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

为了进行亲子鉴定，我们不需要昂贵的、市售的DNA探针，我们从母亲和所谓父亲的大量DNA片段中减去孩子的DNA片段。我们首先消化了母亲-孩子-父亲三人组和不相关的男子的基因组dna。通过填充反应，孩子的DNA消化液被标记为生物素，并与母亲和父亲（或无亲属）的大量消化液（碱性磷酸酶处理）混合。混合物在琼脂糖凝胶上电泳，然后进行碱变性和再缔合。回收1-2 kb片段进行锚定PCR扩增。PCR产物用固定化链霉亲和素捕获，再扩增，dot ELISA检测。由于几乎所有孩子的DNA片段都与父母的相应片段重新结合，并且接头（锚）只能连接到孩子的再生DNA片段上，因此只有当不相关的男人的片段被包括在混合物中时，才能恢复可扩增的片段。然而，在父系包含的情况下也可以检测到dna。为了减少要减去的孩子的片段的数量，我们在液相中通过短暂的杂交形成来富集孩子的DNA片段以进行串联重复序列，并将富集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法。这一过程再次以失败告终。我们当时认为，富集的片段可能作为dna指纹探针。我们将其重新扩增并同时用地高辛标记，并成功地将重新pcr产物用于父系病例三人组的DNA指纹图谱。因此，“没有昂贵DNA探针的亲子鉴定”的最终目标已经实现，尽管方式不同。

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.