How to use this page

• Ape Plasmid Editor Manual It was created and is maintained by Wayne Davis from the University of Utah. The current stable version, as of June 2010, is 1.17 and the software runs on Windows, Mac, and Unix. The software is free but donations are encouraged.
• Bio125 Molecular Biology and Genomics, Spring 2014. Analyze 4 primers on pMSH2.

Please help us by adding new digital resources useful to the community here. Keep this page organized by following the
format and alphabetical organization already in place.

ApE- A plasmid Editor - Jorgensen Lab Make copies of the DNA Sequences for Cut-Outs, one per group; it is helpful if the plasmid DNAs (page 1) are printed on different colored paper from the mammal DNAs (page 2) to help distinguish them during the activity. Then cut the DNA.

All of the links below are organized first by whether they are general reference or a more specified research tool.
The sub-categories are organized alphabetically by research topic. Note that many of the tools listed may fit into
multiple categories, but are only listed once. Tools of this type may be tagged with other relevant categories by
adding a link to another section of the page after the description. Use the _*link format of the following DNA example:

QIIME (Quantitative Insights Into Microbial Ecology) software
Powerful suite of open source bioinformatics tools for performing microbiome analysis from raw sequences. *RNA

This allows for quick navigation around the page and the use of the * in Ctrl + F searches to differentiate between
sections and page body content.

Please help keep this page up to date by fixing old or broken links and by deleting listings that no longer work. If
you think that an item has been placed in the wrong category, feel free to move it. Please be cautious when downloading
software, and make sure to help the community by removing links with harmful content. Content with questionable scientific
value should also be removed.

All information resources are assumed to be online, but research tools should be tagged with ^web for online resources, ^software
for downloadable items, and ^app for items used on phones and tablets. Please tag any additional categories in this
way if needed. Additional suggestions for format changes may be executed directly or discussed on the talk page.

Information resources

References, texts, and information resources.

Databases

Search engines and data repositories. There are thousands available (see MetaBase). The few items listed here are some of the broadest and most powerful

General Search/Reference

• BioNumbers
  • The database of useful biological numbers. (link to database)

• CDC Disease Conditions
  • Filtered index of diseases from the CDC database

• EMBL-EBI
  • Hugely extensive collection of resources, databases, and tools related to diverse aspects of bioinformatics and molecular biology, often containing everything one might need

• Entrez (NCBI)
  • Search all NCBI databases

• MetaBase
  • The database of biological databases - External link

• Tree of Life
  • Collaborative encyclopedia of biodiversity

• Wolfram Alpha
  • Computational server for finding/calculating summary data about a wide range of topics, plus useful widgets like reagent tables and gene lookup

Genome and Metabolism

• BioCyc
  • Collection of thousands of pathway/genome databases for many organisms, plus software tools for understanding their data

• Colibri by Institut Pasteur
  • E. coli genome browser; get sequences, see the position of your gene in the chromosome, see the function of your gene, and other fun stuff. You can also search for protein sequences/motifs within the E. coli genome.

• EcoGene
  • Database of all E. coli genes and sequences (Entrez data is pulled from here)

• EcoCyc
  • Comprehensive biochemical pathway and gene function site for E. coli

• ENCODE
  • Parts list for functional elements in the human genome

• Ensembl
  • Eukaryotic genome browser

• Ensembl Genome
  • Sister to Ensembl, not limited to Eukaryotes

• Inventory of Composable Elements (ICE) - The public instance of the JBEI Registry
  • A DNA part, plasmid, microbial strain, and Arabidopsis Seed online repository with physical sample tracking capabilities
  • A site where you can explore the various features of the JBEI Registry software, and even get some work done!
  • Try out the integrated online tools, including DNA sequence editing and annotation (Vector Editor) and auto-aligning sequencing trace files against a template.
  • Follow a link to the underlying open-source software source code.

• KEGG
  • Genomic database geared towards high-level functions of the biological system

• Mammalian Gene Collection
  • Access to sequence-validated, full-length, protein-coding, mammalian cDNA clones

• MetaCyc
  • Multi-organismal member of BioCyc collection; catalogs entire universe of metabolism

• SGD
  • Saccharomyces genome database (yeast genome)

• UCSC Genome Bioinformatics
  • Sequence reference for a large number of genomes

Protein

• Antibody Resource
  • Enter a protein to search for antibodies and ELISA kits

• BRENDA
  • Comprehensive enzyme information system

• ConSurf
  • Server for the identification of functional regions in proteins

• Genome3D
  • Consensus structural annotations and 3D models for sequences of model organisms (built upon numerous other useful related resources)

• Technical suppport on protein crystallization by Hampton Research
  • Focused on protein crystallization but contains a lot of generally useful information about various reagents with respect to proteins.

• UniProt
  • Extensive database of protein sequence and functional information

RNA

• Appendix by Ambion, Inc.
  • Website with many useful nucleic acid parameters.

• GreenGenes
  • 16S rRNA database and workbench (most recently updated data can be downloaded here)

• Ribosomal Database Project
  • Quality-controlled, aligned and annotated Bacterial and Archaeal 16S rRNA sequences, and Fungal 28S rRNA sequences, and a suite of analysis tools

• SILVA
  • Massive, searchable rRNA database (especially strong for microbes)

Vectors

• Addgene's Vector DB
  • Addgene is a a non-profit plasmid repository where scientists can archive and share their plasmids. Addgene assists with data submission and all tech transfer issues. Plasmids can be requested from Addgene for a fee to cover expenses.
  • AddGene Vectors can be directly imported into Genome Compiler software platform, in which you can easily edit and visualize it.

Lab Techniques

• Barrick Lab of UT Austin
  • Tons of helpful resources like protocols, guides, links, and tools for lab work

• BenchFly
  • Search, view, upload, create, and host scientific protocol videos

• Current Protocols in Molecular Biology
  • Handbook of protocols. (Links to Wiley Online Library)

• Gene expression in E. coli by Ehrmann lab
  • Tips and information on gene expression in E. coli

• Molecular Cloning by Sambrook and Russell
  • Handbook of protocols. Subscription only. External link

• OWW Materials
  • Useful information for making or obtaining reagents, enzymes, buffers, etc.

• On Being a Scientist by National Academy of Science
  • NAS report on responsible conduct in research.

• OWW Protocols
  • Growing collection of laboratory protocols and techniques

• Polony Protocols by Church and Mitra Lab
  • Polonies are colonies of PCR amplicons derived from a single molecule of nucleic acid.

• Protocol-online by Dr. Long-Cheng Li
  • A database of research protocols in a variety of life science fields. It has a popular discussion forum.

• Protocols.io^appweb
  • Huge, open access life science protocol repository for discovery and sharing of scientific methods

• VADLO Search Engine
  • VADLO is a search engine for Life Sciences Protocols, Online Tools, Databases, Software, and Biomedical Powerpoint Lectures. It also has Daily research cartoons, called *'Life in Research' Cartoons.

Microbial physiology

• CyberCell Database (CCDB) by Institute for Biomolecular Design
  • Several tables describing statistical data on E. coli compiled from several sources.

• EcoSal by ASM Press
  • An incomplete web version of the publication Escherichia coli and Salmonella: Cellular and Molecular Biology. (Subscription required)

• Metabolic Pathways Poster PDF by SigmaAldrich
  • Free digital version of the paper Metabolic Pathways map. Because it is a PDF, it is also searchable.

• MicrobeWiki
  • Student edited resource on microbes and microbiology (curated pages reviewed by microbiologists)

Teaching

• Khan Academy (Biology)
  • Selection of science videos and short courses, including the popular Crash Course series

• PhET
  • Interactive simulations for science and math

• ScienceHack
  • Search engine for science videos with a review system

Research Tools

Tools and applications to aid in research tasks.

General

• Benchling^web
  • Online apps for cloning, molecular biology, and analysis

• Bioinformatics Toolbox from DNA2.0 ^web
  • Contains many Javascript tools to do common tasks.

• Cancer Genome Anatomy Project^web
  • Resources and tools related to the characterization of cancer gene expression profiles

• Experiment^web
  • Crowdfunded scientific research

• Genome Compiler^websoftware
  • A powerful, free online & downloadable genetic engineering all-in-one platform for molecular & synthetic biologists

• FastPCR^software
  • An integrated tool for PCR primers or probe design, in silico PCR, oligonucleotide assembly and analyses, alignment and repeat searching

• Galaxy^web
  • Easy-to-use Bioinformatics manipulation tools for UCSC data

• iGEM Parts Registry^web
  • Formerly curated by MIT, this is an open repository of BioBricks; the place for all your standard biological parts.

• Ingeneue by George von Dassow, Eli Meir, Edwin Munro, and Garret Odell at the Center for Cell Dynamics^software
  • An open-source java program for modeling gene regulatory networks. Users can rapidly build networks by specifying their topology, initial conditions, connectivity, and known parameters. Ingeneue can then search/explore paramter space for desired behavior, simulate the effects of noise and mutation, and generate statistics/time graphs of the system.

• MABL^web
  • Reconstruct and analyze phylogenetic relationships between molecular sequences

• OpenCFU^software
  • Local cell colony counting software

• MCDS^software
  • All-in-one molecular cloning and genetic engineering design, simulation & management tool for complex synthetic biology and metabolic engineering project.

• PaR-PaR Laboratory Automation Platform^web
  • PaR-PaR allows researchers to use liquid-handling robots effectively, enabling experiments that would not have been considered previously. After minimal training, a biologist can independently write complicated protocols for a robot within an hour.

• Pinecone by Serotiny ^web
  • High-level design, analysis and transmission of protein constructs. Pinecone matches users' designs with CRO's or DNA synthesizers to produce genetic starting material (dsDNA, plasmid or purified protein). Facilitates mutation and combinatorial protein sets that benefit from manufacturing economies of scale.

• PubChase^softwareapp
  • Generates research article recommendations unique to each user. Site also has a special academic community.

• Science Gateway^web
  • Collection of science-related links, including links to journals, catalogs, and tools. See their about page.

Calculators

• [GdmCl] and [Urea] from refractive index by Sosnick lab ^web
  • GdmCl and urea concentration calculator from index of refraction.

• OligoCalc Oligonucleotide Calculator ^web
  • Calculate a number of parameters for your nucleotide polymers

• Statistics to use from Saint John's University. ^web

DNA

• Affymetrix Tools^software
  • Microchip and SNP analysis software. DevNet Tools and other compatible programs also listed for download and use

• Artemis by the Sanger Center ^software
  • A free DNA sequence viewer and annotation tool (Java based). The Sanger Center also develops a number of other miscellaneous tools for download

• BioEdit^software
  • Windows-only sequence alignment editor (no longer maintained, but free to download)

• BLAST^web
  • Finds regions of similarity between biological sequences.

• CLC Sequence Viewer^software
  • Software environment enabling users to make a large number of bioinformatics analyses, combined with smooth data management, graphical viewing, and output options.

• Gene Design by Boeke lab ^web
  • Collection of online tools for codon optimization and shuffling, restriction site editing, and so on.

• GeneDesigner by DNA2.0 ^software
  • Combine genetic building blocks by drag-and-drop, codon optimize, restriction site editing, sequence oligo design etc. See BMC Bioinformatics 2006 Jun 6;7(1):285 for more detail.
  • Free to download and works on Mac or PC. User agreement is somewhat restrictive, i.e. you cannot sell genes designed using the tool without permission.

• GeneWarrior^web
  • Free/simple online toolset for sequence manipulation

• Genome Compiler^websoftware
  • The Complete set of tools for sequence viewing, annotation and alignment. Free online & downloadable and supports all file formats

• IDT SciTools^web
  • A number of web tools and calculators to assist in the design and execution of molecular biology research

• Jalview^software
  • Program for multiple sequence alignment editing, visualization, and analysis

• NEB Cutter by New England Biolabs, Inc. ^web
  • Tool for finding restriction sites, et cetera.

• PerlPrimer^software
  • Open source PCR primer design. Written in Perl/Tk.

• Primer3^web
  • Tool that lets you pick & evaluate primers from a DNA sequence

• QIIME (Quantitative Insights Into Microbial Ecology) ^software
  • Powerful suite of open source bioinformatics tools for performing microbiome analysis from raw sequences.

• Rare Codon Calculator (RaCC) by NIH MBI Laboratory for Structural Genomics and Proteomics ^web
  • Finds rare codons in a coding sequence.

• Search of rare codons in nucleotide sequence by 'Practical Molecular Biology' ^web

• Sequence file format converter from NIH ^web
  • Web tool for converting between sequence file formats.

• Sequence Manipulation Suite^web
  • Quickly access a host of tools for analyzing and manipulating DNA, RNA, and protein sequences

• SIDD^web
  • Stress induced DNA duplex destabilization. Finds destabilized sites in superhelical DNA.

• UNAFold^web
  • Replacement for mFold for predicting nucleic acid folding. Downloadable and some applications are available online also.

• USEARCH^software
  • High throughput sequence analysis (orders of magnitude faster than BLAST) and other software tools

RNA

• RBS Calculator in Genome Compiler
  • Control translation initiation rate and predict protein production levels.

• Promoter prediction^web
  • both prokaryote and eukaryotic promoter prediction

• UNAfold by Michael Zuker. ^web
  • Apps for predicting RNA and DNA folds, calculating Tm's and free energies. Runs mfold + UNAfold servers

• Sfold^web
  • Statistical Folding and Rational Design of Nucleic Acids. Predicts accessible RNA sites

• Vienna RNA^software
  • RNA secondary structure prediction and design

• XRNA^software
  • Java tools for creating RNA secondary structure diagrams

Protein

• RBS Calculator in Genome Compiler
  • Control translation initiation rate and predict protein production levels

• AGADIR by Serrano lab ^web
  • An algorithm to predict the helical content of peptides.

• AVID by Keating lab ^web
  • An integrative framework for discovering functional relationships among proteins.

• Backbone-dependent rotamer library by Dunbrack lab ^web
  • Libraries of sidechain rotamer from protein structures

• Cn3D by NCBI ^web
  • A helper application for your web browser that allows you to view 3-dimensional structures from NCBI's Entrez retrieval service. It doesn't read PDB files but can be more straightforward to use than DeepView.

• Contact Order Calulator by Baker lab ^web
  • Calculator to determine a protein's contact order

• Dang by Richardsons' lab ^software
  • A command-line tool that generates a table of several useful geometric measurements for each residue or base from a PDB file.

• DeepView by GlaxoSmithKline & Swiss Institute of Bioinformatics ^software
  • Awesome program for viewing and studying protein structure.

• ExPASy Proteomics server by the Swiss Institute of Bioinformatics ^web
  • Collection of links to many pages to calculate parameters of your favorite proteins
  1. Compute pI/Mw
     • A tool which allows the computation of the theoretical pI (isoelectric point) and Mw (molecular weight) for a list of Swiss-Prot and/or TrEMBL entries or for user entered sequences.
  2. ProtParam
     • A tool which allows the computation of various physical and chemical parameters for a given protein stored in Swiss-Prot or TrEMBL or for a user entered sequence.
  3. Translate
     • Translate is a tool which allows the translation of a nucleotide (DNA/RNA) sequence to a protein sequence.

• GETAREA by Sealy Center for Structural Biology ^web
  • Solvent accessible surface areas, atomic solvation energies, and their gradients for macromolecules

• Metazome^web
  • Proteome-level phylogeny and genomics

• Modeller by Sali lab ^software
  • Program for homology or comparative modeling of protein three-dimensional structures by satisfaction of spatial restraints.

• PAIRCOIL2 by Keating and Berger labs ^web
  • Tool to predict the parallel coiled coil fold from sequence using pairwise residue probabilities.

• ProteinProspector by UCSF Mass Spectrometry Facility ^web
  • Proteomics tools for mining sequence databases in conjunction with Mass Spectrometry experiments.

• PyMOL^software
  • Molecular graphics system with an embedded Python interpreter designed for real-time visualization and rapid generation of high-quality molecular graphics images and animations. The latest version does not run on OSX 10.3. (from Kathleen).

• VMD by Theoretical and Computational Biophysics Group at UIUC ^software
  • Molecular visualization program for displaying, animating, and analyzing large biomolecular systems using 3-D graphics and built-in scripting. Generates pretty high resolution pictures of protein structures.

• Zinc Finger Tools by Barbas lab ^web
  • Design Zinc Finger DNA binding proteins

Vectors

• ApE A plasmid Editor ^software
  • Simple tools for plasmid design and manipulation

• GeneDesigner by DNA 2.0 ^software
  • Design genes de novo with a powerful and intuitive user interface

• Genome Compiler^websoftware
  • A free all-in-one platform for intuitive vector design and automatic assembly. Supports main construction techniques, manual and auto sequence annotation, alignment, primer design and compatible to all file formats.

• j5, DeviceEditor, and VectorEditor^web
  • j5: DNA assembly design automation for (combinatorial) flanking homology (e.g., SLIC/Gibson/CPEC/SLiCE/yeast) and type IIs-mediated (e.g., Golden Gate/FX cloning) assembly methods
  • DeviceEditor: a visual DNA design canvas that serves as front-end for j5
  • VectorEditor: a visual DNA editing and annotation tool

• PlasMapper^web
  • Generates and annotates a plasmid map based on sequence data

• Serial Cloner^software
  • Light and intuitive tools for diverse molecular biology manipulations

• SnapGene Viewer^software
  • Free version of SnapGene. Allows mapping of DNA up to 1 Gb in length. Convenient viewing and annotation tools for genetic constructs.

• Vector NTI^software
  • Powerful but expensive vector editor. Supports most sequence types and easily manipulates genetic constructs.

Writing/Composition/Organization

• CambridgeSoft^software
  • Numerous proprietary applications for research and composition (ChemBioDraw is usually accessible via academic institution email)

• Coredemia^web
  • Community for sharing and discussion of research papers

• Google Documents by Google^web
  • Sharable, web-based word processing, spreadsheets, and presentations

• Evernote^softwareapp
  • Powerful cross-platform note-taking, composition, and organization with cloud syncing.
• InstaGrok^software
  • Tool for seeking, collecting, and mapping information

• Inkscape^web
  • Free, feature-rich drawing program with support for SVG and PDF images

• Mendeley^softwareapp
  • Powerful and straightforward local and cloud-based reference manager

• Origin^software
  • Scientific graphing and data analysis software

• Qiqqa^software
  • Organize, annotate, search, and cite references from a PDF library (loads of extra features)

• ReadCube^software
  • Reference manager, similar to Mendeley or Evernote. Can enhance PDFs and search PubMed from within the app.

• ThinkFree Office Online by ThinkFree ^web
  • Beta suite of office applicatins offered over the web

• Vessel^webapp
  • Digital lab notebook for organizing and sharing experiments, findings, protocols, and more.

• WriteBoard by 37signals ^web
  • Permits writing of shareable, web-based text documents

• Writely by Upstartle, LLC. ^web
  • A beta word processor run over the web

• Zoho^web
  • Sharable, web-based word processing, spreadsheets, and presentations (alternative to Google Docs)

See also:

Vectors - info/links related to cloning vectors.

Searching the literature - info/links on searching the scientific literature.

Synthetic Biology - info/links on Synthetic Biology.

Associated Data

Supplementary Materials

Abstract

Multichange ISOthermal(MISO) mutagenesis is a new technique allowing simultaneous introduction ofmultiple site-directed mutations into plasmid DNA by leveraging two existingideas: QuikChange-style primers and one-step isothermal (ISO) assembly.Inversely partnering pairs of QuikChange primers results in robust, exponentialamplification of linear fragments of DNA encoding mutagenic yet homologous ends.These products are amenable to ISO assembly, which efficiently assembles theminto a circular, mutagenized plasmid. Because the technique relies on ISOassembly, MISO mutagenesis is additionally amenable to other relevant DNAmodifications such as insertions and deletions. Here we provide a detaileddescription of the MISO mutagenesis concept and highlight its versatility byapplying it to three experiments currently intractable with standardsite-directed mutagenesis approaches. MISO mutagenesis has the potential tobecome widely used for site-directed mutagenesis.

Site-directed mutagenesis (SDM) is one of the most frequently used techniques inmolecular biology. The QuikChange reaction, developed by Stratagene (La Jolla, CA), isthe standard approach for introducing point mutations into plasmids. Its widespread useto make specific coding changes in proteins has driven fundamental discoveries in thefields of genetics, biology, and biochemistry. QuikChange uses reverse complementarymutational primers to replicate a parent plasmid, introducing mutation(s) at the site ofprimer binding. The DNA replication step is a linear cyclic amplification reaction inwhich a pfu polymerase copies the entire plasmid, stopping uponreaching the primer’s 5′ end; the newly synthesized DNA is nicked andcannot serve as a template in later cycles but rather anneals to form nickeddouble-stranded, mutagenized molecules. Enzymatic digestion of methylated template DNAproduced in E. coli using DpnI reduces the background of wild typeparental molecules in the reaction.

Since its invention, only modest improvements to the QuikChange reaction havebeen proposed,^- and much inefficiency still exists in thissystem. First, while performed on a thermal cycler, DNA replication is linear notexponential. Second, strand displacement by the polymerase results in exponentialamplification of the plasmid and encodes a competing byproduct that cannot transformE. coli. Third, it is difficult to introduce mutations at more thanone location in any single reaction. Fourth, the size of the template is limitingbecause the method relies on replication of the entire plasmid. Fifth, because theentire plasmid must be copied, resequencing all nonselectable coding regions isobligatory. Sixth, background depletion by DpnI can be difficult because a large amountof DNA (50 ng) is recommended for the QuikChange reaction and hemimethylatedheteroduplex DNA is resistant to DpnI digestion.^, Seventh, becauseQuikChange primers are perfectly complementary, primer dimerization is highly favorable;long nick-bridging primers may minimize this and improve amplification. Finally, deletions and insertions largerthan a single codon are generally beyond the scope of the classic QuikChange reaction;however, modified QuikChange protocols attempt to address this shortcoming.

Herein we describe a simple and robust protocol for site directed mutagenesisthat overcomes all of the above challenges of the standard QuikChange reaction. We callour approach Multichange ISOthermal (MISO)mutagenesis since it is capable of introducing multiple DNA modifications in a singlereaction and incorporates a DNA assembly strategy named onestep isothermal (ISO)assembly. MISO mutagenesis isa completely different strategy from QuikChange; however, it still leverages the elegantdesign of QuikChange primers, which are reverse complementary sequences, usually 40 bpin length, that encode desired base changes centrally. In the simplest application ofMISO mutagenesis, two pairs of QuikChange primers are inversely partnered toexponentially amplify two linear, double-stranded PCR products (Figure 1a). The resulting PCR products encode the desiredmutations at each end and moreover share ~40 bp of terminal homology. Theone-step isothermal (ISO) reaction works by using a master mix of three enzymes toseamlessly assemble DNA pieces whose ends contain 30–40 base pairs ofoverlapping sequence (Figure 1b). Briefly, a5′ exonuclease chews back double-stranded DNA molecules to expose complementarysingle-stranded DNA overhangs. Homologous segments then specifically anneal. Next, apolymerase fills in the gapped molecules, and a ligase covalently seals nicks. Thus, PCRproducts with homologous ends, such as those generated through inverse partnering ofQuikChange primers, may be enzymatically joined in vitro using theone-step ISO assembly protocol to generate the desired mutagenized plasmid.

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

To demonstrate a robust capability for multi-site directed mutagenesis, we testedMISO mutagenesis with a set of 6 QuikChange primers encoding 8 base changes. Theseprimers were originally designed to incorporate eight lysine-to-arginine point mutationsinto a 6.5-kb plasmid using a combination of iterative QuikChange reactions and overlapextension PCR.^, We inversely partnered the six primer pairs(Figure 2a) to exponentially amplify sixdouble-stranded DNA fragments, ranging in size from 140 bp to 5.3 kb. The fragments weregel purified, subjected to one-step ISO assembly, and transformed into competentE. coli cells. As a control, one 140-bp fragment was omitted fromthe reaction, and the resulting assembly produced no colonies. Colony PCR reactions on96 individual transformants using two diagnostic primer pairs (Supplementary Figure 1a and b) revealedthat 92/96 clones had assembled correctly (Supplementary Figure 1c). Sequencing of 24correctly assembled constructs revealed that 100% contained all 8 desiredmutations. Thus, in a single round of experimentation we successfully generated alysine-free version of a protein of interest for assessment of post-translationalmodification status. These datasupport MISO mutagenesis as a tremendously improved strategy for multi-site directedmutagenesis.

Three applications of MISO mutagenesis. (a) Simultaneous introduction of eightpoint mutations into the mGOAT coding sequence. Six different pairs ofQuikChange-style primers were partnered (shown by colors) to generate six PCRproducts ranging in size from 140 bp to 5.3 kb and encoding eightlysine-to-arginine substitutions. One-step isothermal assembly reactionefficiently generated the desired lysine-free construct (see Supplementary Figure 1). (b)Introduction of a single point mutation into an existing 15.3-kb constructwithout vector segment amplification. QuikChange-style primers were partneredwith non-mutagenic primers complementary to plasmid ends to generate two PCRproducts (200 bp, 800 bp). Separately, pLD401 was digested with AscI and BstBI,and the large vector fragment was gel purified. The three DNA fragments, withhomologous ends, were subjected to one-step isothermal assembly to construct themutagenized plasmid. Only the region of the plasmid produced by PCR requiredconfirmatory resequencing. (c) Simultaneous introduction of a base substitution,deletion, and insertion into yeast shuttle vectors. One standard set ofQuikChange primers (starred) plus three other primer pairs were partnered (shownby color) to generate four overlapping PCR products. One-step ISO assemblyallowed deletion of two BsmBI sites from a noncoding region, recoding of oneBsaI site in the bla gene, and insertion of a BsaI-flanked RFPgene to generate a new cloning site. [Open gray arrows = codingsequence; stars = point mutations; scissors = unique restrictionenzyme sites; orange circles = undesirable restriction enzymerecognition sites; dashed lines = deleted region.]

Another limitation of QuikChange is the size of the template plasmid. As thereaction mandates replication of the entire construct, the upper limit for QuikChange is~7–10 kb. Further, many large plasmids carry a significant fraction ofcoding region, and any that cannot be functionally validated following QuikChange mustbe entirely resequenced to verify accuracy. Here we demonstrate additional versatilityof MISO mutagenesis to overcome these two issues by coupling MISO mutagenesis with atraditional restriction digestion. To introduce a single point mutation into a 15.3-kbplasmid of which ~9 kb encodes protein sequence (Figure 2b), we identifiedunique restriction enzymes sites flanking the desired base substitution by 200 bp and800 bp and designed primers to anneal beyond these boundaries. In individual PCRreactions, we inversely partnered these primers with two QuikChange-style primersencoding the mutation. Separately, the backbone was digested with the appropriaterestriction enzymes, and the three fragments were gel purified, thereby quicklygenerating three overlapping pieces of DNA amenable to one-step ISO assembly. Weconfirmed introduction of the mutation in five out of five unique transformants bysequencing. Further, one of these constructs was sequence verified at both overlappingjunctions. Here, not only did MISO mutagenesis allow efficient installation of themutation of interest, it also greatly reduced the amount of sequence validationrequired.

DNA modifications of biological relevance are not limited to base substitutions.Rather, the introduction of insertions and deletions into plasmids is often desirable,for instance, to generate fusion proteins, co-expression systems, or to delete proteindomains. To this end, we next used MISO mutagenesis to couple the introduction of apoint mutation with simultaneous deletion of a DNA segment and insertion of a 1-kbsequence into a series of yeast shuttle vectors (Figure2c). Briefly, we neededto recode a single BsaI site within the bla gene, remove two BsmBIsites from a noncoding region of the vector backbone, and construct a new cloning sitewith BsaI sites flanking a red fluorescent protein (RFP) to generate new host plasmidsamenable to Golden Gate assembly. Wedesigned QuikChange primers for BsaI recoding plus three additional pairs of primerswith overlapping overhangs (Figure 2c), which wereused to exponentially amplify four fragments with homologous ends. Gel purification ofall amplification fragments prior to one-step ISO assembly yielded ~99%red colonies, and 20 were miniprepped and the assembly tested by restriction digestswith BsaI and BsmBI (SupplementaryFigure 2). We discovered that 19/20 clones yielded the expected digestionpattern; the single incorrect clone derived from an assembly error in which oneRFP-flanking BsaI site was not intact (Supplementary Figure 2). This highlights that one-step assembly reactionsare prone to mis-assemblies and junctions must be sequence verified. Thus, afterfunctional verification by restriction digestion, we further sequenced the BsaI-RFPjunctions of 8 correctly assembled clones to verify elements that could not beinterrogated by digest; no undesired mutations were found. Taken together, theapplication of MISO mutagenesis presents a simple strategy for making many types ofbiologically relevant DNA modifications in a single round of experimentation.

MISO mutagenesis overcomes many specific technical problems associated withtraditional QuikChange mutagenesis. Since primers are inversely partnered in separatereactions, the problem of primer dimerization is circumvented. Further, MISO mutagenesisaffords exponential rather than linear amplification, thus allowing easy verification ofproduct generation by gel electrophoresis. Background is reduced almost completelythrough template removal by gel purification and/or DpnI digestion. Like QuikChange,however, MISO mutagenesis is limited by DNA sequences that are challenging to amplify byPCR, or may be toxic, unstable, or otherwise not tolerated in bacteria. The error rateassociated with oligonucleotide synthesis is another problem common to both approaches.Limitations specific to MISO mutagenesis derive largely from the ISO-assembly step.First, as demonstrated here, mis-assembly errors can occur during one-step ISO assembly,and it is likely that mis-assemblies will be exacerbated by repetitive or GC-richsequences in homologous regions. It is possible that implementation of alternativeenzymatic assembly strategies may overcome some types of assembly errors.^, Second, the introduction of mutations that are relatively closetogether (e.g., 50–80 bp) may be difficult to achieve using MISO mutagenesis, asthis would require exceedingly long complementary mutagenic primers to encode bothmutations or alternatively generating a very short PCR product. Thus, MISO mutagenesisis likely best applied when desired mutations are close enough to be encoded together onone primer or distant enough to generate a reasonably sized PCR product using twomutagenic primers. Finally, the number of pieces that can be put together by one-stepISO assembly defines the upper limit of DNA modifications introduced during a singleround of MISO mutagenesis. Of course, most of these limitations can be overcome byperforming MISO twice.

Overall, we believe that MISO mutagenesis is a versatile and efficient strategyfor making the most common types of DNA sequence modifications in plasmids. Thisapproach is accessible and cost-effective for all laboratories as it seamlesslyincorporates QuikChange-style primers, which are both familiar and ubiquitous inmolecular biology, and requires no expensive primer purification. Implementation of theone-step ISO assembly protocol is also straightforward as step-by-step instructions toprepare the three enzyme reaction mixture have been comprehensively detailed. Alternatively, kits with all therequired reagents preassembled are available from NEB. Thus, MISO mutagenesis representsan excellent solution for laboratories that infrequently perform SDM in plasmid DNA tothose where it is routine. Notably, single mutations can also be installed using MISOmutagenesis by inversely partnering QuikChange-style primers with a second pair ofnon-mutagenic primers or by employing the approach outlined in (Figure 2b). Indeed, we have used this approach to successfullysalvage failed QuikChange reactions (data not shown).

METHODS

Primer Design

Primers used in this study are listed in Supplementary Table 1. Formutagenic primers, 40 nucleotide exact reverse complementary sequences weredesigned with the base substitution placed centrally, as per the QuikChangemanual (Stratagene). In the case of insertions/deletions, the desired constructwas assembled in silico using the free plasmid editor ApE(http://biologylabs.utah.edu/jorgensen/wayned/ape/), keepingtrack of the junction locations. Primers were then designed to consist of twoparts: an annealing sequence (20–30 nt, T_m ≈ 55°C) and an ‘overhanging’ sequence to generate thehomologous region. In our hands, the minimal homologous region for ISO assemblyis ~30 bp, although 40 bp is recommended,^,and we have succeeded in assembling fragments that overlap by as much as 200 bp.As an example, the primers used to generate Figure2c are diagramed in Supplementary Figure 3. In all cases, overlapping regions wereconfirmed to be unique to each assembly reaction.

PCR Amplification of DNA Fragments

Phusion Polymerase (NEB, F530L) was used to generate all PCR productsdescribed here, although any high fidelity polymerase is appropriate for use.PCR reactions were prepared as follows: 5–10 ng template DNA, 200μM concentration of each dNTP (Takara, 4030), 0.2μM concentration of each primer (Supp. Table 1), 1x Phusion HFbuffer, 0.02 U/μL Phusion DNA polymerase in a finalvolume of 50 μL. Applied Biosystem Veriti 96-WellThermal Cyclers were used for amplifications with an extension time of 30 s/kb.PCR products were either gel purified using the Zymoclean Gel DNA Recovery Kit(Zymo Research, D4002) as per the manufacturer’s instructions orpurified using the DNA Clean & Concentrator kit (Zymo Research, D4003)with or without DpnI treatment (NEB, R0176) for 1 h at 37 °C.

One-Step ISO Assembly

One-step ISO assembly reagents (5X ISO Buffer and Reaction Master Mix)are described in detail elsewhere. PCR products were combined in equimolar amounts in 5μL and mixed with 15 μL ofReaction Master Mix by gentle tapping. One-step isothermal assembly wasperformed at 50 °C in a preheated PCR block for 30 min, and 2μL of each assembly reaction was transformed into50 μL of competent DH5α E.coli cells.

Supplementary Material

Acknowledgments

This work was supported in part by a National Science Foundation (grant MCB-1026068)and a DARPA contract (N66001-12-C-4020) to J.D.B.

ABBREVIATIONS

MISO	multichange isothermal
ISO assembly	one-step isothermal assembly
PCR	polymerase chain reaction
DNA	DNA
nt	nucleotide
bp	base pair

Footnotes

Author Contributions

L.A.M., Y.C., and M.T. are co-first authors of this work and wrote themanuscript. J.C., L.A.M., and M.T. created the figures. L.A.M., J.C., A.M.N.,Y.C., L.D., and M.T. contributed experimental data. The work was performed inthe lab of J.D.B.

The authors declare no competing financial interest.

Supporting Information

Supplementary figures andtable. This material is available free of charge via the Internet athttp://pubs.acs.org.

References

Ape Plasmid Editor Manual Pdf

Ape Gene Editor