DIRECT ELECTROPHORETIC DETECTION OF THE ALLELIC STATE OF SINGLE DNA-MOLECULES IN HUMAN SPERM BY USING THE POLYMERASE CHAIN-REACTION
DIRECT ELECTROPHORETIC DETECTION OF THE ALLELIC STATE OF SINGLE DNA-MOLECULES IN HUMAN SPERM BY USING THE POLYMERASE CHAIN-REACTION
复制标题
DOI：
10.1073/pnas.87.12.4580
复制
发表时间：
1990-06-01
影响因子：
11.1
通讯作者：
ARNHEIM, N
中科院分区：
文献类型：
--
作者：
LI, HH;CUI, XF;ARNHEIM, N
我们已经开发出一种程序，允许检测聚合酶链反应（PCR）产物来自一个单一的目标DNA分子在人类精子，而不使用放射性探针。利用这种方法，可以同时扩增单个精子中存在的三个遗传位点。扩增过程是特异性的，也是有效的，并且允许在聚丙烯酰胺凝胶电泳后通过溴化乙锭染色检测PCR产物。当使用长度不同的等位基因特异性PCR引物时，不同等位基因的PCR产物的大小在长度上也不同，从而允许在每个基因座处的等位基因状态被精确地确定。使用这种方法对单个精子进行的研究应该有助于在很小的物理距离上测量人类的遗传重组。快速简单地直接分析来自一个细胞的PCR产物的等位基因状态的能力对于遗传疾病的产前诊断也是有用的，特别是在植入前从体外受精卵中取出的单个卵裂球的分析中。
We have developed a procedure that allows the detection of polymerase chain reaction (PCR) products derived from a single target DNA molecule in a human sperm without using radioactive probes. With this method, three genetic loci present in a single sperm can be amplified simultaneously. The amplification procedure is specific as well as efficient and permits detection of the PCR product by ethidium bromide staining after polyacrylamide gel electrophoresis. When allele-specific PCR primers that differ in length are used, the size of the PCR products of different alleles also vary in length, allowing the allelic state at each locus to be determined electrophoretically. Studies on individual sperm by using this procedure should facilitate the measurement of genetic recombination in humans over small physical distances. The ability to directly analyze the allelic state of PCR products from one cell rapidly and simply will also be useful for the prenatal diagnosis of genetic disease, especially in the analysis of single blastomeres taken from in vitro fertilized eggs prior to implantation.