Fetal nucleated erythrocyte discrimination from maternal cell on DNA diagnosis.

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Fetal nucleated erythrocytes have been reported to be present in material peripheral blood. These fetal nucleated erythrocytes are recognized double staining with Hematoxylin and Benzidine among the maternal lymphocytes. These fetal nucleated cells are excellent source on prenatal genetic disease DNA diagnosis, however, the numbers of fetal nucleated erythrocytes are very small. The problem of using such rare cells on DNA diagnosis is the cross contamination from maternal lymphocyte into the fetal cell preparation. The most trustworthy evidence that shows no cross contamination is the DNA typing about highly polymorphic gene on actual diagnosis source DNA.For this purpose, we developed the stable HLA-DR DNA typing method on a single cell derived slightly amount DNA samples.Single cell was prepared single cell manipulation method under the microscope with capillary suction. DNA was prepared by alkaline solubilization. DNA was amplified by PCR with random 15mer oligonucleotide primer for … More 50 cycles ( Primer Extension Preamplification : PEP) . A half of amplified DNA was separated for DNA diagnosis and the other half of DNA was used for HLA-DR DNA typing.Human leukocyte antigen(HLA) system is consists several loci and in this study we used HLA-DRB gene for typing HLA-DR gene was highly polymorphic and DNA typing method was fully established. A half of PEP expanded single cell DNA was re-expanded by PCR with HLA-DRB primer. The amplified results was examined with agarose gel electrophoresis. On the 65% of samples we found expected 238bp DNA amplification and 25% samples showed smear electophoresis pattern. Amplified HLA-DRB DNA was typed with HLA-DRB-DNA-PCR-SSO methods. Hybridization pattern with allotype specific oligonucleotide probe DNA showed HLA-DRB types not only on the samples which showed 238bp band but also the samples which showed smear electrophoresis pattern. Total typing efficiency of this single cell DNA HLA-DRB typing method was 90%. We considered that this typing efficiency was valid actual clinical typing and DNA diagnosis. Less