ABSTRACT
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 8[th] 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 8[th]-38[th] weeks of gestation. DNA was extracted from 350 micro l of maternal plasma with two salting-out and phenol-chloroform methods. The absorption in A[260] and purity [A[260]/A[280]] of extracted DNA were detected by ultraviolet spectrophotometer. Three micro l 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
ABSTRACT
In order to establish a reliable non-invasive method for sex determination in a bovine fetus in a routine setting, the possibility of identifying specific sequence in the fetal X and Y-chromosomes has been evaluated in maternal plasma using conventional multiplex polymerase chain reaction [PCR] analysis. The aim of this study was to provide a rapid and reliable method for sexing bovine fetuses. In this experimental study, peripheral blood samples were taken from 38 pregnant heifers with 8 to 38 weeks of gestation. DNA template was extracted by phenol-chloroform method from 350 micro l maternal plasma. Two primer pairs for bovine amelogenin gene [bAML] and BC1.2 were used to amplify fragments from X and Y chromosomes. A multiplex PCR reaction has been optimized for amplification of 467 bp and 341 bp fragments from X and Y bAML gene and a 190 bp fragment from BC1.2 related to Y chromosome. The 467 bp fragment was observed in all 38 samples. Both 341 and 190 bp fragments were detected only in 24 plasma samples from male calves. The sensitivity and specificity of test were 100% with no false negative or false positive results. The results showed that phenol-chloroform method is a simple and suitable method for isolation of fetal DNA in maternal plasma. The multiplex PCR method is an available non-invasive approach which is cost efficient and reliable for sexing bovine fetuses