论文部分内容阅读
目的:修正可溶性型补体受体(sCR1)PCR过程中造成的碱基突变,保证基因序列氨基酸编码的准确性,为蛋白表达和基因转染打下基础。方法:采用掺尿苷寡聚核苷酸介导的定点突变法,对sCR1活性区基因中错配的碱基加以修正,用点杂交方法筛选突变子阳性克隆,测序鉴定。结果:40℃洗膜,放射自显影示除阴性对照和1个样品外,阳性对照和其余19个克隆均显阳性黑斑;56℃洗膜,放射自显影示5个样品为阳性。阳性克隆测序结果正确。结论:掺尿苷寡聚核苷酸介导的定点突变法是一种快速而可靠的DNA突变修正方法,但仍存在较高的假阳性,点杂交有助于阳性克隆的筛选。
OBJECTIVE: To correct the base mutation caused by sCR1 during PCR, to ensure the accuracy of amino acid coding of gene sequence and to lay the foundation for protein expression and gene transfection. Methods: The mismatched bases in sCR1 active region gene were modified by site-directed mutagenesis with uridine oligodeoxynucleotide. The mutant positive clones were screened by dot blot hybridization and sequenced. Results: The membrane was washed at 40 ℃. The positive control and the other 19 clones showed positive spots except for the negative control and one sample. At 56 ℃ for washing, the autoradiography showed 5 positive samples. Positive clone sequencing results correct. CONCLUSION: Site-directed mutagenesis with uridine oligomer incorporation is a rapid and reliable method for DNA mutation correction, but there is still a high false positive rate. Dot blot hybridization is helpful for the screening of positive clones.