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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指纹识别需要使用特定的DNA探针。通用探针系统是将插入克隆载体的未标记的初级探针和特定于该载体的标记次级探针结合在一起。该系统省时省力，因为每次使用探针时都不需要标记探针，只要探针插入同一向量即可。作为通用探针系统的第一步，我们从亚基因组文库中分离出三个DNA指纹探针，该文库是通过将来自人骨髓瘤细胞（YA） DNA EcoRI消化的1-2 kb片段插入到噬菌体pUC118中构建的。对其中一个探针（以插入物长度命名为YA1.8）的测序显示，尽管它不包含明显的重复序列，但它检测到DNA指纹。这表明该探测器与已知的微型卫星探测器截然不同；因此，我们在DDBJ注册，注册号为d29635。接下来，我们分离了重组噬菌体的单链dna，并将其与来自父亲-母亲-孩子三人组或无亲缘关系个体的dna杂交。我们能够检测DNA杂交，即DNA指纹，用市售的，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。