Structural Biochemistry/The Stepwise Process of How DNA is Cloned and Inserted into the Vector
Cloning in plasmid vectors
The success and easiness of cloning a DNA fragment into a plasmid vector depends on several factors. Cloning is significantly more successful when there is only one DNA fragment to be ligated into the plasmid vector. Compatibility of the ends of the two molecules is extremely essential. Cohesive complementary ends generate more efficient cloning then blunt ends. However, the highest cloning efficiency is achieved with DNA digested by two different restriction enzymes. Ligating templates prepared by this procedure is called directional cloning, because the insert DNA can only be ligated into the vector in a single orientation. This method dramatically decreases background level of non-recombinants. Another popular application is the cloning of PCR products produced by Taq polymerase. This polymerase adds a extra A at the 3'-end of amplified DNA, which assists the cloning of PCR products into a special vector.
Preparation of the DNA insert
The DNA is digested with suitable restriction enzymes to produce compatible ends for cloning. To guarantee that the correct restriction fragment is cloned, the DNA insert should be purified on an appropriate percentage agarose gel by gel electrophoresis. The DNA is then extracted and purified from the agarose gel to enhance ligation efficiency.
DNA inserts can be prepared from genomic DNA through PCR amplification. Designing appropriate forward and reverse primers is an critical step in this process. By designing appropriate flanking primers, a target sequence can be amplified from genomic DNA. Once the PCR reaction has been completed and the target sequence amplified, as stated above, the products can be run through a 1-2% agarose gel and then run through a gel purification kit to purify the specific sized fragments in question. The DNA eluate can then be sequenced for further confirmation of the correct DNA sequence to be cloned. Then the amplified DNA product can be subjected to restriction digests or ligation.
Preparing the vector for cloning
The strategies for preparing the DNA inserts for cloning can be applied to the vector also, with the exception of introducing a restriction enzyme site by PCR.
Cloning vectors can also be prepared for cloning by making sure that the vector is in correct concentrations for the highest efficiency yield. Also, it is important to be sure that the vector is appropriate for ligation with the DNA fragment to be cloned. Similar restriction enzyme sites or digests should be applied to be sure that the fragment can "fit in" to the vector appropriately. Also, specific vectors can be chosen based on certain characteristics of the DNA fragment to be inserted such as A-overhangs or blunt-end cloning.
Ligation of plasmid vector and insert DNA
After the insert DNA and vector have been prepared for ligation, estimate the concentration of each by agarose gel electrophoresis along with molecular weight standards of a known concentration. Different vector:insert DNA ratios can be tested to find the optimal ratio for ligation. Generally, a 1:1 or 1:3 molar ratio works pretty well. Addition of DNA ligase (generally T4 DNA ligase) and ligase buffer containing ATP is required for this ligation step.
Specifically designed vectors can also be purchased where specific polymerase enzymes are already on the entry sites of the vector for easier more efficient cloning of fragments into entry vectors, but at a higher price.
Transformation of DNA into bacteria
Usually this step is subsequent to ligation, transforming the vector-DNA insert into competent cells. The transformed cells are plated on LB plates with selective antibiotics and grown overnight. The vectors with the inserts are screened by various methods: blue/white color screening, antibiotic screening, etc. The correct clones are selected and grown, producing huge amounts of vector with the DNA insert. Plasmid DNA minipreps are then done to obtain these correct vectors of interest from the bacteria.
Cells can be transformed with the cloned vectors through electroporating competent cells or through chemically competent heat sensitive cells. Electroporating involves mixing electrocompetent cells with the cloned vectors and then running an electric current through the cells allowing them to take up the DNA. Cells are then shaken at an appropriate growth temperature in growth media to stimulate growth and then plated on antibiotic-selective LB plates to select for successfully transformed cells that took up the antibiotic resistant cloned vectors. Chemically competent cells are similar to electro-competent cells except they take up the cloned vectors through heat shock rather than electrical current.