title: Maldi-TOF mass spectrometry for analyzing cell-free fetal DNA in maternal plasma.
abstract: 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.
authors.affiliation: Centre for Emerging Infectious Diseases, Chinese University of Hong Kong, Hong Kong, China.
authors.forename: Chunming
authors.initials: C
authors.lastname: Ding
mesh_terms: D002474:Cell-Free System; D004247:DNA / Q000097:blood* / Q000302:isolation & purification; D017931:DNA Primers; D005260:Female; D005333:Fetus / Q000378:metabolism*; D018507:Gene Expression Regulation, Developmental; D024282:Genes, sry; D005820:Genetic Testing; D006801:Humans; D040582:Inheritance Patterns; D008297:Male; D008431:Maternal-Fetal Exchange; D009154:Mutation; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide; D011247:Pregnancy; D011296:Prenatal Diagnosis / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D017931:DNA Primers; D004247:DNA