Cell-free fetal DNA plasma extraction and real-time polymerase chain reaction quantification

Isolation, quantification, and genetic analysis of circulating plasma DNA have clinical applications in prenatal diagnosis, oncology, organ transplantation, posttrauma monitoring, and infectious disease. Recent technology has allowed the rapid isolation and purification of DNA from whole blood, plasma, serum, buffy coat, tissues, stool, and urine. With the advent of real-time polymerase chain reaction (PCR) amplification, extracted DNA not only can be easily identified to aid in clinical diagnoses, but also can be readily quantified to analyze ongoing clinical dynamics and aid in the medical prognoses of patients. Historically, identification of unique cell-free fetal DNA sequences has relied on the detection of paternally specific Y chromosome sequences owing to their relative ease in identification. However, any DNA sequence that is unique to the fetus has the potential to be amplified and quantified using real-time PCR. Our laboratory specializes in extraction of fetal DNA from maternal plasma with subsequent quantification with real-time PCR of paternally inherited sequences, such as the Y chromosome gene, SRY. The successful isolation and quantification of this DNA from plasma is dependent on three distinct protocols: plasma harvesting from whole blood, DNA extraction from cell-free plasma, and real-time PCR amplification and quantification of the SRY sequence.