Introduction
Slicing off, of a predetermined region of a DNA strand and introduction of a selected is a crucial undertaking in the field of molecular biology. This process facilitates various gene manipulation procedures such as cloning and mutagenesis. In addition, it allows for generation of chimeric genes. The main areas targeted in this process are the gene regulatory sites and sequences involved in gene coding. Alteration at any of these areas results in substantial changes in gene functions. As such, these changes provide room for research and understanding of the composition, utility and sequencing of biological moiety.
The traditional approach to mutagenesis entails initiation of mutations at specific sites by use of special compounds called mutagenic agents. Mutagenesis may also occur randomly. Even though traditional approach in screening and identification of single mutations of genes is a wearisome and a not so feasible undertaking, it is carried out regularly. As a result of this, novel methods have been designed for initiation of mutagenesis. An example of such methods what scientists have named site-directed mutagenesis, which is now, a mainstay approaches in manipulation of genes. The approach entails production of mutant biological entities especially proteins and or DNA with specified modifications in the amino acid or nucleotide sequences.
Site directed mutagenesis could be performed in three ways: cassette mutagenesis, primer extension mutagenesis and processes founded on PCR.
In this research, primer extension mutagenesis is used on a derivative plasmid cloning vector pUC19M. This method needs a single strand of DNA initially. To achieve this, the plasmid DNA is first taken through a denaturation process by use of heat to obtain a single stranded DNA. The process entails use of a dual primer system that consists of a “mutagenic primer” that slot in the wanted changes in the plasmid DNA while the other is a “selection primer” that forges itself to the exclusive restriction site and acts as a shield to prevent digestion of plasmid DNA by the restriction enzyme Ndel.
The double stranded DNA obtained are converted into mutS E.coli strains. In addition, the mutant plasmid are isolated from the pool of resultant plasmids and converted to E.coli cells. The plasmid DNA used had been obtained from distinct transformant and taken through a thorough screening process to achieve the wanted changes.
Materials and methods
The process of mutagenesis of pUC19M was carried out as indicated below:
14µl of initially denatured puc19m DNA, was used. Unto this, 2µl of 10x forging buffer was added. The two primers, mutagenic and selection were then added in quantities of 2µl of each primer. The mixture was the heated for 5 minutes to a temperature of 600C. The primers were then taken through an annealing process by instantly storing the tubes with the annealing mixture in a temperature-regulated icebox. Primer dependent DNA synthesis was achieved through addition of 3µl of the buffer for synthesis. Furthermore, 1µ of T4 DNA polymerase, 1µl DNA ligase were added together with5µl of water to the buffer for synthesis. To achieve formation of a second strand of DNA, the reaction mixture was nurtured at 3700C for a period of 2 hours after which it was transferred to the icebox.
A 5-fold dilution was carried out on the 2µl of the reaction mixture, after which 1µl of it was added to 100µl of E.coli strain BMH 71-18 mutS via a process called electroporation. To 100μl of BMH 71-18 mutS cells, 1µl of the diluted DNA was added and nurtured in the icebox for half an hour. The cells were then subjected to heating at a temperature of 42 minutes for 60 seconds after which L-broth was added in quantities of 950µl instantly and then shaken while being incubated at 370C for three quarter of an hour.
Thereafter, 50µl/ml concentration of ampicillin was added as 4ml of the L-broth and shaken overnight. Lysis was then carried out to extract the plasmid DNA by centrifugation then suspension of 1.5ml of cells. All this was carried out in a 250µl resuspension solution, unto which a lysis solution had been added prior to commencement of the process. 10µl of a solution of alkaline protease was incorporated into the mixture and taken through a series of four inversions to allow for maximum mixing. Progression of cell lysis was halted through addition of 350µl of neutralizing solution.
The supernatant was separated and this contained the plasmid DNA. This supernatant was then put into eppendorf tubes to await purification.
The spin column was then attached to the collection tube. After the set up, the supernatant was decanted into the spin column and centrifugation was done at very high speeds for 60 seconds. The flow through from the spin column was washed away and reattachment of the spin column carried out. A washing solution (750µl ethanol) was put into the sample and the process of centrifugation repeated at high speeds. The flow through was again washed of the column before reattachment. The washing solution, this time at 250µl was added and the sample taken through the centrifugation process for about two minutes at very high speeds.
The spin column was cautiously then taken to a micro centrifuge tube with a capacity of 1.5ml, to avoid any transfer of the washing solution. After this, nuclease free water (100µl) was then added to the spin column and centrifuged for 60 seconds at high speeds. As a result of this, any column devoid of DNA was discarded while those that had were retained and the DNA quantified. In our case, it was found out that the tube had 10µl of plasmid DNA. To this amount, 2µl of buffer (x10) was added together with 1μl NdeI for digestion and 7μl sterilized water. The whole concoction was then nurtured for 2 hours at 370C.
The last of these transformation process entailed recovery and resuspension of JM101 cells in calcium chloride to allow expedited uptake of plasmid DNA by the cells as the solution fosters permeability of the cell membrane. Once the cells were primed for DNA transformation, a two-tube separation was done with one eppendorf tube carrying 5μl of the NdeI digested DNA while the other had the unprocessed initial plasmid DNA. Both times were then cooled for half an hour and then incubated in 950µl of the L-broth for quarter an hour for a period of 45 minutes. The cells that had undergone the transformation process, were then cultured on agar plates that had ampicillin, X-gal and IPTG at 370C for an overnight stay before the number of colonies and percentage of blue colonies were counted.
It is important that primers in the synthesis reaction are phosphorylated at 5΄end
Phosphorylation of 5’ ends assists in ligating of the cloned DNA to the vector DNA that is dephosphorylated. The process is usually carried out in the initial synthesis process since T4 polynucleotide kinase may be unable to carry out phosphorylation of the 56’ region in the process of PCR extension resulting in formation of incomplete PCR derivatives.
In the first transformation (electroporation) it is critical that the host must be mut S, but this is not essential at the second transformation
This experiment utilizes guanine instead of thiamine in the daughter strand of the mutagenic primer. Any modifications in the daughter strand is identified and corrected by the MutS. This protein is usually referred to as a mis-match repair protein. Since this repair might interfere with the plasmid DNA, selection of a host that is devoid of this mis-match is necessary. This host is required for the replication of the plasmid DNA in the second stage of transformation.
Explain the need for Nde1 digest prior to the second transformation. How much of a purification of mutated plasmids resulted from this digest
The main principle of the experiment is based on mutagenesis of plasmid DNA with a distinctive restriction site. In this experiment, a plasmid with genes conferring ampicillin resistance was used. The plasmid has an Ndel 183 restriction site. This is the basis of the criteria for the choice of the mutated plasmid. However, in this experiment, digestion of the mutated plasmid was not carried out at this specific restriction site; instead, the non-mutated bacterial plasmid was digested into a linear form.
When designing the primers for this type of mutagenesis, what features of them is essential?
Primers are designed with regard to the type of mutation needed. Phosphorylation of the primers is the rate-limiting step since, phosphorylation at the 5’ position assists in ligating the primers onto the vector DNA. This is crucial since, T4 DNA polynucleotide kinase is unable to integrate them in the process of primer extension. Formation of dimmers by primers is dependent on one essential factor: Melting point temperature (Tm). 100% binding to the vector DNA is not feasible and as such, it is essential to integrate the mutagenic base pairs at the center of the sequence. Better results can be achieved by use of High Performance Liquid Chromatography.
Results and Discussion
The results of the scored plates for 5µl of NdeI and 2µl of original plasmid are shown in the table below.
Table 1: Digested NdeI.
Table 2: Original plasmid.
Ampicillin plates demonstrate the sites that were efficacious with directed mutagenesis. This is indicated by the occurrence of blue colonies. The mutagenic pair brought in a single base pair replacement of the stop codon. The replacement was done by incorporation of trp codon to facilitate total translation of lacZ gene and as such, it feasible to transform the mutant lacZ gene contained in pUC19M to wild form. The blue color on the plates is as a result of lacZ gene translation due to digestion of lactose to glucose by β- galactosidase on X-gal plates a confirmation that mutagenesis occurred. White colonies indicate the failure of digestion of lactose on the X- gal plates.
The frequency of transformation was not high as elaborated by low percentage of the blue colonies on the Ndel digested plates. The original plasmid plate, blue colonies were formed having a percentage yield of 11.11%. This was excessively high since the expectation was not to form any blue colonies since, it has a mutant lacZ gene. As such, there was incomplete translation of lacZ gene, which ideally should have resulted in failure of formation of blue colonies.