Universal Probe System for DNA Fingerprinting

DNA指纹需要使用特定的DNA探针。通用探针系统是已插入克隆载体中的未标记的初级探针和特定于该载体的标记的二级探针的组合。作为通用探针系统的第一步，我们从一个亚基因组文库中分离了三个DNA指纹探针，该文库是从人骨髓瘤细胞DNA(命名为YA)的EcoRI酶切中插入1-2kb片段而构建的亚基因组文库中分离出来的。对其中一个探针进行测序，根据插入片段的长度命名为YA1.8，结果显示，尽管它没有明显的重复序列，但它检测到了DNA指纹。这表明该探针与已知的小卫星探针有根本的不同，因此，我们将其注册在DDBJ，登录号为D29635。接下来，我们分离了重组噬菌体的单链DNA，并将它们与来自父亲-母亲-孩子三人组或无关个体的DNA进行了杂交。我们能够用商业上可以买到的碱性磷酸酶标记的二级探针或地高辛标记的通用测序引物来检测DNA杂交，即DNA指纹。

DNA fingerprinting requires the use of a specific DNA probe. A universal probe system is a combination of an unlabeled primary probe that has been inserted in a cloning vector and a labeled secondary probe that is specific to the vector. This system is time- and labor-saving in that one does not need to label probes every time one uses them, as long as they are inserted in the same vector.As first step to a universal probe system, we isolated three DNA fingerprinting probes from a subgenomic library that had been constructed by insertion into the phagemid pUC118 of 1-2 kb fragments from the EcoRI digest of DNA from human myeloma cells (named YA). Sequencing of one of the probes, termed YA1.8 after the length of the insert, revealed that although it contained no apparent repetitive sequence, it detected DNA fingerprints. This suggests that the probe is radically different from the known minisatellite probes ; therefore, we registered it with DDBJ under accession no.D29635.Next, we isolated single-stranded DNAs of recombinant phagemids, and hybridized them with southern blots of DNAs from father-mother-child trios or unrelated individuals. We were able to detect the DNA hybrids, that is, DNA fingerprints, with a commercially available, alkaline phosphatase-labeled secondary probe or a digoxigenin-labeled universal sequencing primer.