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

对于没有昂贵的商业DNA探针的亲子鉴定，我们从母亲和所谓父亲的大量多余DNA片段中减去孩子的DNA片段。我们首先消化了母亲-孩子-父亲三人组和无关男性的基因组DNA，然后通过填充反应用生物素标记孩子的DNA消化物，并将其与大量过量的母亲和父亲（或无关男性）的碱性磷酸酶（碱性磷酸酶处理）混合。将混合物在琼脂糖凝胶上沉淀，然后进行碱变性和再结合。1-2回收kb片段用于锚定PCR扩增。PCR产物用固定化的链霉亲和素捕获，再扩增，用斑点ELISA检测，因为几乎所有的子DNA片段与父母的相应片段重新结合，并且接头（锚）只能连接到子的复性DNA片段，只有当无关的人的片段包括在混合物中时，可回收的片段才应该是可回收的。然而，DNA在亲子鉴定的情况下也是可检测的。为了减少要减去的子片段的数量，我们通过在液相中短暂的杂交体形成来富集子DNA片段的串联重复序列，并使富集的片段经受凝胶内竞争性再结合，即，如上所述的凝胶内基因组减去。手术再次以失败告终。我们当时认为，富集片段可能作为DNA指纹探针cocktall。我们将其重新扩增并同时用地高辛标记，并成功地将其用于亲子鉴定案例三人组的DNA指纹分析。因此，“没有昂贵的DNA探针的亲子鉴定”的最终目标已经实现，尽管形式不同。

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.