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        MALDI-TOF Mass Spectrometry for Analyzing Cell-Free Fetal DNA in Maternal Plasma

        互联网

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        The discovery of cell-free fetal DNA in the plasma and serum of pregnant women has opened a new window for noninvasive prenatal diagnosis. Robust detection and quantification have been achieved when the fetal DNA sequence of interest does not have a maternal counterpart (e.g., Y chromosomal DNA, RhD gene when the mother is RhD negative) by techniques such as real-time polymerase chain reaction (PCR). However, detection of subtle fetal mutations is difficult due to the overwhelming maternal DNA background. A method combining PCR, base extension reaction, and matrix-assisted laser desorption ionization/time of flight mass spectrometry (MALDI-TOF MS) allowing DNA detection with single base specificity and single DNA molecule sensitivity is described. DNA sequence is amplified by PCR first. Then, a third primer (extension primer) is designed to anneal to the region immediately upstream of the mutation site. Depending on the specific mutation and the ddNTP/dNTP mixtures used, either one or two bases are added to the extension primer to produce two extension products from the wild-type DNA and the mutant DNA. Last, the two extension products are detected by high-throughput MALDI-TOF MS. In addition, with an improved base extension method called single allele base extension reaction, fetal DNA can be robustly detected even when overwhelming maternal background DNA is present.
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