排除法PCR加变性梯度凝胶电泳鉴别未知基因

E-PCR with DGGE approach for indentifying subclass genes

通过鉴别未知的cry4亚组基因来确定排除法PCR加变性梯度凝胶电泳新方法。方法 :应用排除法PCR的组基因和亚组基因引物扩增杀蚊毒素基因 ,cry4。这些引物是根据已知的cyr4基因的共有或特有DNA片段来设计的。组基因引物扩增产物被用于变性梯度凝胶电泳。平行变性梯度凝胶是由 8%的聚丙烯酰胺加上 2 0 %到 80 %的变性剂组成。结果 :已知的和未知的cry4亚组基因被组基因引物扩增的产物在变性梯度凝胶电泳被分离开。尽管它们之间仅有两个碱基对不同 (T在位置 2 2 4和G在位置 394)。应用组基因和亚组基因引物扩增 ,新发现的基因可被分类到次亚组基因水平。从 5个苏云金芽孢杆菌亚菌中发现三个未发表的cry4亚组基因。结论 :排除法PCR加变性梯度凝胶电泳是一个高度敏感、特异性和可靠性强的新方法 ,可用于鉴别各种未知的亚组基因。

To establish E-PCR with DGGE method as a novel approach for identifying cry4 subclass genes in mosquitocidal strains of B thuringiensis Methods: cry4 ,a mosquitocidal protoxin gene,was amplified by E-PCR with family and type primers that were designed from known conserved and unique regions,respectively The E-PCR family products were samples for DGGE The linear DGGE was performed in 8% polyacrylamide with 20% to 80% denaturant Results:E-PCR family products amplified from known and unknown subclass cry4 genes were separated in DGGE,although their DNA sequences differ in only two base pairs(T in position 224 and G in position 394) When using two pairs of family and type primers,the novel subclass genes were identified in tertiary level A total of three novel subclass cry4 genes were found from five mosquitocidal B thuringiensis strains Conclusion:E-PCR with DGGE approach is a highly sensitive,specific,and reliable method for identifying potential novel subclass genes