The rapid diagnosis of freemartin by polymerase chain reaction

详细信息    查看全文

• 作者：Oh ; S.J. ; Yang ; B.S. ; Jeon ; I.S. ; Seong ; H.H. ; Im ; K.S. ; Lee ; J.H. ; Yoon ; D.H. ; Kim ; T.H. ; Park ; Y.Y.
• 刊名：Theriogenology
• 出版年：1997
• 出版时间：January 1, 1997
• 年：1997
• 卷：47
• 期：1
• 页码：271
• 全文大小：51.8 K

文摘

In cattle, over 90 % of females in heterosexual twins are freemartins. The sterility of freemartins is due to lack of normal female internal reproductive organs. The common methods for the detection of freemartin are karyotyping and blood typing. Karyotyping requires considerable laboratory work for detection of possible XX/XY chimerism. Blood typing is a very convenient and fast technique, but requires many blood samples from both twins, which are not always available. The purpose of this study was to detect the freemartin with the amplification of Y-chromosome DNA by polymerase chain reaction. Genomic DNAs of 15 freemartins were isolated from 500μl of the blood and amplified with the pairs of Y-chromosome specific DNA primers (BOV97M and BRY4a). The reaction mixtures contained 26.5μl of water, 5μl 10 × PCR buffer, 8μl dNTPs (1.25mM each), 3μl MgCl₂(1.5mM), 2.5 unit Taq DNA polymerase and 100ng genomic DNA. Samples were covered with 20μl mineral oil and amplified for 30 cycles at the following temperatures: denaturation 94°C for 30 sec, annealing 55°C for 30 sec and extension 72°C for 30 sec. In order to detect the sensitivity of this method, XX/XY chimerism was made artificially by dilution of bull's blood in cow's blood in the following ratios: 0.01 % , 0.05 % , 0.1 % , 0.5 % , 1.0 % and 5.0 % . In these artificially made chimeric samples, male and female was detected even in the samples with 0.01 % of bull blood.