Analysis of Cell-Free Fetal DNA from Maternal Plasma and Serum Using a Conventional Multiplex PCR: Factors Influencing Success

Recent technology enables the use of cell-free fetal DNA in maternal plasma and serum for noninvasive prenatal geneticdiagnosis. This study was designed to evaluate factors most likely to influence the success of a simple, cost efficient, reliable andreplicable conventional PCR technique in the clinical routine of prenatal genetic diagnosis of selected cases. The results stronglysuggest that DNA extraction and PCR cycle optimization are 2 major success-limiting steps and the maternal plasma is a better choiceover serum for DNA extraction for such prenatal genetic diagnosis. In addition, the use of a ready-to-use PCR mixture containingheat-activated Taq polymerase significantly reduced the risk of nonspecific amplification and of primer dimerization formed at lowtemperatures during PCR setup and the initial PCR cycle eliminating false positive results and insufficient PCR amplification,respectively. Thus the ease, rapidity and effectiveness shown by the presented system requiring only optimization of routine PCRprocedure and no additional sophisticated equipment could theoretically reduce the cost and number of invasive procedures requiredfor prenatal diagnosis of X-linked recessive genetic disorders and of fetal RhD status