因子：
--
通讯作者：
Richard A. Gibbs
中科院分区：
文献类型：
--
作者：
Zhidong Xu;Yongjia Yu;Abraham W. Hsie;C. Caskey;Belinda J.F. Rossiter;Richard A. Gibbs
我们建立了一种用聚合酶链反应（PCR）检测中国仓鼠细胞次黄嘌呤-鸟嘌呤磷酸核糖转移酶（hprt）位点缺失突变的快速筛查方法。从自发和紫外线（UV）光和X射线诱导的hprt缺陷突变体中提取DNA。使用两对引物分别扩增含有完整外显子3和外显子9编码序列的276 bp和344 bp片段。使用Taq DNA聚合酶进行PCR 40个循环，并使用溴化乙锭染色的琼脂糖凝胶上的电泳直接分析PCR产物中各扩增DNA的存在。用这种方法，我们分析了39个独立衍生的hprt缺陷突变体。发现10个自发突变体中有4个在外显子9中有缺失。紫外线产生的突变体主要是野生型扩增模式（10/14）。X射线诱导的大多数缺失模式（11/15），其中6个仅发生在外显子9，5个发生在外显子3和9。这些观察结果与经典的观点一致，即紫外线主要诱导错义突变，X射线产生高比例的缺失突变。hprt基因3'端缺失突变最多，提示该区域可能存在缺失热点。用于缺失检测的PCR测定具有在不使用放射性同位素的情况下可在少于4小时内完成的优点。该检测方法可用于常规缺失筛查。
We have developed a rapid screening method using the polymerase chain reaction (PCR) for detecting deletion mutations at the hypoxanthine-guanine phosphoribosyltransferase (hprt) locus in Chinese hamster cells. DNA was extracted from spontaneous and ultraviolet (UV) light- and X-ray-induced hprt-deficient mutants. Two primer sets were used to amplify 276 bp and 344 bp fragments containing the entire exon 3 and exon 9 coding sequence, respectively. The PCR was performed using Taq DNA polymerase for 40 cycles, and the PCR product was directly analyzed for the presence of the respective amplified DNA using electrophoresis on agarose gels stained with ethidium bromide. With this assay, we have analyzed 39 independently derived hprt-deficient mutants. Four of ten spontaneous mutants were found to have deletions in exon 9. UV light produced mutants with predominantly wild-type amplification patterns (10/14). X-ray induced mostly deletion patterns (11/15); six of these occurred only in exon 9, and five occurred in both exons 3 and 9. These observations are consistent with the classical notion that UV light induces predominantly missense mutations and X-ray produces a high proportion of deletion mutations. Deletion mutations occurred most frequently at the 3' end of the hprt gene, suggesting the possible existence of hot spots for deletions in this region. The PCR assay for deletion detection has the advantage that it can be completed in less than 4 hr without using radioisotopes. This assay should be useful for routine deletion screening.
登录
查看更多内容
影响因子：
11.1
作者：
J. T. Stout;C. Caskey
通讯作者：
J. T. Stout;C. Caskey
人类次黄嘌呤鸟嘌呤磷酸核糖转移酶缺乏症的分子调查。
DOI：
10.1172/jci112275
发表时间：
1986
期刊：
The Journal of clinical investigation
影响因子：
--
作者：
Wilson,JM;Stout,JT;Palella,TD;Davidson,BL;Kelley,WN;Caskey,CT
通讯作者：
Caskey,CT
小鼠和中国仓鼠的次黄嘌呤鸟嘌呤磷酸核糖基转移酶基因：cDNA重组体的构建和序列分析。
影响因子：
14.9
作者：
Konecki,DS;Brennand,J;Fuscoe,JC;Caskey,CT;Chinault,AC
通讯作者：
Chinault,AC
次黄嘌呤磷酸核糖转移酶基因的结构、表达和突变。
DOI：
10.1073/pnas.81.7.2147
发表时间：
1984
影响因子：
11.1
作者：
Melton,DW;Konecki,DS;Brennand,J;Caskey,CT
通讯作者：
Caskey,CT
中国仓鼠卵巢细胞中含有二氢叶酸还原酶基因的 DNA 修复结构域的表征。
DOI：
--
发表时间：
1986
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Bohr,VA;Okumoto,DS;Ho,L;Hanawalt,PC
通讯作者：
Hanawalt,PC