Results: 2

1.
Figure 1

Figure 1. From: Multichange Isothermal Mutagenesis: a new strategy for multiple site-directed mutations in plasmid DNA.

Overview of Multichange ISOthermal (MISO) mutagenesis. (a) QuikChange-style primer pairs (A, A′; B, B′) encode reverse complementary 40-nucleotide primers with a base substitution (star). Inverse partnering of primer pairs [A+B′] and [B+A′] in separate PCR reactions yields exponential amplification of two linear pieces of DNA with homologous ends. After template removal (DpnI digestion or gel purification), the mutagenized plasmid is assembled using one-step isothermal assembly.5 (b) One-step isothermal assembly relies on the concerted action of three enzymes. A 5′ exonuclease chews back double-stranded DNA, exposing complementary single strands that anneal. Then a polymerase fills in the gaps, and a ligase seals the nick.

Leslie A. Mitchell, et al. ACS Synth Biol. ;2(8):473-477.
2.
Figure 2

Figure 2. From: Multichange Isothermal Mutagenesis: a new strategy for multiple site-directed mutations in plasmid DNA.

Three applications of MISO mutagenesis. (a) Simultaneous introduction of eight point mutations into the mGOAT coding sequence. Six different pairs of QuikChange-style primers were partnered (shown by colors) to generate six PCR products ranging in size from 140 bp to 5.3 kb and encoding eight lysine-to-arginine substitutions. One-step isothermal assembly reaction efficiently generated the desired lysine-free construct (see Supplementary Figure 1). (b) Introduction of a single point mutation into an existing 15.3-kb construct without vector segment amplification. QuikChange-style primers were partnered with non-mutagenic primers complementary to plasmid ends to generate two PCR products (200 bp, 800 bp). Separately, pLD401 was digested with AscI and BstBI, and the large vector fragment was gel purified. The three DNA fragments, with homologous ends, were subjected to one-step isothermal assembly to construct the mutagenized plasmid. Only the region of the plasmid produced by PCR required confirmatory resequencing. (c) Simultaneous introduction of a base substitution, deletion, and insertion into yeast shuttle vectors. One standard set of QuikChange primers (starred) plus three other primer pairs were partnered (shown by color) to generate four overlapping PCR products. One-step ISO assembly allowed deletion of two BsmBI sites from a noncoding region, recoding of one BsaI site in the bla gene, and insertion of a BsaI-flanked RFP gene to generate a new cloning site. [Open gray arrows = coding sequence; stars = point mutations; scissors = unique restriction enzyme sites; orange circles = undesirable restriction enzyme recognition sites; dashed lines = deleted region.]

Leslie A. Mitchell, et al. ACS Synth Biol. ;2(8):473-477.

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