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目的探讨运用PCR (polymerase chain reaction)方法快速筛选DNA文库的可行性。方法以猪α-1,3GT cDNA片段为探针,用cDNA上的特异性序列合成的引物,采用噬菌斑原位杂交和PCR相结合的方法,对猪的gDNA文库进行α-1,3GT基因筛选,经酶切、Southern Blot、测序和荧光原位杂交定位。结果经过两次杂交和一次PCR即得信号很强的7个阳性单克隆,插入子均在8 kb以上,且包含第三内含子,其中3个片段测序证实含第三、第四外显子;荧光原位杂交证实该基因位于染色体1q2.10-q2.11。结论PCR可以应用于快速筛选DNA文库。
Objective To explore the feasibility of using PCR (polymerase chain reaction) to screen DNA library rapidly. Methods The porcine α-1,3GT cDNA fragment was used as a probe, and primers specific to the cDNA sequence were synthesized. The porcine gDNA library was subjected to α-1,3GT by using plaque in situ hybridization and PCR Gene screening, Southern blot, Southern blot, sequencing and fluorescence in situ hybridization. Results Seven positive clones with strong signal were obtained by two hybridizations and one PCR. The inserts were all over 8 kb and contained the third intron. Three of the three fragments were sequenced to confirm the presence of the third and fourth exon Son; Fluorescence in situ hybridization confirmed that the gene is located on chromosome 1q2.10-q2.11. Conclusion PCR can be used to rapidly screen DNA libraries.