| Budget Amount *help |
¥2,500,000 (Direct Cost: ¥2,500,000)
Fiscal Year 1992: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 1991: ¥1,800,000 (Direct Cost: ¥1,800,000)
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| Research Abstract |
Sex-determination of domestic animal embryos has already been performed by the polymerase chain reaction (PCR) method using the several primers. As those methods were developed by the foreign countries, no use of them are available in commercial-use in our country. SRY, a single copy gene has recently been identified as encoding the testis determining factor in eutherian mammals. So, we have attempted to develop new primers of its amplification and to improve the PCR method.
We designed a pair of new primers, (5'-CCCATGAATGCATTCATGGTGTGGT-3', 5'-TTATAGTTCGGGTATTTCTCTCTGTG; SRYF,SRYR), and deviced the high sensitive PCR method to amplify male specific DNA. We adopted method is two step cycle procedures in PCR. The first step is low annealing temperature and the second step is high annealing temperature. Mismatch to the sequences of primers are overcomed by this method, and sensitivity of amplification is improved. Male specific DNA are easily amplified in cattle, horse, pig, mouse and rat using same primers. In female animals, non-specific artifacts are observed in PCR products. These are useful as female specific makers. It is possible to detect the male specific signal, even the use of 40 pg crude DNA as template.
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