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Universal Probe System for DNA Fingerprinting

DNA 指纹识别通用探针系统

基本信息

批准号：
04670358
负责人：
TAMAKI Yoshihiro
金额：
$ 1.34万
依托单位：
Oita Medical University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1992
资助国家：
日本
起止时间：
1992 至 1993
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-04670358/
关键词：
DNA fingerprint Universal probe Paternity testing

项目摘要

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.

DNA指纹分析需要使用特定的DNA探针。通用探针系统是已插入克隆载体中的未标记的一级探针和对该载体具有特异性的标记的二级探针的组合。该系统省时省力，因为不需要在每次使用探针时都标记探针，只要将它们插入同一载体即可。我们从亚基因组文库中分离了三个DNA指纹探针，所述亚基因组文库是通过将来自人骨髓瘤细胞（命名为YA）的DNA的EcoRI消化物的1- 2kb片段插入噬菌粒pUC 118中而构建的。对其中一个探针（在插入片段的长度之后被称为YA 1.8）进行测序，发现尽管它不包含明显的重复序列，但它检测到了DNA指纹。这表明该探针与已知的小卫星探针完全不同，因此，我们将其在DDBJ注册为登录号D29635。接下来，我们分离重组噬菌粒的单链DNA，并将其与来自父-母-子三人组或无关个体的DNA进行Southern印迹杂交。我们能够检测的DNA杂交，即DNA指纹，与市售的，碱性磷酸酶标记的第二探针或地高辛标记的通用测序引物。