Invasive methods for prenatal diagnosis, include
chorionic villus sampling (CVS) and amniocentesis
that entail a risk of fetal loss and mortality. In
1997, Lo et al. (
The aim of this study was to establish a rapid and reliable method for sexing of bovine fetuses.
This has prompted us to improve another non-invasive method of bovine fetal sex determination using multiplex PCR amplification of the X chromosome (467 bp amelogenin gene) and the Y chromosome (341 bp amelogenin gene fragments and BC1.2-sequence (that is derived from a male-specific bovine DNA sequences) simultaneously.
In this experimental study, peripheral blood samples were taken from 38 pregnant heifers with gestational age of 8 to 38 weeks. Five normal heifers which had no history of pregnancy and five normal male cows served as positive control. Maternal peripheral blood sample (10 ml) were collected and put into tubes containing ethylenediaminetetraacetic acid (EDTA) (20 mM). The tubes were centrifuged at 1000 r/minute for 10 minutes with the brake and acceleration powers set to zero. Then tubes were centrifuged at 1200 r/minutes for 10 minutes with the brake and acceleration powers set to zero. Approximately 0.5 ml of supernatant (ie, the plasma) was left in the tube to ensure that the buffy coat was not disturbed. Tubes were centrifuged at 2000 r/minutes for 5 minutes with the brake and acceleration powers set to zero. 350 μl of supernatant and samples were stored at -20˚C for further processing.
Maternal plasma (350 μl) and an equal volume of Tris-EDTA (TE) buffer were mixed in a 1.5 ml Eppendorf tube by addition of 5 μl proteinase K solution (20 mg/ml). The mixture was digested at 56 ˚C for 3 hours, and then 350 μl of equilibrium phenol plus chloroform was added respectively. The tubes were centrifuged at 12000 r/minutes for 12 minutes and then the supernatant was transferred to a fresh tube. Equal volume of chloroform and isoamyl alcohol (24: 1) were added. After centrifugation (at 12000 r/minutes for 12 minutes), 1:10 of 3 mol/l sodium acetate and 2 volumes of 100% ethanol were added and the mixture was stored at -20˚C for 14 hours. Tubes were then centrifuged at 12000 r/minutes for 8 minutes at room temperature. The supernatant was discarded, DNA was purified and deposited with 70% ethanol before being dried in the airing closet. Tubes was dried at 65˚C for 3 minutes and were finally dissolved in 20 μl TE. Tube was placed in dry bath at 65˚C for 40 minutes and then stored at 4˚C.
The concentration and purity of extracted DNA were identified by an ultraviolet spectrophotometer (Nanodrop 2000 Thermo). The results were read at 260 nm and 280 nm respectively.
In this study, two primer pairs were used: one set was derived from a male specific bovine DNA sequence termed, " BC1.2" (
The template DNA (plasma DNA) was diluted at different densities. When the concentration of template DNA extract of maternal plasma for mixture PCR was 200-300 ng/ml, there was the clearest band (
The results in figure 1B show three clear bands at 190 bp, 341 bp and 467 bp in the template DNA extracted from heifers bearing male fetus (lane 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.
The DNA template was extracted from the plasma of 38 pregnant heifers (8 to 38 weeks). Fetus-derived Y sequence bAML gene and BC1.2 fragment were detected in 24 cases of maternal plasma samples from the 38 cases. The sensitivity of PCR product in 38 pregnant herifers was 100%.
The discovery of cell-free fetal DNA in maternal plasma in 1997 has opened up new possibilities for non-invasive diagnoses (
In this study, we developed a multiplex PCR system for prenatal identification of fetal sexing. Though PCR methods cannot be compared with real-time PCR approach (
Results of fetal sex prediction by non-invasive approach using the conventional multiplex PCR analysis of maternal plasma DNA in 38 pregnant heifers at various gestational ages
|Samplesno||Gestational age(weeks)||Result ofmultiplex PCR||Birthoutcome||Samplesno||Gestational age(weeks)||Result ofmultiplex PCR||Birthoutcome|
Unlike other described conventional PCR systems for prenatal fetal sex determinations on maternal plasma (
Like the results of similar studies in human (
One of the benefits of this study and similar studies (
The results have shown that phenol-chloroform method is a simple and suitable method for isolation of fetal DNA in maternal blood. Furthermore, multiplex PCR technique is cost-efficient, reliable and available for non-invasive sex determination in bovine fetus.