INTRODUCTION: Site directed mutagenesis is molecular biology technique that is used to make specific, intentional and precise changes to the DNA sequence of a gene or any gene products. It is also used to determine the structure and biological activity of DNA, RNA and protein molecules. It is also called as site-specific mutagenesis or oligonucleotide-directed mutagenesis. This method has also found use in understanding important genetic process like DNA conformational analysis and also functioning of processes like transposition and site-specific recombination.
History/Background: Numerous methods have been developed earlier to mutate DNA.
Initially all of they approaches focused on the generation of random mutations in chromosomal DNA for example induced by X-rays and chemicals. While these provided a valuable tool for classical genetic studies, they were limited as they could not target the mutation to a specific gene. Techniques for randomly mutagenizing a genome required screening or selection from massive numbers of mutants to obtain the desired mutation. The ability to control DNA through the use of plasmid vectors became an attraction for newer technologies, which allowed specific changes in discrete, manageable segments of the genome with relatively little effort.
Site-directed mutagenesis method was first benefited from recombinant DNA technology in 1970s, when isolated genes were exposed to conditions such as chemical agents or nucleotide analogs to localize their mutagenic effects. During this time, the use of plasmid vectors for DNA replication greatly enhanced the study of mutations. (Cosby and Lesley). Site-directed mutagenesis can be grouped generally into three categories of which oligonucleotide-directed mutagenesis is by far the most commonly used method.
Mutagenesis is carried out with one single stranded template (usually M13) by annealing a synthetic primer in which defined changes can be incorporated. Second strand synthesis is then initiated with a polymerase and is then transformed into a host like E. coli were the clones are recovered and checked for appropriate mutagenic change by DNA sequencing. Once the mutagenic clone is identified the fragment is subcloned into a vector for further analysis. In detail the procedure employs two mutagenic oligonucleotide primers, one primer contains the desired mutation and the second contains a mutation with a unique, nonessential restriction site.
The two primers are annealed to circular single-stranded DNA and direct synthesis of a new second strand containing both primers. The resulting DNA is transformed into a mismatch repair defective (mutS) E. coli strain, which increases the probability that the two mutations will segregate together during the first round of DNA replication. Even though the site directed mutagenesis is considered as one of the important discovery in molecular biology, it also got risk as the results are mostly unpredicted and surprising. Also, size of template plays a major role in determination of success of site directed mutagenesis. Noll,Kranz 2009) METHOD: The experiment was performed in five steps and the procedure was started with synthesis of the second strand of DNA. In the first part, pUC19M DNA was supplied which was denatured by heating at 1000C for 3 minutes directly by the technician in an eppendorf tube. To another eppendorf tube 14 ? l of denatured pUC19M DNA was transferred to which 2 ? l of 10x annealing buffer (to help the primers to get annealed to the single stranded DNA),2 ? l of mutagenesis primer (to disable the mutation and make the LacZ gene functional) and 2 ? of selection primer (to alter the restriction site which was found on the plasmid) was added. The sample was then heated to 600C for 5 minutes to facilitate the annealing process to take place and was chilled on ice. The tube was later spun to collect the liquid. To 20 ? l of sample in the tube 3 ? l of synthesis buffer (to provide conditions for the synthesis of second strand) , 1 ? l of T4 DNA polymerase ( for helping hybridization of the two DNA strands together), 1 ? l of T4 DNA ligase (to connect the two strands together) and 5 ? l of water was added.
The tube was then mixed and spun briefly and incubated at 370C for 2 hours. The sample was then incubated for 1 week to perform the next step in the second week. The second step of the experiment involved transformation into a host, E. coli. This was performed by diluting 2 ? l of the reaction mixture with 8 ? l of sterile water. To 1 ? l of this diluted DNA 50? l of E. coli BMH 71-18 mutS component cells were added and incubated on ice for 30 minutes. The cells were then heat shocked for 1 minute at 420C and 950 ? l of liquid broth was added and further incubated for 45 minutes with shaking at 370C.
To this mixture 4 ml of liquid broth and 25 ? l of ampicillin (10 ? g/? l) was added to make 5 ml of 50 ? g/ml solution which was shaken overnight in an automatic shaker and stored in refrigerator for a week to perform the next step. After the plasmid was transformed into E. coli the next step involves isolation of plasmid DNA in step 3. The sample from the second week was centrifuged for 5 minutes to collect out 1-10 ml of the pellet and the supernatant was discarded. The pellet was then thoroughly resuspended with 250 ? l of Cell Resuspension Solution (CRS), to prevent any enzymatic activity.
Also 250 µl of Cell Lysis Solution (CLS), to help lysis and get the plasmid DNA was added and mixed thoroughly. Another 10 µl of Alkaline Protease (AP) solution was added to eliminate membrane protein and the mixture properly mixed. The mixture was incubated for 5 minutes at room temperature and then 350 µl of Neutralization Solution (NS) was added to neutralize the pH and mixed. The total mixture of solution was then centrifuged for 10 minutes at room temperature to produce the clear lysate. A spin column with a collection tube was then used to perform affinity chromatography for the binding of plasma DNA.
To do this the sample centrifuged in earlier step was used and the cleared lysate obtained was decanted into spin column and then centrifuged for 1 minute at room temperature, the flow is then discarded and column is reinserted in the collection tube. The column was then washed with 750 ? l of Wash Solution (WS) and centrifuged for 1 minute and discarded the flow through. This was then washed again with 250 ? l of WS and centrifuged for 2 minutes at room temperature to successfully isolate the plasmid DNA on the column. The DNA is then eluted, by transferring the column to a sterile 1. 5 ml microcentrifuge tube carefully.
To this tube 100 ? l of Nuclease free water was added and centrifuged again for 1 minute. The column is then discarded and the DNA sample is transferred into a new eppendorf tube and stored below -200 C and then digestion is performed. The last part of step 3 involves digestion of the plasmid, which was performed by setting up 10 ? l of plasmid pool with 2 ? l of the buffer solution, 7 ? l of sterile water and 1 ? l of the NdeI enzyme. The reason for the addition of NdeI digest to the plasmid DNA is that the old strand DNA has restriction site for NdeI, so the NdeI will cut the strand from the restriction site and become linear DNA.
On the other hand, the new pUC19M DNA does not have restriction site for NdeI therefore NdeI will not bind to it and it will stay circular. This total 20 ? l volume in eppendorf tube is then incubated for 2 hours at 370 C and refrigerated for a week to perform step 4 in the following week. Step 4 of the experiment involved final transformation in which 10 ml of late log phase JM101 cells were taken and spin down in a sterile centrifuge tube at 5000 rpm for 10 minutes.
The supernatant was discarded and the cells were recovered in 4 ml of 100 mM CaCl2 this was done to increase the osmotic pressure inside the cells which would open the pores allowing the entry of the plasmid along with solvents, this was then mixed and resuspended and spun at 2500 rpm for 10 minutes. The cells were again recovered in 100 ? l of CaCl2 and mixed and the tube was allowed to stand on ice for 30 minutes. After recovering the cells successfully, 50 ? l of cell suspension was added to 2 eppendorf tubes one with 5 ? l of NdeI digested DNA from the earlier week and to 2 ? of original plasmid, which was not digested in another tube. The tubes were mixed and made to stand on ice for 30 minutes. The tubes with the cells were then heat shocked at 420 C for 1 minute followed by addition of 950 ? l of Liquid–broth. The tubes were then further incubated for 45 minutes at 370 C. Following the incubation 100 ? l of sample of each tube were added to the labeled two L-agar plates carefully which had ampicillin, X-gal and IPTG and incubated at 370 C overnight and the total number of colonies were counted the following week.
The Step 5 of the experiment involved counting of the white and blue colonies obtained on the plates. Discussion and results: The aim of the experiment was to reconfirm the mutation in pUC19. As pUC19M (variant of pUC19) obtained from E. coli, which already had mutation in the LacZ gene, which would have colourless colonies on X-gal plates and through site directed mutagenesis a complete translation of this gene is performed and reverting the mutation in pUC to wild type to produce blue colonies. In this way the efficiency of the assay can be estimated without sequencing.
The E. coli cells deficient in mis-match repair, this mis-match ensures the strand being copied matches the parental strand as it recognizes the methylation pattern on the parental strand which was necessary for the first transformation as if E. coli had mis-match repair it would have fixed the mutations put in by the primers which is not necessary in the final transformation as the parental strands have been digested with NdeI. The selection primer in the experiment includes a mutation, which is designed in a way to eliminate the unique restriction site.
The mutagenic primer and selection primer are annealed to the denatured pUC19M plasmid, followed by elongation with DNA polymerase. Elongation produces a double stranded DNA molecule, which is transformed into mutS E. coli, resulting in two types of plasmids the parental wild-type plasmid and the mutated plasmid. These plasmids are digested with the restriction enzyme (NdeI). The parental wild-type plasmid is cleaved at the unique restriction site, thus only selecting for the mutated plasmid. The mutated plasmid is then finally transformed into E. coli and cells are recovered.
DNA from each transformants were isolated and allowed to grow on L-agar plates and later screened by sequencing for desire change. It is important that the primers are phosphorylated at 5’ end as the desired mutation is introduced at this end and also to link the two oligonucleotides (DNA ligase to ligase at ends). It is important for the host to be mutS, as E. coli mutS cells are deficient in mis-match repair. The mis-match repair mechanism in E. coli would detect our desired mutation in the NdeI restriction site and would thus restore the mutation. Thus electroporation into mutS allows for our desired mutation to be reserved.
The final transformation do not need to have mutS cells as the parental plasmids with the non-desirable non-functional lacZ gene are already lysed and the desired mutated plasmids have already undergone potential cleavage by the NdeI restriction enzyme. The experiment was consistently efficient and rapid. The mutations may be introduced at any site within any circular DNA molecule which serves one of the advantages. Unfortunately, the yield of the experiment was only 10% as only few pairs have observed the blue colonies while other could only observe white colonies on the plates.
The aim of the experiment was still achieved but with a low yield. The majority of the class was not able to find any blue colonies and the probable reason may be the transformation and the digestion steps may not have performed very accurately. In conclusion, our results suggest that we were able to successfully perform site-directed mutagenesis by eliminating a unique restriction site (NdeI). Using a selection primer to alter the NdeI restriction site and a mutagenic primer to alter the dysfunctional lacZ gene of pUC19M, we successfully selected for the phenotype of the mutated plasmid pUC19 thus suggesting the correct genotype.
However, the correct genotype could not be determined without sequencing. REFERENCES: * In Vitro Site-Directed Mutagenesis using the unique Restriction Site Elimination (USE) Li Zhu, Methods In Molecular Biology Vol 57. Invitro Mutagenesis Protocols. * Site-Directed Mutagenesis by Complementary-Strand Synthesis Using A Closing Oligonucleotide and Double- Stranded DNA Templates S. N. Slilaty, M. Fung, S. H Shen, and S Lebel (1990) Analytical Biochemsitry 185: 194-200. * Site-directed mutagenesis of virtually any plasmid by eliminating a unique restriction site. Deng W. P and Nickoloff J.
A (1992) Analytical chemistry 200: 81-88. * Site directed mutagenesis Paul CARTER, Bichecm. J. (1986) 237, 1-7. * Site-directed mutagenesis of multi-copy-number plasmids: Red/ET recombination and unique restriction site elimination. S. Noll, G. Hampp, H. Bausbacher, N. Pellegata and H. Kranz Biotechniques 46: 527-333 (2009). * Site-directed mutagenesis Neal Cosby and Scott Lesley (1997) Promega Notes magazine Number 61, p. 12. * A simple method for site-directed mutagenesis using the polymerase chain reaction. A. Hemsley, N. Arnheim, M. D Toney and D. J Galas (1989) Nucleic Acid Research.
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