1) Overlap extension PCR
重叠延伸PCR
1.
1 as the template,the PSD-95 mutant genes were amplified by overlap extension PCR,and then were cloned into the plasmids of PSD-95-pcDNA3.
1为模板,采用重叠延伸PCR法获得突变型PSD-95的局部或全长cDNA片段,并克隆至PSD-95-pcDNA3。
2.
According to the amino acids sequence of OC-IΔD86 gene and Escherichia coli codon usage, we synthesized this gene by overlap extension PCR method with 7 oligonucleotides DNA fragments.
根据OC-IΔD86基因序列,设计合成了7条寡核苷酸片段,通过重叠延伸PCR技术合成了OC-IΔD86基因,利用设计好的BamH I/Xho I酶切位点将OC-IΔD86基因克隆到原核表达载体pet21b中,在1mmol/L的IPTG诱导后5h,OC-I?D86融合基因在大肠杆菌中得到表达,表达产物处于可溶状态,其表达量占总蛋白的11。
3.
How to create two separate site-specific mutagenesis in a DNA fragment using overlap extension PCR was explored.
探讨如何利用重叠延伸PCR对同一靶DNA片段中的两个不同位点实施联合突变。
2) overlapping extension PCR
重叠延伸PCR
1.
Cell culture-adaptive mutations were carried out by the method of overlapping extension PCR OE-PCR at the sites of E1202G T1280I and S2197P in the genes of ns3 and ns5a.
方法:根据HCV全长基因组cDNA序列及突变位点设计引物,运用重叠延伸PCR法对ns3和ns5a基因的E1202G、T1280I和S2197P位点进行细胞培养适应性突变,将突变后的片段分别克隆入pBluescriptIIKS(+)、pRSET-A载体,经测序正确后,分别连入含有HCV全长cDNA的质粒的H/FL相应位置,置换原未突变片段,并进行PCR及酶切鉴定。
3) SOE PCR
重叠延伸PCR
1.
SOE PCR and its Application in Genetic Engineering;
重叠延伸PCR技术及其在基因工程上的应用
2.
【Method】 Based on the DNA sequence from GenBank,seven single-strand DNA were designed for amplification of SUMO4 through SOE PCR(gene splicing by overlap extension PCR).
【方法】根据Gen-Bank提供的核酸序列,设计7条单链DNA,采用重叠延伸PCR方法,合成SUMO4基因编码区。
3.
The tat41-101 and tat1-101 gene fragments, encoding Tat41-101aa and Tat1-101aa respectively, were amplified by PCR and used to generate fusion gene tat(B41-101N)by splic-ing by overlap extension PCR(SOE PCR).
方法在HIV-1HXB2株天然Tat蛋白N-端添加Tat41-101位氨基酸(aa),采用PCR法从HIV-1HXB2株tat基因中扩增分别编码Tat41-101aa和Tat1-101aa的tat41-101和tat1-101两个基因片段,重叠延伸PCR法扩增其融合基因ta(tB41-101N),并构建其原核表达质粒pET32a-ta(tB41-101N),经双酶切及测序验证后,转化大肠杆菌BL21(DE3),IPTG诱导表达。
4) splicing overlap extension PCR
重叠延伸PCR
1.
Methods: The VH-linker-VL, namely scFv was prepared by amplifying the VH and VL genes of plasmid pGEM-T-VH and pGEM-T-VL with splicing overlap extension PCR(SOE PCR).
方法:设计以SfiⅠ、NotⅠ为酶切位点、以(Gly4Ser)3为linker的2对引物,从抗人PAF单克隆抗体可变区基因的克隆载体中扩增VH和VL基因,用重叠延伸PCR在VH和VL基因间引入连接短肽,构建VH-linker-VL的scFv基因。
2.
Methods The VH-linker-VL, namely scFv gene was prepared by amplifying the VH and VL genes of plasmid pGEM-T-VH and pGEM-T-VL with splicing overlap extension PCR (SOE PCR).
方法 从抗TfR单克隆抗体重链和轻链可变区基因的克隆载体 pGEM T VH和 pGEM T VL中扩增重链可变区 (VH)和轻链可变区 (VL)基因 ,用重叠延伸PCR的方法 ,在VH和VL基因间引入连接短肽 (Linker) ,构建VH Linker VL的单链抗体 (singlechainFv ,scFv)基因。
3.
Then, the complete gene of AgB was cloned by splicing overlap extension PCR method using 35 nucleotides overlap between the upper and downer fragments and inserted into the vector pVAX1.
采用重叠延伸PCR法(splicing overlap extension PCR method,SOE-PCR)把具有35个相同碱基的上下半段扩增为全长基因,并构建到真核表达载体pVAX1,将酶切、PCR、测序鉴定的阳性质粒经脂质体转染BHK-21细胞。
5) overlap extention PCR
重叠延伸PCR
1.
K88ac-STⅡ fused gene amplified by overlap extention PCR was cloned into T vector.
利用重叠延伸PCR技术将K88ac STⅡ融合基因克隆于T载体上,将重组基因质粒转化到受体菌DH10B中,蓝白斑筛选阳性菌落,通过PCR、NcoI/XcoI酶切后测序,与genbank报道的K88ac结构基因序列进行比对,证明所克隆的目的片段为K88ac STII融合基因。
6) splicing by overlap extension
PCR重叠延伸技术
1.
Methods The PCR product of signal and mature peptides of HD-5 were linked by splicing by overlap extension(SOE).
方法通过PCR重叠延伸技术得到HD-5信号肽及成熟肽的嵌合分子,并将其插入真核表达质粒pVITRO3。
2.
The PCR product of signal and mature peptides of GLP2 was linked by SOE (splicing by overlap extension) and the gene, which coded the second amino acid in the N-terminal of GLP2, was mutated from GCT to GGT by PCR.
方法通过RT-PCR方法获得含GLP2编码序列的高血糖素原cDNA片段;通过PCR重叠延伸技术得到GLP2信号肽及成熟肽的嵌合分子,并将编码GLP2成熟肽N-末端第二位丙氨酸的序列(GCT)突变为编码甘氨酸的序列(GGT);将其插入真核表达质粒pVITRO3,转染至肠上皮细胞(Caco-2),观察其对细胞增殖活性的影响。
补充资料：多肽链延伸因子
多肽链延伸因子
多肽链延伸因子 polypeptide chain elonga－tion factor
蛋白质生物合成中参与多肽链延伸过程的蛋白质因子。从大肠杆菌等原核细胞可纯化获得三种延伸因子（elongation factor），即eftu，efts和efg，分子量大约分别为4.7万，3.6万和8.3万。eftu与gtp结合成eftu-gtp，然后再与氨酰trna结合，形成三复合体（ternary complex）氨酰-trna-eftu－gtp。这种三复合体与核糖体的氨酰trna部位（a部位，aminoacyl site）结合，继而gtp被分解，eftu以eftu－gtp的形式从核糖体上游离出来。游离的eftu－gdp与efts反应，再生成eftu－ts，然后与gtp结合成eftu－gtp。另一方面结合在p部位（peptide transfer）。多肽链即可延长一个氨基酸残基。接着肽基trna从a部位转移（translccation）到p部位，p部位的trna从核糖体上脱离下来。该反应由efg（g因子，亦称为移位酶）催化，再将一分子的gtp水解。如图所示，由于上述反应的逐次反复进行，而使多肽链延伸反应得以进行，每延长一个氨基酸残基，就水解两分子gtp。动物细胞或其他真核细胞基本上也以同样机制进行多肽链的延伸，并且也分离到了对应于原核细胞的各因子。
说明：补充资料仅用于学习参考，请勿用于其它任何用途。