scholarly journals MALS: an efficient strategy for multiple site-directed mutagenesis employing a combination of DNA amplification, ligation and suppression PCR

2009 ◽  
Vol 9 (1) ◽  
pp. 83 ◽  
Author(s):  
Alexey A Fushan ◽  
Dennis T Drayna
Keyword(s):  
Dna Amplification ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Multiple Site ◽  
Efficient Strategy ◽  
Suppression Pcr

scholarly journals The consequences of deglycosylation of recombinant intra-melanosomal domain of human tyrosinase

2017 ◽  
Vol 399 (1) ◽  
pp. 73-77 ◽  
Author(s):  
Monika B. Dolinska ◽  
Yuri V. Sergeev
Keyword(s):  
Oculocutaneous Albinism ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Multiple Site ◽  
Insect Larvae ◽  
Melanin Biosynthesis ◽  
Human Tyrosinase

AbstractTyrosinase, a melanosomal glycoenzyme, catalyzes initial steps of the melanin biosynthesis. While glycosylation was previously studiedin vivo, we present three recombinant mutant variants of human tyrosinase, which were obtained using multiple site-directed mutagenesis, expressed in insect larvae, purified and characterized biochemically. The mutagenesis demonstrated the reduced protein expression and enzymatic activity due to possible loss of protein stability and protein degradation. However, the complete deglycosylation of asparagine residuesin vitro, including the residue in position 371, interrupts tyrosinase function, which is consistent with a melanin loss in oculocutaneous albinism type 1 (OCA1) patients.

Activity and spectroscopic properties of bacterial D-amino acid transaminase after multiple site-directed mutagenesis of a single tryptophan residue

Biochemistry ◽  
1989 ◽  
Vol 28 (2) ◽  
pp. 510-516 ◽  
Author(s):  
A. Martinez del Pozo ◽  
M. Merola ◽  
H. Ueno ◽  
J. M. Manning ◽  
K. Tanizawa ◽  
...  
Keyword(s):  
Amino Acid ◽  
Spectroscopic Properties ◽  
Site Directed Mutagenesis ◽  
Tryptophan Residue ◽  
Directed Mutagenesis ◽  
Multiple Site

scholarly journals Darwin Assembly: fast, efficient, multi-site bespoke mutagenesis

2017 ◽  
Author(s):  
Christopher Cozens ◽  
Vitor B. Pinheiro
Keyword(s):  
Total Population ◽  
Cost Effective ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Multiple Site ◽  
Biotechnological Applications ◽  
Wild Type ◽  
High Quality ◽  
Working Day ◽  
Library Transformation

ABSTRACTEngineering proteins for designer functions and biotechnological applications almost invariably requires (or at least benefits from) multiple mutations to non-contiguous residues. Several methods for multiple site-directed mutagenesis exist, but there remains a need for fast and simple methods to efficiently introduce such mutations – particularly for generating large, high quality libraries for directed evolution. Here, we present Darwin Assembly, which can deliver high quality libraries of over 108 transformants, targeting multiple (> 10) distal sites with minimal wild-type contamination (lower than 0.25% of total population) and which takes a single working day from purified plasmid to library transformation. Darwin Assembly uses commercially available enzymes, can be readily automated, and offers a cost-effective route to highly complex and customizable library generation.

scholarly journals Multiple site-directed mutagenesis via simple cloning by prolonged overlap extension

BioTechniques ◽  
2020 ◽  
Vol 68 (6) ◽  
pp. 345-348
Author(s):  
Rasmus Hejlesen ◽  
Ernst-Martin Füchtbauer
Keyword(s):  
Dna Sequences ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Multiple Site ◽  
Overlap Extension

We describe the application of simple cloning by prolonged overlap extension for multiple site-directed mutagenesis in the same plasmid. We show that it is possible to use this technique with very short PCR templates. The technique is ideally suited for the generation of longer donor DNA sequences for CRISPR/Cas9-mediated homologous repair.

Parallel assembly for multiple site-directed mutagenesis of plasmids

2012 ◽  
Vol 430 (1) ◽  
pp. 65-67 ◽  
Author(s):  
Pu Yan ◽  
XinZheng Gao ◽  
Wentao Shen ◽  
Peng Zhou ◽  
Jun Duan
Keyword(s):  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Multiple Site ◽  
Parallel Assembly

scholarly journals T4 DNA polymerase improves the efficiency of multiple site-directed mutagenesis

2016 ◽  
Vol 30 (4) ◽  
pp. 721-725 ◽  
Author(s):  
Wei Guo ◽  
Shuhui Sun ◽  
Binghua Xie ◽  
Xiao-Jing Zhu ◽  
Zhong-Min Dai
Keyword(s):  
Dna Polymerase ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Multiple Site

scholarly journals A high-efficiency method for site-directed mutagenesis of large plasmids based on large DNA fragment amplification and recombinational ligation

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Kewei Zhang ◽  
Xiaomei Yin ◽  
Kaituo Shi ◽  
Shihua Zhang ◽  
Juan Wang ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
High Efficiency ◽  
Dna Amplification ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Filamin A ◽  
Large Plasmids ◽  
Dna Fragment ◽  
Conventional Methods ◽  
Insertion Mutations

AbstractSite-directed mutagenesis for large plasmids is a difficult task that cannot easily be solved by the conventional methods used in many laboratories. In this study, we developed an effective method for Site-directed Mutagenesis for Large Plasmids (SMLP) based on a PCR technique. The SMLP method combines several effective approaches, including a high-efficiency DNA polymerase for the large DNA amplification, two independent PCR reactions and a fast recombinational ligation. Using this method, we have achieved a variety of mutants for the filamin A gene (7.9 kb) cloned in the pcDNA (5.4 kb) or the pLV-U6-CMV-EGFP (9.4 kb) plasmids, indicating that this method can be applied to site-directed mutagenesis for the plasmids up to 17.3 kb. We show that the SMLP method has a greater advantage than the conventional methods tested in this study, and this method can be applied to substitution, deletion, and insertion mutations for both large and small plasmids as well as the assembly of three fragments from PCR reactions. Altogether, the SMLP method is simple, effective, and beneficial to the laboratories that require completing the mutagenesis of large plasmids.

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