Why is site directed mutagenesis important

The mutated residue and the two neighboring residues are shown on top of the DNA sequence. For each primer set, the top DNA sequence is the normal sequence, and the bottom sequence is the sequence of the mutated plasmid.

The mutated bases are shown in lower case and red color. Each tandem repeat of the primer is marked with a different background-color and numbered in roman numerals. Note that in the color-marked regions, a few bases are missing from the ends of the primer-copies. These few bases apparently participated in primer-primer annealing. Sequence traces from the ABI Sequencer are shown only for two repeats of the primers to allow visualization of the direct sequencing result.

The roman numerals below the traces relate to the roman numerals of the repeats in the respective sequence above the trace. To overcome the problem of primer-primer annealing observed with the double primer-procedure, we developed an alternative single-primer PCR procedure outlined in Figure 1.

We first tested this protocol using the same primer-sets that did not work with the double-primer method. Seeing the success of the protocol we continued to employ it to generate all the mutants needed for our studies. After each transformation we obtained consistently several hundred colonies. We routinely checked five colonies at random from each transformation by direct sequencing of the plasmid. In 26 transformations that we carried out, the number of plasmids with the designed mutated sequence averaged 3.

Plasmids that did not have the expected mutated sequence had the original sequence without a mutation. So far, we have not observed tandem repeats of a primer as we observed in the double-primer PCR method Figure 2. In 26 SDM experiments, the length of the primers ranged between 31—36 nt. These ranges slightly exceed the primer-design guidelines noted in Methods. Since each mutation has to be located at a specific position, we could not always find a suitable primer sequence within the guidelines.

Although, we exceeded slightly the guideline range, all the primers worked successfully. The method has been used in two other laboratories Prof. Nathan Dascal and Prof. The agarose gel in Figure 3 shows the DNA products of one sample at successive steps of our procedure. Plasmid alone, prior to PCR, shows two major bands. Additional smaller bands represent non-specific PCR products.

Ethidium bromide stained gel is shown in inverted color black to white , without any additional digital image editing. Therefore, the newly synthesized full size linear plasmid strands do not appear as strong bands on the agarose gel. In routine application of our parallel PCR SDM method, we carry out all steps of the method successively without visualizing intermediate products during the whole procedure, i. We combine the contents of the two PCR tubes, and proceed with the denaturation and reannealing protocol in Table 3.

After this step the DNA is directly taken for transformation into competent E. Site-directed mutagenesis methods are crucial in analyzing structure-function relationships. But, the large number of methods published in the literature attest to the difficulty of executing these methods reliably and efficiently. We observed that widely used double-primer protocol resulted in insertion of multiple copies of primer probably due to primer-primer annealing Figure 2.

The site-directed mutagenesis method we present here circumvents the use of primer pairs in the same PCR. We have tested the method in our studies on ENaC subunits, and we have also submitted the method for independent testing by two other laboratories as noted in the Results.

So far we have observed no instance of multiple primers in the clones we have isolated. The stability of the mutant enzyme was significantly reduced, and it had no detectable activity. No protein was detected for the insertion variant.

The other three mutations manifested enzymes with similar expression levels in the soluble fraction, as compared to the wild-type level.

GALE deficiency exists in a continuum, from generalized to peripheral via intermediate Openo, Schulz et al. If GALE is deficient in all tissues, it is classified as generalized; and, if it is only deficient in red and white cells but normal in other tissues, it is known as peripheral deficiency. It is possible that the presence of bi-allelic amorphic mutations is incompatible with life Sanders, Sefton et al. Infants with generalized deficiency develop disease on a lactose-containing milk diet, while infants with peripheral disease remain well, at least in the newborn period.

Genomic GALE is about 5 kb in length, with multiple alternatively spliced transcripts. Others have reported a few cases Alano, Almashanu et al. The V94M mutation has been reported in the homozygous state as being associated with generalized disease Wohlers, Christacos et al. In-depth studies of the V94M mutation through SDM in the yeast system showed that this mutation severely damages the specific activity of the enzyme predominantly at the level of V M without affecting its abundance and thermal stability Wohlers, Christacos et al.

In the same study, the G90E mutation was shown to have zero enzymatic activity, rendering the mutant enzyme to high temperature and protease Wohlers, Christacos et al. Other missense mutations have not yet been reported in patients, but they have been studied in vitro or in model systems.

The LP mutation encodes an enzyme that experiences severe proteolytic degradation during expression and purification. Also the authors showed that enzymes resulting from the N34S , G90E and DG mutations exhibited increased susceptibility to digestion in limited proteolysis experiments Timson Two other mutations, DG and LM , that which are associated with intermediate epimerase deficiency, manifested enzymes with near normal GALE activity, but with compromised thermal stability and protease-sensitivity Wohlers, Christacos et al.

Three other mutations associated with intermediate forms S81R , TM and PL were analyzed for their kinetic and structural properties in vitro and their effects on galactose-sensitivity of S.

All three mutations result in impairment of the kinetic parameters, principally the turnover number, k cat , compared to the wild-type enzyme.

However, the degree of impairment was mild compared with that seen with the mutation V94M Chhay, Vargas et al. Studies are limited by the fact the many patients are compound heterozygotes and by the observation that dominant-negative interactions may be involved in some of these cases.

Although the Leloir pathway is evolutionarily conserved and is indispensable for productive galactose metabolism, the catalytic mechanisms of the GAL enzymes are largely unknown. Several groups have attempted to combine the techniques of SDM, analytical biochemistry and X-ray crystallography to advance the understanding of the catalytic mechanisms of the different GAL enzymes. This family of proteins was first identified by three highly conserved motifs among the four kinases mentioned above by sequence alignment and analysis.

Interestingly, two different catalytic mechanisms have been proposed for this family. This catalytic base can then abstract a proton from the hydroxyl group of the substrate converting the weakly nucleophilic hydroxyl group into the more strongly nucleophilic alkoxide ion, which then attacks the electron-deficient phosphorus atom in ATP Fig.

In such systems, it is common to find positively-charged lysine or arginine residues close to the catalytic site to help stabilize the negative charges on the enzyme and the substrates.

Studies on MVK suggest this enzyme follows this mechanism. The crystal structure of MVK reveals an aspartate residue in the rat enzyme positioned to act as an active site base.

There is also a lysine residue 13 in rat MVK , which is close to both the putative catalytic aspartate residue and the hydroxyl group of the substrate Fu, Wang et al. Replacement of the lysine residue with a methionine by SDM resulted in a reduced, but non-zero, rate V max was reduced approximately fold Potter, Wojnar et al.

Similar results were observed when the equivalent lysine residue 18 was changed to methionine in yeast mevalonate diphosphate decarboxylase Krepkiy and Miziorko and Miziorkoet al. These results are consistent with this positively-charged residue playing an assisting, but non-vital, role in catalysis. Crystal structures of GALK put it into this mechanism by revealing there are aspartate and arginine residues in the active center close to the galactose C1 hydroxyl group Asp and Arg 37 in the human structure, Asp and Arg 36 in Lactococcus lactis Thoden and Holden and Holdenet al.

Similarly, changing Arg 37 of human GALT to alanine resulted in a nearly inactive enzyme; and lysine resulted in compromised k cat and K M for galactose Tang, et al. In this mechanism, the latter is stabilized by the formation of a hydrogen bond to a neighboring asparagine residue Asn , which is not conserved in the superfamily. The reaction follows the double displacement mechanism as shown in Fig.

The most characteristic feature of the reaction is forming a covalent UMP-enzyme intermediate Arabshahi, Brody et al. The intermediate was isolated by gel permeation chromatography in reaction mixtures containing the enzyme and radiolabeled UDP-Glu, and the radiolabeled intermediate could react with gal-1P or glu-1P to form the corresponding radiolabeled UDP sugar Wong, Sheu et al.

This intermediate is very fragile in slightly acidic solutions but quite stable in strong basic solutions Wong, Sheu et al. Further degradation study of this intermediate confirmed that the nucleophile in GALT, to which the uridylyl group is bonded in the uridylyl-enzyme intermediate, is imidazole N3 of a histidine residue Yang and Frey et al.

Catalytic mechanisms proposed for GHMP kinase. A positively charged residue R 2 , sits close to the catalytic residue and stabilizes the alkoxide ion. Double displacement reactions of GALT. GluP is subsequently released, whereas the enzyme remains bound to UMP. Substituting each of the 15 histidine residues in E. In order to identify which of these two residues is the catalytic residue, two more specific mutations were introduced by SDM, HG and HG , which resulted in loss of function of the enzyme because of the missing imidazole ring of histidine, which might be filled and salvaged by adding exogenous imidazole ring.

Print Page. Traditional PCR When PCR is used for site-directed mutagenesis, the primers are designed to include the desired change, which could be base substitution, addition, or deletion Figure 1.

Figure 1. Site-directed mutagenesis by traditional PCR. Primers incorporating the desired base changes are used in PCR.

As the primers are extended, the mutation is created in the resulting amplicon. Figure 2. Site-directed mutagenesis by primer extension. A Insertion: Primers B and C contain the complementary sequence that will be inserted blue line. The complementary ends of the first round amplicons hybridize and the PCR creates the final product with the desired insertion.

B Deletion: Primers B and C are located on either side of the sequence to be deleted, and contain sequence from both sides of the deletion black or gray additions that match the black or gray original sequence. The overlapping regions of these amplicons hybridize and the PCR creates the final product with the desired deletion.

Figure 3. Site-directed mutagenesis by inverse PCR. Metrics details. Mutagenesis plays an essential role in molecular biology and biochemistry.

It has also been used in enzymology and protein science to generate proteins which are more tractable for biophysical techniques. The ability to quickly and specifically mutate a residue s in protein is important for mechanistic and functional studies.

Although many site-directed mutagenesis methods have been developed, a simple, quick and multi-applicable method is still desirable.

This modified protocol used a new primer design that promoted primer-template annealing by eliminating primer dimerization and also permitted the newly synthesized DNA to be used as the template in subsequent amplification cycles. These two factors we believe are the main reasons for the enhanced amplification efficiency and for its applications in multi-site mutagenesis. Furthermore the new protocol required significantly less parental DNA which facilitated the Dpn I digestion after the PCR amplification and enhanced the overall efficiency and reliability.

Site-directed mutagenesis is the cornerstone of modern molecular biology allowing exquisite control of protein sequence. This technique is essential in functional study, genetic engineering, biochemistry and protein engineering. The last category is particularly diverse, including the humanization of antibodies, introduction of new catalytic activities and creation of proteins more suited to biophysical predominantly structural characterization.

The process while extremely useful and simple does have some limitations [ 13 ]. As the primers completely overlap, self annealing is quite favorable and care in primer design is required to avoid self pairing competing with template annealing. This constraint leads to a lower PCR amplification efficiency. Although increasing the amount of parental template DNA can help to alleviate this problem it can introduce other complications.

Addition of short around 8 base non-overlapping ends to the primers has been reported and this modification simplified primer design [ 13 ]. Schematic presentations of mutagenesis PCR amplification processes. B Using the new primer design to generate single-site mutation, deletion or insertion. C Using the new primer design to generate double mutations, deletions or insertions.

In our structural proteomics lab, site-directed mutagenesis, truncation and deletion mutagenesis are routine methods used by variety of personnel with different expertise in molecular biology. Our modified method uses primers containing extended non-overlapping sequences at the 3' end significantly larger than suggested in [ 13 ] and primer-primer complementary sequences at the 5' end Figure 2.

We used this modified method to make various mutations, including insertions 18 residues and deletions 25 residues in a cloned vraR gene of methicillin resistant Staphylococcus aureus MRSA [ 21 ]. We have also used four primers to create multiple-site mutations, deletions and insertions. This modified procedure has proven to be highly efficient. Schematic diagram of the primer design for site-directed mutagenesis.

Primer designs are shown for site-directed mutation A , deletion B and insertion C. Triangles, DEL and INS indicate the locations of the mutations, deletion and insertion respectively in the primer sequences. Our new primer design scheme minimized the primer-primer dimerisation and enabled the primers to use the PCR products as the template. The schematic presentation of our new primer design is shown in Figure 2.

Each primer pair contains non-overlapping sequences at their 3' end and primer-primer complementary overlapping sequences at the 5' end.

The mutation sites can be placed either in the complementary region or non-overlapping region. Transformation of E. DNA sequencing showed that in each mutagenesis reaction all four transformants contained the desired mutations Figure 3B. Under these conditions neither control experiment gave colonies which harbored the desired mutants despite five attempts.