Rapid PCR method for site-directed mutagenesis on double-stranded plasmid DNA.
نویسندگان
چکیده
منابع مشابه
New insights into the QuikChangeTM process guide the use of Phusion DNA polymerase for site-directed mutagenesis
The QuikChange™ site-directed mutagenesis method is popular but imperfect. An improvement by using partially overlapping primers has been reported several times; however, it is incompatible with the proposed mechanism. The QuikChange™ method using complementary primers is proposed to linearly amplify a target plasmid with the products annealing to produce double-stranded DNA molecules with 5'-o...
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A general, simple and efficient method for preparing site-specific mutations in double-stranded plasmid DNA without the need for special plasmids, bacterial strains or reagents is described. Only one synthetic oligonucleotide for each mutation is required, subcloning is unnecessary and a high efficiency of mutation (58-97%) was obtained. If two synthetic oligonucleotide primers are used, two se...
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BACKGROUND: Among all common techniques in sitedirectedmutagenesis, λ Red recombinase system has beenwidely used to knock out chromosomal genes in bacteria. In thismethod, there is always the risk of DNA Linear digestion byhost's restriction enzymes that leads to the low frequency ofrecombination. OBJECTIVES:To overcome this, we constructeda recombinant vector to disrupt phoP gene in Salmonella...
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T 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 U N C O R R E C Polymerase chain reactions (PCR) coupled with mutagenic primer-based site-directed mutagenesis have been extensively employed for probing the structure–function relationship of proteins and/or nucleic acids. Many methods to obtain single-basepair changes, deletions, and insertions have...
متن کاملSite-directed mutagenesis using uracil-containing double-stranded DNA templates and DpnI digestion.
DpnI can cleave fully methylated parental DNA while leaving hemi-methylated DNA intact. Based on this observation, we developed a rapid site-directed mutagenesis method using uracil-containing, double-stranded (ds)DNA templates and DpnI digestion. A 38% mutation efficiency was achieved by DpnI treatment of the mutagenic strand-extension reaction, and it increased to 70%-91% when uracil-containi...
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ورودعنوان ژورنال:
- BioTechniques
دوره 20 1 شماره
صفحات -
تاریخ انتشار 1996