Microdissection And Microcloning Of Swine Chromosome 12

Using the Guangxi Bama miniature swine, one of local breed of our country, as materials, The microdissection and microcloning of single swine chromosome 12 was carried out. The chromosome 12 specific DNA library of swine was established and analyzed. The research will be useful for intensive molecular linkage map construction of chromosome 12 and the isolation of important genes located on chromosome 12.1. Establishment of the method for single chromosome microdissection using microglassneedles, as well as amplification with the dissected chromosome .Freshly made mitosis metaphase spreads of swine by from culture of peripheral blood lymphocytes was used for microdissection. The single chromosome 12 was scraped from metaphase spread and transferred into an eppendorf tube with a microglassneddle. After digested with proteinase K and restriction enzyme, the DNA of chromosome 12 was amplified by two rounds of linker adaptor PCR. The size of the amplified DNA varied from 250 to 1600 bp. The PCR products originated from the genome of swine, which were verified by Southern hybridization. Compared with previous reports, there are some advantages in our method: the performance is easier, the dissection is more precise and the cost is low. This is the first report about the microdissection and amplification of swine chromosome 12.2.Two microsatellite located on chromosome 12 were used for verification of the PCR product origination. It was confirmed that the amplification DNAs came from chromosome 12. On the other hand, it showed that the microclone library contained abundant single sequence repeats and have potential usage for exploring chromosome specific microsatellite markers.3.Construction of specific DNA library from chromosome 12 of swine. The chromosome 12 DNA library was established by cloning the second round PCR product into pGEM-T easy vectors. The size of clone inserts varied from 250 to 1600 bp (with an average of 500 bp approximately). The library will provide plenty of chromosome-specific probes for genetic mapping. Otherwise our research can provide experience for similarity4.A microsatellite contained 12 (GT) repeats acquired from the microcloning library ofswine chromosome 12.