Introduction
Programmed removal of a region of DNA and the introduction of predetermined DNA sequence is an important process in molecular biology that allows for manipulation of genes to yield the desired phenotypic expressions. Site directed mutagenesis is one of the methods applied in modern genetic studies to manipulate nucleotide sequences in cloning of DNA in animal, human and cellular models (Rapley 54). Genes involved in regulatory processes and coding is targeted in such manoeuvres to achieve the desired phenotypic manifestations. Any changes in such genes, brings about a myriad of significant alterations in gene function that enable scientists to study and research on the gene structure, function and coding (Braman 23-25).
Mutagenesis and cloning are made possible through insertion of foreign DNA moieties in a native DNA strand. Special chemicals are used in facilitation of mutagenesis and are as such called mutagenic agents. In some cases, mutagenesis is a spontaneous process that occurs without any prior contact with a mutagenic agent. To regulate the process of mutagenesis, researchers have designed methods that allow for accurate instigation of the mutagenic process. Site directed mutagenesis is one such method and it entails initiation of changes in the nucleotide sequences to bring about desired changes in both protein and DNA moieties (Burton and Freifelder 38).
Earlier on, the procedure utilized single stranded DNA, but with site directed mutagenesis, double stranded DNA moieties can be used. This is achieved through manipulation of the base pair sequences via introduction of foreign base pairs or parts of DNA in a native DNA strand. Some of the mechanisms by which site directed mutagenesis can be achieved include but are not limited to cassette mutagenesis, PCR engineered mutagenesis and primer leeway mutagenesis.
Site directed mutagenesis provides an insight on the function and role of different biomolecules in the body such as proteins, genes and even some unusual amino acids. The lack of predictability of this method is a major setback in its use in modern biotechnological research. In this investigation, the process of mutagenesis is carried out on an imitative plasmid cloning vector pUC19M by utilizing a single strand DNA through primer extension mutagenesis.
The pUC19M is known to have a restriction site referred to as the NdeI restriction site that impedes the phenotypic expression of LacZ gene. Inhibition of phenotypic expression is exhibited by the failure of production of β-galactosidase (Kendrew 122). When pUC19 is cultured in X-gal plates, a blue colour is expected to be produced because of cleavage of X-gal. However, introduction of a restriction site such as the NdeI restriction site in pUC19M inhibits expression of LacZ resulting in formation of white colonies. The plasmid used in this experiment is pUC19M.
Since a single stranded DNA is required to initiate mutagenesis, the plasmid DNA is first denatured using heat to obtain a single strand of DNA. A mutagenic primer and a selection primer are required for the successful incorporation of plasmid DNA into the native DNA strand. Once the double strand DNA is obtained, it is transformed into strains of E.coli termed mutS. Furthermore, sequestration of mutant plasmid is done and the sequestrated plasmid DNA incorporated into E.coli cells.
Method
Second strand synthesis
Approximately 14µl of originally denatured pUC19M DNA prepared and 2µl of 10x forging buffer was added unto it. 2µl of the two primers (mutagenic and selection), were then added and the mixture heated for 5 minutes to attain a temperature of 600C. Annealing was achieved by promptly storing the mixture in the tubes in a pre set icebox. To achieve second strand DNA synthesis, 1µ of T4 DNA polymerase, 1µl DNA ligase and 5µl of water were added to 3µl of the buffer and the whole mixture was incubated at 370C for two hours and then moved to the icebox.
Transformation
2µl of the mixture was taken through a five-fold dilution process and 1µl of the resultant solution added to 100µl of E.coli strain BMH 71-18 mutS through a technique called electroporation. The mixture was then incubated for 30 minutes. Thereafter, the cells were heated at 420C for 1 minute and 950µl of L-broth added immediately with constant shaking for 45minutes at 370C. 4ml of Ampicillin at a concentration of 50µl/ml was added to the broth and the mixture shaken overnight.
Isolation of plasmid DNA
To achieve lysis, centrifugation then suspension of 1.5ml of cells, was carried out in a 250µl resuspension solution containing a lysis solution. To allow for thorough mixing, 10µl of alkaline protease solution was added to the mixture with quadruple inversions. The lytic procedure was brought to a stop by addition of 350µl of a neutralizing solution.
The supernatant containing the plasmid DNA was then isolated from the mixture and transferred to eppendorf tubes. To enable decantation, the spin column was mounted onto the collection tube and centrifugation initiated at 12000rpm for one minute. After this, the remnants of the spin column were washed by use of 750µl of ethanol and centrifugation repeated. The process was again repeated but with the use of 250µl of ethanol and centrifugation for 2 minutes.
The spin column was with caution transferred to 1.5ml micro centrifuge tube to prevent transfer of any of the washing solution used in the experiment. 100µl of nuclease free water was put into the spin column, and centrifugation carried out for one minute. This facilitated disregarding of any column without DNA to allow quantification of DNA retained in the columns. A digest was set up using 10µl of the plasmid DNA pool with NdeI in a total volume of 20µl and then Incubated for 2hrs at 370C.
Final Transformation
Final transformation entailed the recovery and resuspension of JM101 cells in calcium chloride to enable rapid uptake of plasmid DNA by the cells due to increased permeability of the cell membrane. Priming of the cells was done to allow for DNA transformation, after which, separation of the solution into tubes was done with one eppendorf tube containing 5μl of the NdeI digested DNA and the other containing natural original plasmid DNA. The two tubes were then cooled for thirty minutes and then nurtured in 950µl of the L-broth for forty-five minutes. The transformed cells were then cultured on agar plates containing ampicillin, X-gal and IPTG at a temperature of 370C overnight. The number of colonies was counted and percentage of blue colonies calculated.
Questions
1) It is important that primers in the synthesis reaction are phosphorylated at 5΄end. Why?
This promotes ligation of the cloned DNA to the vector DNA. The vector DNA is usually dephosphorylated. The process occurs during the synthesis of the second DNA strand when T4 polynucleotide kinase may be incapable of bringing out phosphorylation of the 5’ region in the course of PCR extension causing formation of partial PCR by-products.
2) In the first transformation (electroporation), it is critical that the host must be mutS, but this is not essential at the second transformation. Why?
MutS is a mismatch repair protein that carries out any repairs during electroporation (first transformation), to maintain the viability of the daughter strand. MutS is not required in the second transformation since it may impede proper expression of the plasmid DNA and as such, a host that does not have MutS is required for accurate replication of the plasmid DNA during the second transformation.
3) Explain the need for Nde1 digest prior to the second transformation. How much of a purification of mutated plasmids resulted from this digest.
In this experiment, ampicillin resistance gene containing plasmids were used. These contain a specific restriction site termed the Ndel 183 restriction site (mutated plasmid). However, digestion of the mutated plasmid was feasible at the specific restriction site and as such, the non-mutated bacterial plasmid was digested to form a linear derivative (Campbell and Farrell 102).
4) When designing the primers for this type of mutagenesis, what features of them is essential?
The type of mutation required is a major factor in designing primers. The crucial step in the whole process is the 5’ phosphorylation of the primers since this step is essential for flawless ligation of the primers to the vector DNA. This is of utmost importance since, due to the inability of T4 DNA polynucleotide kinase to merge them in the course of primer extension. Melting point temperature is the rate-limiting factor for formation of dimmers by primers. Total binding to the vector DNA is impossible and as such, it is vital to incorporate the mutagenic base pairs at the midpoint of the sequence.
Works Cited
Braman, Jeff. In Vitro Mutagenesis Protocols. New York, NY: Humana Press, 2002.
Burton, Tropp, and Freifelder, David. Molecular Biology: Genes to Proteins. Sudbury, MA: Jones & Bartlett Learning, 2007.
Campbell, Mary, and Farrell, Shawn. Biochemistry. Stamford, CT: Cengage Learning, 2006.
Kendrew, John. The Encyclopaedia of Molecular Biology. London: Blackwell Science, 1994.
Rapley, Ralph. The Nucleic Acid Protocols Handbook. New York, NY: Humana Press, 2000.