An Improved Method to Extract DNA from 1 ml of Uncultured Amniotic Fluid from Patients at Less than 16 Weeks’ Gestation

<div><p>The aim of this study was to develop an improved technique for DNA extraction from 1 ml of uncultured AF from patients with a gestational age less than 16 weeks and to allow the use of array-CGH without DNA amplification. The DNA extraction protocol was tested in a series of 90 samples including 41 of uncultured AF at less than 16 weeks of gestation. Statistical analyses were performed using linear regression. To evaluate the sensitivity and the specificity of array-CGH on 1 ml of uncultured AF, five samples with an abnormal karyotype (three with aneuploidy, two with structural abnormalities) and five with a normal karyotype were studied. This protocol was reproducible and we were able to show a great improvement with higher yield of DNA obtained from all patients, including those with a gestational age less than 16 weeks (p = 0.003). All chromosomal abnormalities were detected and characterized by array-CGH and normal samples showed normal profiles. This new DNA extraction protocol associated with array-CGH analysis could be used in prenatal testing even when gestational age is less than 16 weeks, especially in cases with abnormal ultrasound findings.</p> </div

Prospective study of amniotic fluid (AF) by array-CGH.

<p>AMA, advanced maternal age; AU,abnormal ultrasound.</p

Summary of karyotype, array-CGH results, indication and DNA isolated from 1 ml of uncultured amniotic fluid at less than 16 weeks’ gestation.

<p>AMA, Advanced maternal age; IUGR, intrauterine growth retardation; AF, amniotic fluid; R, risk evaluation after triple test screening’.</p

Retrospective study of amniotic fluid (AF) by array-CGH.

<p>AF cffDNA, amniotic fluid supernatant cell-free fetal DNA.</p

Genome views of five number and structural abnormalities characterized by array-CGH, extracted from 1 ml of uncultured amniotic fluid from patients at less than 16 weeks of gestation.

<p>(cases 1 to 5).</p

Summary of DNA yield, purity (260/280 and 260/230 reading) extract by this optimized protocol of the 90 uncultured amniotic fluid.

<p>Summary of DNA yield, purity (260/280 and 260/230 reading) extract by this optimized protocol of the 90 uncultured amniotic fluid.</p

Results of multiple linear regression analysis performed to assess the independent relationship between DNA level, the study (ours vs. Bi et al. 2008) and gestational age.

<p>The multiple linear regression analysis model adjusted for gestational age (R² = 0.42).</p

Comparison of DNA yield and gestational age with Bi et al. [2008] study.

<p>Comparison of DNA yield and gestational age with Bi et al. [2008] study.</p

Genome views of 12 chromosomal abnormalities characterized by array-CGH, extracted from 1 ml of uncultured amniotic fluid from patients at more than 16 weeks of gestation.

<p>(cases 11 to 22).</p