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Paternity Testing by In-Gel Competitive Reassociation of Polymorphic DNA Fragments

通过多态性 DNA 片段的凝胶内竞争性重组进行亲子鉴定

基本信息

批准号：
05557031
负责人：
TAMAKI Yoshihiro
金额：
$ 3.14万
依托单位：
Oita Medical University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Developmental Scientific Research (B)
财政年份：
1993
资助国家：
日本
起止时间：
1993 至 1994
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-05557031/
关键词：
Competitive Reassociation Subtraction PCR Paternity Testing DNA polymorphism ゲル内競合的再会合法

项目摘要

For paternity testing without an expensive, commercially available DNA probe, we subtracted the child's DNA fragments from a large excess of the DNA fraqments of the mother and the alleged father. We digested first the genomic DNAs of the mother-child-father trio and the unrelated man. The child's DNA digest was labeled with biotin by a fill-in reaction, and mixed with a large excess of the digests (alkaline phosphatase-treated) of the mother and the father (or unrelated man). The mixture was electrophoresed on an agarose gel, followed by alkali-denaturation and reassociation. 1-2 kb fragments were recovered for anchored PCR amplification. The PCR product was captured with immobilized streptavidin, reamplified, and detected by dot ELISA.Since practically all of the child's DNA fragments reassociate with the parents' corresponding fragments, and the adaptors (anchors) can be ligated only to the child's renatured DNA fragments, amplifiable fragments should be recoverable only when the unrelated man's fragments were included in the mixture. However, DNAs were detectable in the case of paternity inclusion as well. To reduce the number of the child's fragments to be subtracted, we enriched the child's DNA fragments for tandemly repetitive sequences by brief hybrid formation in the liquid phase, and subjected the enriched fragments to the in-gel competitive reassociation, that is, in-gel genomic subtraction as described above. Again, the procedure ended in failure. We thought then that the enriched fragments might serve as a DNA-fingerprinting probe cocktall. We re-amplified and simultaneously labeled them with digoxigenin, and successfully used the re-PCR product in the DNA fingerprinting of paternity case trios. Thus the final goal of "paternity testing without an expensive DNA probe "has been attained, although in a different fromat.

为了在没有昂贵的商业DNA探针的情况下进行亲子鉴定，我们从母亲和所谓的父亲的大量DNA片段中减去了孩子的DNA片段。我们首先消化了母亲-孩子-父亲三人组和无血缘关系的男子的基因组DNA。通过填充反应，孩子的DNA摘要被标记上生物素，并与母亲和父亲(或无血缘关系的男人)的大量过量消化(碱性磷酸酶处理)混合。该混合物在琼脂糖凝胶上进行电泳，然后进行碱变性和再结合。回收1-2kb片段用于锚定聚合酶链式反应扩增。扩增产物用固定化链霉亲和素捕获，再扩增，并用Dot ELISA检测。由于几乎所有儿童的DNA片段都与父母的相应片段重新结合，而且接头(锚)只能与儿童复性的DNA片段连接，因此只有在混合物中包含无关男性的片段时，才能回收可扩增的片段。然而，在父子关系纳入的情况下也可以检测到DNA。为了减少要减去的儿童片段的数量，我们通过在液体中形成简短的杂交形成来丰富儿童的DNA片段以获得简单的重复序列，并使浓缩的片段进行凝胶内竞争性再结合，即如上所述的凝胶内基因组减去。再一次，手术以失败告终。我们当时认为，这些丰富的片段可能会作为一个DNA指纹探针罗托尔。我们对它们进行了二次扩增，同时用地高辛标记了它们，并成功地将该产物用于亲子鉴定案例三人组的DNA指纹图谱。因此，“不需要昂贵的DNA探针进行亲子鉴定”的最终目标已经实现，尽管是在不同的情况下。