1992 Fiscal Year Final Research Report Summary

Sex-determination of domestic animals using male specific DNA fragments

Research Project

Project/Area Number	03660271
Research Category	Grant-in-Aid for General Scientific Research (C)
Allocation Type	Single-year Grants
Research Field	畜産学(含草地学)
Research Institution	Hokkaido University
Principal Investigator	UEDA Junji Hokkaido Univ., Anim.Sci., Associate Prof., 農学部, 助教授 (50002374)
Co-Investigator(Kenkyū-buntansha)	MORI Tadashi Hokkaido Univ., Anim.Sci., Res. Associate, 農学部, 助手 (30230072) SHIMIZU Hiroshi Hokkaido Univ., Anim.Sci., Professor, 農学部, 教授 (90001453)
Project Period (FY)	1991 – 1992
Keywords	Sex-determination / Domestic animal / Y-chromosome / DNA / PCR / SRY / Cattle / Pig
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.