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目的探讨DNA片段差异性分级分离法对孕妇血浆中胎儿游离DNA富集的效果。方法取孕中期孕妇静脉血5ml,分别提取血浆中游离DNA和白细胞DNA,用琼脂糖凝胶电泳分离DNA片段,回收<400、400~1000、1000~3000bp部分凝胶中的DNA,扩增16个短串联重复序列(STR)遗传标志位点,同时扩增和检测孕妇白细胞基因组DNA和胎儿羊水细胞基因组DNA作对照,然后,用ABI310基因分析仪检测。结果共检测3例孕妇外周血标本48个STR位点,其中有意义位点(即胎儿父源性等位基因可与孕妇等位基因区分的STR位点)31个。从血浆总DNA、<400、400~1000和1000~3000bpDNA组中成功检测到胎儿等位基因的位点数占有意义位点数的百分比分别为6.45%、29.03%、38.71%和9.68%,各组检出成功率的差异有统计学意义(χ2=13.432,P=0.004),而<400和400~1000bpDNA组成功率较高。结论孕妇血浆DNA中短片段组分(<1000bp)胎儿DNA含量相对较高,有望应用于无创性胎儿产前诊断。
Objective To investigate the effect of DNA fractional fractionation on the enrichment of fetal DNA in pregnant women. Methods Venous blood from pregnant women of 5ml at the second trimester was used to extract free DNA and leukocyte DNA respectively. The DNA fragments were separated by agarose gel electrophoresis and the DNA in <400, 400-1000 and 1000-3000bp partial gels was recovered and amplified 16 A short tandem repeat (STR) genetic marker locus, while amplification and detection of pregnant women, leukocyte genomic DNA and fetal amniotic fluid genomic DNA as a control, and then detected by ABI310 gene analyzer. Results A total of 48 STR loci were detected in peripheral blood samples of 3 pregnant women, among which 31 were significant sites (STR loci where the fetal parental alleles could be distinguished from the pregnant women alleles). The percentages of the loci of the fetal alleles that were successfully detected in <400, 400-1000 and 1000-3000 bp DNA groups from plasma total DNA were 6.45%, 29.03%, 38.71% and 9.68%, respectively The difference of the success rate was statistically significant (χ2 = 13.432, P = 0.004), while <400 and 400 ~ 1000bp DNA composition of higher power. Conclusion The fetal DNA content of short DNA fragments (<1000bp) in plasma of pregnant women is relatively high, which is expected to be used in prenatal diagnosis of noninvasive fetuses.