Oligonucleotide-Directed Mutagenesis by Elimination of a Unique Restriction Site (USE Mutagenesis)

2019 ◽  
Vol 2019 (1) ◽  
pp. pdb.prot097782 ◽  
Author(s):  
Matteo Forloni ◽  
Alex Y. Liu ◽  
Narendra Wajapeyee
Keyword(s):  
Restriction Site ◽  
Directed Mutagenesis ◽  
Unique Restriction Site ◽  
Site Use ◽  
Unique Restriction

Oligonucleotide-directed Mutagenesis by Elimination of a Unique Restriction Site (USE Mutagenesis)

2006 ◽  
Vol 2006 (1) ◽  
pp. pdb.prot3742
Author(s):  
Joseph Sambrook ◽  
David W. Russell
Keyword(s):  
Restriction Site ◽  
Directed Mutagenesis ◽  
Unique Restriction Site ◽  
Site Use ◽  
Unique Restriction

scholarly journals Site-directed mutagenesis of multi-copy-number plasmids: Red/ET recombination and unique restriction site elimination

BioTechniques ◽  
2009 ◽  
Vol 46 (7) ◽  
pp. 527-533 ◽  
Author(s):  
Stephan Noll ◽  
Gabriele Hampp ◽  
Hanna Bausbacher ◽  
Natalia S. Pellegata ◽  
Harald Kranz
Keyword(s):  
Copy Number ◽  
Restriction Site ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Unique Restriction Site ◽  
Unique Restriction

scholarly journals Study of the organization of the genomes of Escherichia coli, Brucella melitensis and Agrobacterium tumefaciens by insertion of a unique restriction site

Microbiology ◽  
1995 ◽  
Vol 141 (10) ◽  
pp. 2425-2432 ◽  
Author(s):  
E. Jumas-Bilak ◽  
C. Maugard ◽  
S. Michaux-Charachon ◽  
A. Allardet-Servent ◽  
A. Perrin ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Agrobacterium Tumefaciens ◽  
Restriction Site ◽  
Brucella Melitensis ◽  
Unique Restriction Site ◽  
Unique Restriction

scholarly journals Modification of recombinant human epidermal growth factor (rh-EGF) expression vector by site-directed mutagenesis for therapeutic protein production

2019 ◽  
Vol 24 (1) ◽  
pp. 8
Author(s):  
Achmad Rodiansyah ◽  
Riyona Desvy Pratiwi ◽  
Sabighoh Zanjabila ◽  
Asrul Muhamad Fuad
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Expression Vector ◽  
Restriction Site ◽  
Stop Codon ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Human Epidermal Growth Factor ◽  
Epidermal Growth ◽  
Ecori Restriction

Recombinant human epidermal growth factor (rh-EGF) has high value in therapies for h-EGF deficiency-related diseases. The expression of the h-EGF gene was designed by using the pET21b(+) vector and Escherichia coli BL21(DE3) as the expression host. In a previous study, the sequence of a 6xHis tag without any restriction sites was fused to the h-EGF gene, yet it was not possible to obtain a purified and single rh-EGF by this approach. In this study, we modified the rh-EGF expression vector using site-directed mutagenesis (SDM) to remove the sequence of the 6xHis tag. The vector modification was carried out by inserting a stop codon and the EcoRI restriction site, along with deleting the 6xHis tag sequence. The results of PCR showed non-specific bands, while 2-step cycles PCR produced one non-specific band, and 3-step cycles PCR produced two non-specific bands. After purification of the PCR products, the SDM-recombinant plasmids treated for template plasmid-free product were transformed into E. coli DH5a. Even though the transformation efficiency was low, the planned gene mutations including the deletion of the 6xHis tag and insertion of the stop codon and EcoRI restriction site in plasmid pET21b(+) were successfully carried out. When using this modified vector in expression studies, rh-EGF of a similar size to that of the rh-EGF standard and approximately 1 kDa smaller than the rh-EGF-6xHis of the previous study was obtained.

scholarly journals Straightforward Procedure for Laboratory Production of DNA Ladder

2012 ◽  
Vol 2012 ◽  
pp. 1-4 ◽  
Author(s):  
Vo Thi Thuong Lan ◽  
Pham Thi Thanh Loan ◽  
Pham Anh Thuy Duong ◽  
Le Thi Thanh ◽  
Ngo Thi Ha ◽  
...  
Keyword(s):  
Molecular Biology ◽  
Tandem Repeat ◽  
Restriction Site ◽  
Restriction Enzymes ◽  
Dna Fragments ◽  
Repeated Dna ◽  
Dna Ladder ◽  
Time Saving ◽  
Partial Digestion ◽  
Unique Restriction

DNA ladder is commonly used to determine the size of DNA fragments by electrophoresis in routine molecular biology laboratories. In this study, we report a new procedure to prepare a DNA ladder that consists of 10 fragments from 100 to 1000 bp. This protocol is a combination of routinely employed methods: cloning, PCR, and partial digestion with restriction enzymes. DNA fragments of 100 bp with unique restriction site at both ends were self-ligated to create a tandem repeat. Once being cloned, the tandem repeat was rapidly amplified by PCR and partially digested by restriction enzymes to produce a ladder containing multimers of the repeated DNA fragments. Our procedure for production of DNA ladder could be simple, time saving, and inexpensive in comparison with current ones widely used in most laboratories.

scholarly journals An easy-to-use site-directed mutagenesis method with a designed restriction site for convenient and reliable mutant screening

2009 ◽  
Vol 10 (6) ◽  
pp. 479-482 ◽  
Author(s):  
Bao-zhong Zhang ◽  
Xin Zhang ◽  
Xiao-ping An ◽  
Duo-liang Ran ◽  
Yu-sen Zhou ◽  
...  
Keyword(s):  
Restriction Site ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Mutant Screening

scholarly journals PCR-based cloning and immunocytological titration of infectious porcine endogenous retrovirus subgroup A and B

2002 ◽  
Vol 83 (9) ◽  
pp. 2231-2240 ◽  
Author(s):  
Birke Bartosch ◽  
Robin A. Weiss ◽  
Yasuhiro Takeuchi
Keyword(s):  
Restriction Site ◽  
Current Method ◽  
Endogenous Retrovirus ◽  
Endogenous Retroviruses ◽  
Porcine Endogenous Retrovirus ◽  
293 Cells ◽  
Cloning Technique ◽  
Unique Restriction ◽  
Human Xenotransplantation

Two pig endogenous retroviruses (PERV), PERV-A and -B, productively infect human cells and are therefore considered to constitute a potential risk in pig-to-human xenotransplantation. A PCR-based cloning technique to isolate infectious PERV proviruses was established. Overlapping 3′ half and 5′ halves of PERV proviral genomes were amplified using DNA extracted from human 293 cells infected with PERV-A or -B. These clones were fused at a unique restriction site in the overlapping region and tested for their infectivity. Representative constructs possessed the same infectious properties as their parent isolates. We also developed a polyclonal anti-PERV serum by using recombinant PERV capsid protein derived from one of the infectious constructs as immunogen and established an immunocytological method for detection and titration of PERV infection. This detection method proved to be more sensitive than the current method of choice (transfer of MLV-lacZ vectors) for infectivity assessment of PERV. These findings should be considered for future characterization of PERV isolates.

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