高GC含量基因NADK的扩增及其真核表达载体的中国细胞生物学学报.PDFVIP

中国细胞生物学学报 Chinese Journal of Cell Biology 2017, 39(4): 458–462 DOI: 10.11844/cj cb.2017.04.0358 技术与方法 NADK2 高GC含量基因 的扩增及其 真核表达载体的构建 陈静怡 张泸娇 刘丹慧* (温州医科大学检验医学院、生命科学学院, 检验医学教育部重点实验室, 浙江省医学遗传学重点实验室, 温州325035) 摘要 在构建线粒体烟酰胺腺嘌呤二核苷酸激酶(mitochondrial nicotinamide adenine dinucleotide kinase, NADK2)真核表达载体时, 该文发现, 使用Ex Taq DNA聚合酶扩增无法得到其编 ′ NADK2 码区域全长, 5 端缺失一段DNA 片段。对其cDNA序列进行分析发现, 编码区GC含量不均衡。 ′ 起始密码子附近GC含量最高可达92.5%, 而3 端终止密码子附近GC含量最低只有27.5%。之后, 使 Taq NADK2 用LA 及GC缓冲液进行扩增, 得到了 基因ORF(open reading frame)全长, 并克隆入真核表 达载体。该文结果对类似高GC含量DNA 片段的PCR扩增有一定的提示作用。 关键词 NADK2 ; PCR; 高GC含量; LA Taq Amplification of GC-Rich NADK2 Gene and Construction of Its Eukaryotic Expression Vector Chen Jingyi, Zhang Lujiao, Liu Danhui* (School of Laboratory Medicine and Life Sciences, Key Laboratory of Laboratory Medicine, Ministry of Education of China, Zhejiang Provincial Key Laboratory of Medical Genetics, Wenzhou Medical University, Wenzhou 325035, China) Abstract The coding region of mitochondrial nicotinamide adenine dinucleotide kinase (NADK2) gene can’t be amplified with Ex Taq DNA polymerase. The PCR products lacks a fragment in the 5′ terminal. We find that the GC distribution is highly uneven in the open reading frame (ORF) of NADK2 gene. The GC content is as high as 92.5% at its 5′ terminal, whereas it is as low as 27.5% at its 3′ terminal. We then use LA Taq with GC