Quantification of Fetal DNA by Use of Methylation-Based DNA Discrimination
Quantification of Fetal DNA by Use of Methylation-Based DNA Discrimination
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DOI：
10.1373/clinchem.2010.146290
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发表时间：
2010-10-01
影响因子：
9.3
通讯作者：
Ehrich, Mathias
中科院分区：
文献类型：
--
作者：
Nygren, Anders O. H.;Dean, Jarrod;Ehrich, Mathias
背景：检测母体血浆中循环无细胞胎儿核酸已被用于无创产前诊断。大多数应用依赖于胎儿核酸的定性检测来确定胎儿的基因组成。这种方法会导致分析上的困境，因为不含胎儿DNA或被母体细胞DNA污染的样品的检测结果可能会产生误导。我们开发了一种多重方法来分析胎盘中相对于母体血液的高甲基化区域，以评估从母体血浆中分离的循环无细胞DNA的胎儿部分。方法：该试验使用甲基化敏感限制性内切酶去除DNA样本的母体（未甲基化）部分。未消化的胎儿DNA片段然后在合成的寡核苷酸存在下进行共扩增，以允许竞争性PCR。扩增产物通过单碱基延伸和MALDI-TOF MS分析进行定量。结果：使用2个独立的标记（性别决定区Y）-盒14 （SOX14）和t盒3 (TBX3)，我们测量了从孕妇获得的样本中平均151个胎儿DNA拷贝/mL血浆和平均胎儿分数0.13。我们研究了242份从孕妇和非孕妇血浆中分离的DNA样本，观察到分析灵敏度和特异性分别为99%和100%。结论：通过平行研究多个区域，我们减少了测量方差并实现了循环无细胞DNA的定量。我们的研究结果表明，这种基于多重甲基化的反应可以检测和量化从母体血浆中分离的样本中胎儿DNA的数量。(c) 2010年美国临床化学协会
BACKGROUND: Detection of circulating cell-free fetal nucleic acids in maternal plasma has been used in non-invasive prenatal diagnostics. Most applications rely on the qualitative detection of fetal nucleic acids to determine the genetic makeup of the fetus. This method leads to an analytic dilemma, because test results from samples that do not contain fetal DNA or are contaminated with maternal cellular DNA can be misleading. We developed a multiplex approach to analyze regions that are hypermethylated in placenta relative to maternal blood to evaluate the fetal portion of circulating cell-free DNA isolated from maternal plasma.METHODS: The assay used methylation-sensitive restriction enzymes to eliminate the maternal (unmethylated) fraction of the DNA sample. The undigested fetal DNA fraction was then coamplified in the presence of a synthetic oligonucleotide to permit competitive PCR. The amplification products were quantified by single-base extension and MALDI-TOF MS analysis.RESULTS: Using 2 independent markers, (sex determining region Y)-box 14 (SOX14) and T-box 3 (TBX3), we measured a mean of 151 copies of fetal DNA/mL plasma and a mean fetal fraction of 0.13 in samples obtained from pregnant women. We investigated 242 DNA samples isolated from plasma from pregnant and nonpregnant women and observed an analytical sensitivity and specificity for the assay of 99% and 100%, respectively.CONCLUSIONS: By investigating several regions in parallel, we reduced the measurement variance and enabled quantification of circulating cell-free DNA. Our results indicate that this multiplex methylation-based reaction detects and quantifies the amount of fetal DNA in a sample isolated from maternal plasma. (c) 2010 American Association for Clinical Chemistry