Abstract
Due to the low percentage of fetal DNA present in maternal plasma (< 10%) during early gestation, efficient extraction processes are required for successful downstream detection applications in non-invasive prenatal diagnostic testing. In this study, two extraction methods using similar chemistries but different workflows were compared for isolation efficiency and percent fetal DNA recovery. The Akonni Biosystems TruTip technology uses a binding matrix embedded in a pipette tip; the Circulating Nucleic Acids Kit from Qiagen employs a spin column approach. The TruTip method adds an extra step to decrease the recovery of DNA fragments larger than 600 bp from the sample to yield an overall higher percentage of smaller molecular weight DNA, effectively enriching for fetal DNA. In this evaluation, three separate extraction comparison studies were performed - a dilution series of fragmented DNA in plasma, a set of clinical maternal samples, and a blood collection tube time point study of maternal samples. Both extraction methods were found to efficiently extract small fragment DNA from large volumes of plasma. In the amended samples, the TruTip extraction method was ~15% less efficient with overall DNA recovery, but yielded an 87% increase in % fetal DNA relative to the Qiagen method. The average percent increase of fetal DNA of TruTip extracted samples compared to the Qiagen method was 55% for all sets of blinded clinical samples. A study comparing extraction efficiencies from whole blood samples incubated up to 48 hours prior to processing into plasma resulted in more consistent % fetal DNA recoveries using TruTip. The extracted products were tested on two detection platforms, quantitative real-time PCR and droplet digital PCR, and yielded similar results for both extraction methods.
Highlights
Interest in non-invasive prenatal diagnostics (NIPD) or testing (NIPT) is growing rapidly due to its potential to supplement the standard prenatal diagnostic methods of amniocentesis or chorionic villus sampling, which carry significant health risks including fetal deformation and miscarriage [1]
The results show that TruTip is efficient at isolating smaller fragments of DNA from large volumes of plasma
In this study we compared extraction of cell-free fetal DNA (cffDNA) from large volumes of maternal plasma using the Akonni TruTip Kit and Qiagen Circulating Nucleic Acid Kit under simulated conditions, as well as in two separate studies involving clinical maternal blood samples. We determined that both methods are able to efficiently isolate DNA over a range of fetal DNA in a background of female DNA relevant to typical concentrations found in early gestation (6-8 weeks)
Summary
Interest in non-invasive prenatal diagnostics (NIPD) or testing (NIPT) is growing rapidly due to its potential to supplement the standard prenatal diagnostic methods of amniocentesis or chorionic villus sampling, which carry significant health risks including fetal deformation and miscarriage [1]. The presence of cffDNA at low concentrations (≤ 10%) in maternal plasma early in pregnancy when diagnostic testing is most desirable [2,3] necessitates processing and concentration of large sample volumes to yield adequate amounts of DNA for analysis [4]. Scaling to these larger volumes can be challenging for typical isolation approaches involving spin columns, vacuum manifolds or magnetic beads. Additional lysis of maternal white blood cells can occur if samples are not processed expeditiously or with proper cell preservatives, thereby increasing the maternal DNA and resulting in an even lower percentage of fetal DNA
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