Evaluation of two DNA extraction methods from maternal plasma for using in non-invasive bovine fetus gender determination

Fetal DNA in maternal plasma and serum has been shown to be a useful material for prenatal fetal sex determination during early gestational ages. Non-invasive prenatal diagnosis is now possible at 8th week of pregnancy, by maternal blood sample testing. The purpose of this study was to evaluate two DNA extraction methods from mother plasma and its routine clinical application in bovine fetus gender determination with non-invasive method. Maternal blood samples were taken from 40 pregnant cows during the 8th-38th weeks of gestation. DNA was extracted from 350 µl of maternal plasma with two salting-out and phenol-chloroform methods. The absorption in A260 and purity (A260/A280) of extracted DNA were detected by ultraviolet spectrophotometer. Three µl of the extracted DNA with phenol-chloroform method was used as a template. The PCR reaction was carried out to amplify the fragments of X and Y chromosomes of amelogenin, TSPY and BC1.2 genes. The difference between the mean absorption of DNA extracted by phenol-chloroform method and salting-out method was not significant in A260 (p>0.05, p=0.3549), but the difference between mean purity (A260/A280) of DNA extracted by phenol-chloroform method and salting-out method was significant (p<0.001). X chromosome fragment was detected in all 40 samples and Y chromosome fragments were detected in 25 plasma samples which were delivered a male calf. The sensitivity and specificity of test was 100% with no false negative and false positive results. The results showed that phenol-chloroform method is a simple and sensitive method for isolation of fetal DNA in maternal plasma.