Center for Plant Molecular Biology

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Background

1. SNARE proteins

2. Shaker-like potassium channels in plants

3. Protein-protein interaction techniques in yeast

4. Ternary interactions

5. The "2in1" cloning system

6. Ratiometric Bimolecular Fluorescence Complementation (rBiFC)

7. Site-directed Mutagenesis

Site-directed Mutagenesis

Site-directed Mutagenesis (SDM) is an indispensable tool for structure-function studies. Through a simple PCR reaction with overlapping, complementary oligonucleotide pairs virtually any desired mutation can be introduced in a plasmid-borne gene. The reaction mixture is used to transform E. coli and colonies are screened for mutants through sequencing. To avoid recovery of unmutated template DNA, the PCR reaction is incubated with the restriction endonuclease DpnI which specifically targets and cleaves methylated DNA (see Figure 12).

Figure 12: SDM workflow (A) SDM using a primer pair that includes the desired mutation (red “x”). Identification of positively mutated clones can only be done via sequencing. (B) Introducing an additional silent mutation (green “x”) allows pre-screening of colonies via restriction analysis. (Please note: The colour of the colonies is for visualising purposes only. In reality, all colonies look the same, hence it is not possible to distinguish mutated and template plasmid at this stage)

Since PCR does not introduce methylation of the DNA this method reduces the amount of template DNA. However digest with DpnI is sometimes incomplete and does not include cleavage of hemimethylated DNA (template plus mutated strand). An evaluation of all mutated clones generated through SDM in the past five years in our lab (more than 100 different constructs) revealed that a rescue of 20-30% of un-mutated, wildtype plasmids after DpnI digest is inevitable. This means screening more colonies via sequencing which is time-consuming and costly. For that reason some researchers have decided to introduce silent mutations in addition to the desired point mutation which do not alter the protein but allow distinction on DNA level through restriction sites. Designing an oligonucleotide which fulfills this requirement is hard work with pencil and paper and requires skill and time. The silent mutation needs to take into account the frame as well as finding a potential palindromic sequence that is recognized by a certain restriction endonuclease.

Figure 13: Mutating the pore domain of the Arabidopsis potassium channel AKT1 and functional consequences in vivo. (A) Wild type AKT1 in the Gateway® Entry vector pDONR207 is used as template for SDM of the GYG-motif within the pore-domain of the voltage-gated potassium channel AKT1 which is critical for formation of the channel-pore. SDM-Assist suggested primers for a single mutation of the Glycine 255 to Threonine adding a SnaBI-site, and for mutating both Glycines to Alanine introducing an NdeI site within the gene. Both restriction sites were absent in the wild type sequence. (B) DNA restriction analysis of randomly picked colonies after SDM demonstrates that distinction between wild type (lane 1 and 4) and mutated sequences (lane 2 and 3) becomes easily possible through the addition of a restriction site: an additional band appears in mutated plasmids (arrows). Sequence verification (trace file) is incorporated in (A). (C) Functional consequence of the single or double mutation: The yeast strain SGY1528 (Tang et al. 1995) which is disrupted in potassium uptake can only grow on high K+-medium (+100mM) or on low K+-medium (0.5mM) if an exogenous K+ transporter (such as wild type AKT1) is expressed. Disruption of the GYG-motif in the mutant AKT1-G255T and G255A;G257A renders the channel dysfunctional on low K+-medium, the yeast fails to grow. Equal amounts of yeast were dropped as dilution series on CSM-Leu-, His-, Trp- media with final potassium concentrations of 100mM or 0.5mM. Growth on plates with high K+-concentration was monitored after 2 days and on low K+-plates after 8 days at 30°C. AKT1 and mutants were expressed using the yeast vector pMetYC-Dest. (Figure taken from Karnik et al. 2013)

We have programmed a software that allows for simple, reliable and fast detection of SDM primers. SDM Assist will guide the user in three easy steps through the process of primer design allowing to drag and drop the desired mutation directly on the protein sequence, enabling to pick from different “silent” restriction sites and finally offering a range of primers ranked by their thermodynamic parameters. The software is a stand-alone freeware and can be downloaded here. There is also an online video tutorial that provides guidance. For more information download Karnik et al. 2013a or contact us directly.

Figure 11: Screenshots of SDM-Assist while creating primers to mutate the Arabidopsis potassium channel AKT1. (A) Dragging and dropping of Alanine onto the Glycine residues at position 255 and 257 (black arrow) of the AKT1 sequence creates a stretch of 9 amino acids (below, “Mutated site”) that includes Alanine (orange) instead of Glycine (yellow) residues. Pressing the “Mutagenise” button opens a new window (B) that includes all possible nucleotide sequences derived from those 9 amino acids. The table highlights the mutated nucleotides in red and resulting palindromes which create a restriction site in green. The user has to choose one of the mutated sequences and press the button “primerise” to get a list of potential primers. (Figure taken from Karnik et al. 2013a)