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. 2009;25(3):314-9.
doi: 10.1159/000235877. Epub 2009 Sep 22.

Improving enrichment of circulating fetal DNA for genetic testing: size fractionation followed by whole gene amplification

Carolina J Jorgez  1 , Farideh Z Bischoff
Affiliations

Improving enrichment of circulating fetal DNA for genetic testing: size fractionation followed by whole gene amplification

Carolina J Jorgez et al. Fetal Diagn Ther. 2009.

Abstract

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.

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Figures

Fig. 1.
Fig. 1.
a Distribution of % β-globin levels (total DNA) based on fragment size in maternal plasma. Graph represents 31 pregnant women divided by first (n = 12; 1T), second (n = 12; 2T), and third (n = 7; 3T) trimesters. Plasma controls included non-pregnant women (n = 8) and adult men (n = 10). No significant difference was found among groups (p > 0.1). b Distribution of % DYS1 (fetal DNA) based on fragment size in maternal plasma. Graph represents 16 pregnant women divided by first (n = 6; 1T), second (n = 6; 2T), and third (n = 4; 3T) trimesters. Plasma controls included adult men (n = 10). Significant difference in DYS1 levels observed at 100–300 bp fragment size between all trimesters (p = 0.0009).
Fig. 1.
Fig. 1.
a Distribution of % β-globin levels (total DNA) based on fragment size in maternal plasma. Graph represents 31 pregnant women divided by first (n = 12; 1T), second (n = 12; 2T), and third (n = 7; 3T) trimesters. Plasma controls included non-pregnant women (n = 8) and adult men (n = 10). No significant difference was found among groups (p > 0.1). b Distribution of % DYS1 (fetal DNA) based on fragment size in maternal plasma. Graph represents 16 pregnant women divided by first (n = 6; 1T), second (n = 6; 2T), and third (n = 4; 3T) trimesters. Plasma controls included adult men (n = 10). Significant difference in DYS1 levels observed at 100–300 bp fragment size between all trimesters (p = 0.0009).
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