The menu of site-directed mutagenesis offerings just gained a little flavor—MISO (multichange isothermal) mutagenesis. The technique is described in the recently launched journal ACS Synthetic Biology. It was developed out of the frustrations authors Mitchell et al. experienced with circular mutagenesis. Widely used, circular mutagenesis—in its best-known incarnation as the QuikChange reaction—uses reverse complementary primers containing a point mutation. Annealed to the target plasmid, the primers enable linear cyclic amplification of the entire vector by a high-fidelity polymerase. DpnI digestion of the E. coli-synthesized methylated parental DNA favors transformation of the mutated plasmid. Subsequent refinements have included exponential amplification and multi-site mutagenesis; however, the reaction still requires replication of the whole plasmid, limiting the size that can be accommodated. Like circular mutagenesis, MISO uses mutagenic primers, but instead produces linear PCR products that are isothermally assembled. The isothermal assembly reaction comprises a 5’ exonuclease, a polymerase, and a ligase, which together can join PCR products with homologous ends in a half-hour incubation. For making a couple of mutations, the choice between MISO and circular mutagenesis may boil down to preference. But Mitchell et al. demonstrate MISO's advantages by making eight point mutations in a single plasmid at once using six primer pairs. The overlapping amplicons were gel purified, assembled, and transformed; of 96 colonies, 92 had the correctly assembled plasmid and 100% of those sequenced had the expected mutations. MISO mutagenesis also shines with especially large plasmids. To make a point mutant in the coding sequence of a large protein, the authors found two unique restriction enzymes sites, each a few hundred bases from the site to be mutagenized. Two PCR products were prepared, each with one end at the site to be mutated and the other beyond the flanking restriction site. These overlapping amplicons and the doubly digested plasmid underwent isothermal assembly, creating a 15.3-kb plasmid for which only a few hundred bases of coding sequence needed sequence confirmation. By contrast, this size plasmid would be difficult to prepare with circular mutagenesis (the upper limit is ~10 kb), and the entire coding region would require sequencing since it would have been PCR amplified. MISO mutagenesis can also be used to introduce insertions and deletions, making its applications virtually limitless. Hence, for researchers craving improved site-directed mutagenesis, MISO should feed the need.
Mitchell et al. Multichange isothermal mutagenesis: a new strategy for multiple site-directed mutations in plasmid DNA. ACS Synth Biol. [Epub ahead of print, February 27, 2013; doi:10.1021/sb300131w].Add it up
Assays for typing variable-number tandem repeats (VNTRs) have applications in forensics, population studies, and characterization of microbial samples. Usually the number of repeats is determined by PCR amplifying the VNTR locus and sizing by electrophoresis. However, Choi et al. were interested in finding a way to count VNTRs without having to amplify the target DNA. Although most PCR-free detection schemes use electrochemical sensing, the Choi et al. method, described in ACS Nano, relies on a readout even a preschooler could handle: counting the dots. The dots, of course, are gold nanoparticles, and they are attached to a 30-mer oligonucleotide functionalized with an alkyne at the other end for “click” chemistry. After clicking the alkyne group to an azide moiety placed in the midst of an oligo complementary to a VNTR sequence, a T-shaped probe is produced that, when annealed to target DNA, marks each repeat with a gold nanoparticle. Clearly, a must-have for the assay is certainty that just one probe sequence is linked to each nanoparticle, and the authors show that anion-exchange HPLC can separate gold nanoparticle–DNA monoconjugates from byproducts. In a first test, purified probes were incubated with synthetic templates representing one to four repeats of a human VNTR. After probe-target annealing, ligase was added to join any adjacent VNTR probes, and then the reaction was diluted and viewed by transmission electron microscopy to count the distinct nanoparticles in each cluster of dots. At low repeat numbers, the most frequent cluster size is the correct VNTR number. At larger repeat numbers, many clusters have fewer than expected nanoparticles due to incomplete annealing and ligation; however, the maximum number of dots seen per cluster matches the VNTR number. Choi et al. tested the technique in real-world conditions by typing the D1S80 VNTR locus of buccal swab DNA, showing that the maximum number of 18 dots appeared in 20% of clusters, corresponding to the sequencing-determined value. In sum, this direct inspection assay adds up to a conceptually simple, PCR-free VNTR typing system.
Choi et al. Polymerase chain reaction-free variable-number tandem repeat typing using gold nanoparticle-DNA monoconjugates. ACS Nano. [Epub ahead of print, February 18, 2013; doi: 10.1021/nn400004d].