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根据链霉素磁珠和生物素特异结合的特性,用生物素标记的二聚核苷酸重复序列探针从巴氏蘑菇的基因组中分离微卫星序列。将结合于链霉素磁珠上的标记探针同两端连接已知序列人工接头的巴氏蘑菇DNA酶切片段杂交。洗脱未杂交DNA片段后,用磁珠富集的片段建立微卫星文库。挑取522个菌落用对应重复序列为引物进行PCR筛选,得到48个阳性克隆,经测序有32个菌落含微卫星序列。微卫星富集效率为阳性克隆数的67%,总克隆数的6%。除去重复或无效的微卫星序列,在设计出的12对用于鉴别85个巴氏蘑菇的Co60辐射变异株微卫星引物中,有4对引物总共扩增出明显的变异菌株17个。证明有些微卫星位点可用于巴氏蘑菇辐射变异品种的指纹筛选与鉴别。
Based on the specific binding properties of streptomycin beads and biotin, microsatellite sequences were isolated from the genome of P. pastoris with biotin-labeled dinucleotide repeat probes. The labeled probe bound to the streptavidin beads is hybridized with a fragment of a DNA fragment of the bacterium of Parsnipia linked at both ends to an artificial linker of known sequence. After eluting unhybridized DNA fragments, microsatellite libraries were created using magnetic bead-enriched fragments. A total of 522 positive colonies were picked out from 522 colonies using the corresponding repeat sequences as primers. After sequencing, 32 colonies contained microsatellite sequences. The efficiency of microsatellite enrichment was 67% of the positive clones and 6% of the total clones. In addition to the repeated or ineffective microsatellite sequences, out of 12 pairs of microsatellite primers designed for differentiating Co60 radiation from 85 strains of Mushrooms, a total of 17 mutant strains were amplified. Prove some microsatellite loci can be used for fingerprinting and identification of Variation in the radiation of pasteurized mushrooms.