Fetal nucleated erythrocyte discrimination from maternal cell on DNA diagnosis.

已有报道称胎儿有核红细胞存在于材料外周血中。这些胎儿有核红细胞在母体淋巴细胞中被识别为苏木素和联苯胺双重染色。这些胎儿有核细胞是产前遗传病DNA诊断的良好来源，但胎儿有核红细胞的数量很少。使用这种稀有细胞进行DNA诊断的问题是母体淋巴细胞进入胎儿细胞准备的交叉污染。针对实际诊断来源DNA上高度多态基因的DNA分型问题，建立了稳定的单细胞微量DNA DNA分型方法，在显微镜下用毛细管抽吸法制备了单细胞操作方法。采用碱性增溶法制备DNA。用随机15聚体寡核苷酸引物扩增…超过50个循环(引物延伸预扩增：PEP)。将扩增出的DNA一半用于DNA诊断，另一半用于人类白细胞抗原-DR基因分型。人类白细胞抗原系统由多个基因座组成，本研究采用人类白细胞抗原-DRB基因进行基因分型，具有较高的多态性，建立了完整的DNA分型方法。用HLADRB引物对扩增后的单细胞DNA进行扩增，扩增出一半的DNA。用琼脂糖凝胶电泳法检测扩增结果。在65%的样本中，我们发现有238bp的DNA预期扩增，25%的样本显示出涂片电泳式。用人类白细胞抗原-DRB-DNA-聚合酶链式反应-单链构象多态性方法对扩增的人类白细胞抗原-DRB DNA进行分型。与同种异型特异性寡核苷酸探针DNA杂交，不仅在238bp条带的样本上显示出了人类白细胞抗原-DRB的类型，而且在出现涂片电泳图的样本上也显示了人类白细胞抗原-DRB的类型。这种单细胞DNA-DRB配型方法的总配型效率为90%。我们认为这种分型效率是有效的临床分型和DNA诊断。较少

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