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采用磁珠富集法,利用生物素标记的(CA)12寡核苷酸探针从黑斑原(鱼兆)基因组DNA MboI酶切的400—1000 bp片段中筛选CA/GT微卫星位点,洗脱的杂交片段克隆到pMD18-T载体上构建富集微卫星基因组文库后,通过PCR筛选检测出720个阳性克隆,占所有克隆的89.2%,从阳性克隆中随机选取139个进行测序,序列分析发现,124个克隆含有7个以上的重复序列,其中完全的为80个(64.5%),不完全的为40个(32.3%),复合的为15个(3.2%),重复次数范围为7—165次,平均为52次。在124条序列中共59条可以设计引物。
The CA / GT microsatellite loci were screened from the 400-1000 bp fragment digested with MboI by the biotinylated (CA) 12 oligonucleotide probe using magnetic bead enrichment , And the elution hybridized fragment was cloned into pMD18-T vector to construct a microsatellite-rich genomic library. 720 positive clones were detected by PCR screening, accounting for 89.2% of all clones. A total of 139 positive clones were selected and sequenced. Sequence analysis showed that 124 clones contained more than 7 repeats, of which 80 were complete (64.5%), 40 (32.3%) were incomplete, 15 (3.2%) were combined, and the range of repetition 7-165 times, an average of 52 times. A total of 59 primers can be designed in 124 sequences.