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

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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.