Comparison of three methods for DNA extraction from amniotic fluid for fetal sex determination by multiplex polymerase chain reaction (PCR).

ABSTRACT

The objective of this study was to compare three methods used for amniotic fluid DNA extraction. These methods were Proteinase K/Phenol-Chloroform, Proteinase K/7.5M Guanidine-HCl and DNAzolา BD Reagent. Ten samples of uncontaminated amniotic obtained by amniocentesis performed in mothers with advanced maternal age for detection of fetal chromosome abnormality were studied. Each sample was divided into three tubes, 1 ml placed in each, and DNA extraction was performed by all three methods. The quality and quantity of DNA extracted by each method were compared by electrophoresis on 3% agarose gel and spectrophotometric study at 260 & 280 nm. The DNA obtained was subsequently used for fetal sex determination by multiplex PCR method. The primers used for multiplex PCR were specific for X and Y chromosomes. Accuracy of fetal sex determination was compared with the results from amniocyte culture and the sex at birth. The results showed that DNA extracted by DNAzolาBD Reagent was 100% accurate when used to determine fetal sex by PCR; eight samples on 1st PCR and the other two on repeat PCR. DNA extracted by Proteinase K/7.5M Guanidine-HCl also yielded 100% accuracy in fetal sex determination; seven samples on 1st PCR, two samples on 2nd PCR and one sample needed 3rd PCR. The Proteinase K/Phenol-Choroform method yielded only 90% accuracy and one sample failed to determine fetal sex after having repeated PCR three times. The extraction method which gave the maximum amount of DNA was the Proteinase K/Phenol-Chloroform one but the failed to give 100% accuracy in determining fetal sex. The method which produced the least protein contamination was DNAzolฎBD Reagent and gave 100% accuracy in determining fetal sex with smallest number of PCR reactions. In conclusion, DNAzolฎBD reagent method is relatively rapid for amniotic fluid DNA extraction with high accuracy for fetal sex determination but when large amount of DNA is also needed for other purposes, Proteinase K/Phenol-Chlorofrom is recommended as the method of choice.