A low-cost efficient multiplex PCR for prenatal sex determination in bovine fetus using free fetal DNA in maternal plasma

Abstract

Background: 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.

Materials and methods: 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 µl 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.

Results: 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.

Conclusion: 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.

Keywords: Free Fetal DNA; Maternal Plasma; Multiplex PCR; Sex Determination.

Fig 1

A. The concentration of template DNA (plasma DNA) extract of maternal plasma for PCR, respectivel Lane 2, 200 ng/ml; Lane 3, 300 ng/ml; Lane 4, 500 ng/ml; Lane 5, 600 ng/ml; Lane 6, 700 ng/ml; Lanes 1 is a adult male (positive control) genomic DNA and Lane M in figure A represents the 50 base pair ladder. B. Gel electrophoresis of bovine fetal sex prediction by a simultaneous multiplex PCR analysis of maternal plasma. The multiplex amplified products of the bAML sequence on X chromosome, the bAML sequence on Y chromosome and the BC1.2 sequence on Y chromosome are 467 bp, 341 bp and 190 bp in length, respectively. Result of multiplex PCR analysis on plasma DNA samples. Lanes 1-6 demonstrate the results of plasma DNA analysis of the 6 pregnant heifers. Lane 7 is made with adult male, and. Lane 8 is a normal heifer who had no history of pregnancy which served as positive control. Predictions of male pregnancies were made for 1, 2, 4 and 6 and female pregnancies for 3 and 5 respectively. Lane M in figure A represents the 50 base pair ladder.