Improving enrichment of circulating fetal DNA for genetic testing: size fractionation followed by whole gene amplification.
OBJECTIVE
Among the pitfalls of using cell-free fetal DNA in plasma for prenatal diagnosis is quality of the recovered DNA fragments and concomitant presence of maternal DNA (>95%). Our objective is to provide alternative methods for achieving enrichment and high-quality fetal DNA from plasma.


METHODS
Cell-free DNA from 31 pregnant women and 18 controls (10 males and 8 females) were size separated using agarose gel electrophoresis. DNA fragments of 100-300, 500-700 and 1,500-2,000 bp were excised and extracted, followed by whole genome amplification (WGA) of recovered fragments. Levels of beta-globin and DYS1 were measured.


RESULTS
Distribution of beta-globin size fragments was similar among pregnant women and controls. Among control male cases, distribution of size fragments was the same for both beta-globin and DYS1. Among maternal cases confirmed to be male, the smallest size fragment (100-300 bp) accounted for nearly 50% (39.76 +/- 17.55%) of the recovered DYS1-DNA (fetal) and only 10% (10.40 +/- 6.49%) of beta-globin (total) DNA. After WGA of plasma fragments from pregnant women, DYS1 sequence amplification was best observed when using the 100-300 bp fragments as template.


CONCLUSIONS
Combination of electrophoresis for size separation and WGA led to enriched fetal DNA from plasma. This novel combination of strategies is more likely to permit universal clinical applications of cell-free fetal DNA.
Jorgez
Carolina J
CJ
Department of Obstetrics and Gynecology, Baylor College of Medicine, Houston, Texas, USA.
Bischoff
Farideh Z
FZ
D004247:DNA / Q000097:blood* / Q000737:chemistry / Q000302:isolation & purification; D004587:Electrophoresis, Agar Gel; D005260:Female; D005820:Genetic Testing; D006801:Humans; D008297:Male; D021141:Nucleic Acid Amplification Techniques; D011247:Pregnancy; D011296:Prenatal Diagnosis / Q000379:methods*
D004247:DNA