- Mary Johnson Ph. D.mary at labome dot comSynatom Research, Princeton, New Jersey, United States
Polymerase Chain Reaction (PCR) is a fundamental technology in molecular biology. It was first conceived in 1983 by a molecular biologist in an unusual way, while driving his car down a California highway. Over the years, PCR had an immediate impact on cloning approaches which are very important to study the functions of genes in living systems. Cloning of any DNA fragments involves four basic steps: isolate the source and the vector DNA; using restriction enzymes, cut both DNAs creating ends that can connect the source DNA with the vector; ligate the cut ends; finally the DNA is transformed in a host cell like bacteria.
PCR cloning includes numerous approaches: TA cloning , ligation independent cloning (LIC) , recombinase-dependent cloning, and PCR-mediated cloning. What cloning method is more suitable? The utility of any method for PCR cloning is critically based on its reliability and simplicity rather than other factors as convenience, price, or efficiency under optimum conditions. Furthermore, the methods which are easy to monitor and optimize are the most reliable. For example, TA cloning and LIC require end modifications that cannot be monitored.
Among all the PCR-mediated cloning forms, overlap extension PCR cloning is a simple and reliable method to create recombinant plasmids, which has been introduced by Bryksin and Matsumura from the Department of Biochemistry at Emory University (Atlanta, Georgia, ISA) . How does it work? First, using appropriate forward and reverse primers the DNA insert is PCR-amplified to obtain a final PCR product with overlapping regions to the vector. Then, the vector and insert are mixed. After denaturation and annealing, the insert hybridizes to the vector and then is extended by Phusion DNA polymerase – which importantly does not possess strand displacement activity, using the vector as template. After several PCR cycles, the result is a fusion plasmid with two nicks (one on each strand). The new plasmid is then transformed into competent E. coli cells, which seal the nicks with DNA repair enzymes.
The researchers first used green fluorescence protein (gfp) to test this method. The gfp gene was PCR-amplified using specific primers to obtain a final PCR product overlapping with the pQE30 plasmid. The new plasmid obtained was used to transform E. coli while the parental plasmid was destroyed by the restriction endonuclease DpnI. For the overlap extension PCR, they tested five different DNA polymerase showing that Phusion DNA polymerase was more efficient for overlap extension PCR cloning probably due to its superior processivity and fidelity. Other scientists have developed a similar method using the Taq DNA polymerase reporting similar cloning efficiency. But the limit of the Taq DNA polymerase is the less processivity and fidelity compared to the Phusion DNA polymerase.
Furthermore, they showed that high concentration of the insert and relatively low annealing temperatures are critically for the efficiency of the reaction. They also measured the efficiency as a function of temperature cycles showing that the number of clones had a peak at 17-18 PCR cycles, slightly decreasing with further cycles. The insert/plasmid ratio is also important. The researchers tested three different ratio (1:5; 1:50 and 1:250) demonstrating that the 1:250 ratio produced the most recombinant clones.
The main limit of the overlap extension PCR cloning is due to insert length. The scientists observed that the number of colonies was decreased considerably with the increase in insert length. They suggested a limit of 6.7 Kb. Furthermore, the error rate associated with their method was less than 3%.
In conclusion, the tools of PCR and cloning are extremely important to enable the routine study of gene function. Overall, the overlap PCR cloning method has showed to be easy to monitor and optimize as any other long PCR protocols and it does not require restriction endonucleases or DNA ligase. For these reasons it can be used as a simple but efficient way to create recombinant plasmids.
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- Haun R, Serventi I, Moss J. Rapid, reliable ligation-independent cloning of PCR products using modified plasmid vectors. Biotechniques. 1992;13:515-8 PMID 1362067