The amplification of a segment of DNA can be achieved using PCR resulting in multiple copies of the target sequence. This occurs in a three-stage cycle consisting of denaturation, annealing and extension from primers, the product of which increases exponentially because the number of new DNA strands is doubled in each cycle. The process is an enzymatic reaction and includes the following components; two oligodeoxynucleotide primers, each binding to a strand of template DNA, a thermostable DNA polymerase able to work at the high and varied temperatures required during PCR and four deoxyribonucleoside triphosphates (dNTP), used to extend the primers.
History behind PCR
PCR, invented in 1985 by Kary Mullis who received a Nobel Prize for the discovery in 1993, first utilised the Klenow fragment of E. coli DNA polymerase I for the reaction (Klug and Cummings 1997). This enzyme denatures at lower temperatures than those needed to denature template DNA and so additional polymerase had to be added after each cycle, in order to continue amplification of the target sequence. Advances in this process include the use of thermostable DNA polymerases, which resist denaturation at high temperatures and so an initial aliquot if polymerase can last for the successive cycles needed in the PCR as well as the development of thermal cyclers or PCR machines, which rapidly change temperature as needed, in an automated programmable manner, resulting in modern PCR.
The theory of PCR
The DNA to be amplified, the template DNA, is denatured to separate each strand by heating at a temperature between 90-95ï¿½C and this separation of the duplex allows the chosen oligonucleotide primers to anneal to their specific homologue within the template sequence. The primers should have a melting temperature (Tm) that allows annealing at temperatures of 55ï¿½-65ï¿½C and are designed to provide starting point for the synthesis of new DNA strands, complementary to the target DNA. If the primer is shorter than 25 nucleotides, Tm is calculated using the following formula;
Tm = 4(G+C) + 2(A+T) (McPherson et al., 1995). The length of the primers chosen is determined to ensure that they bind with the correct sequence on the template strand. Shorter sequences have more chance of multiple binding sites along the template DNA, so primers must be of a length to reduce the probability of this happening. Kocher and Wilson (1993) report that a primer sequence of 17 base pairs should be long enough to bind to a specific unique sequence within mammalian genomic DNA and thus primers should be selected around this length to reduce non-specific annealing events.
The melting temperatures of the two flanking primers should not differ by more than 5ï¿½C, so the G/C content and length must be choosen accordingly. Primers should include between a 40-60% G/C content, a balanced distribution of G/C and A/T rich domains and have no internal secondary structure, bends or loops along the lenght of the primer. It is also important that they are not complementary to each other at the 3′ ends to ensure that primer-dimers will not form.
Extension of the complementary strands from the primers by addition of nucleotides occurs at a higher temperature of between 70-75ï¿½C and needs the action of an heat stable DNA polymerase. Taq polymerase derived from the hot-springs bacterium Thermus aquaticus, which survives temperatures in excess of 85ï¿½C, was first discovered in YellowStone National Park, USA (Brock 1994). This polymerase is therefore able to withstand the high temperatures needed during the reaction.
Taq has no proof-reading 3′ to 5′ exonuclease activity, unlike DNA polymerase I and so mismatches are incorporated at a rate of about one per 9000bp, attributed to dNTP concentration, very low or very high concentrations may result in a higher error rate and since Taq may be damaged in repeated cycling (Kocher and Wilson 1993). Higher fidelity if required can be provided by other polymerases such as Tli isolated from the bacterium Thermococcus litoralis, which provides a proof-reading activity. Tli DNA polymerase replicates DNA at 74ï¿½C catalysing the polymerisation of nucleotides in the 5′-3′ direction and also possesses a 3′-5′ proofreading activity, resulting in increased fidelity of nucleotide incorporation (New England Biolabs 2002).
The importance of Magnesium chloride (MgCl2)
Cations can influence both specific and non-specific annealing of the primers and so the optimal concentration of MgCl2 has to be selected for each experiment. Taq polymerase requires free magnesium ions for activity as well as the magnesium bound by dNTP’s and the DNA, so the magnesium concentration should be slightly higher than that of the nucleotide concentration (Kocher and Wilson 1993).
Nucleotides chelate Mg2+ forming soluble complexes to produce the actual substrate that the polymerase recognises and utilizes for the extension of the new complimentary stand of DNA. Mg2+ effects annealing of the primers to the template DNA by stabilising the primer-template interaction, owing to the neutralisation of the negatively charged phosphate groups on the DNA backbone, reducing the electro-repulsive forces between (Qiagen). It can therefore also increase non-specific annealing and produce undesirable PCR products.
Initial denaturation and extending step
At the beginning of the PCR cycle is it important that the template DNA is entirely denatured, if this does not occur the primers cannot completely anneal to the target sequence, this inefficient annealing in the first amplification can result in a lower yield of the final product. If the GC ratio is less than 50% within the primers the initial denaturation should occur during an interval of between 1 to 3 minutes, if the GC content is higher than this then the interval should be extended for a longer amount of time.
However, Taq polymerase at the high temperatures required to denature the DNA can become unstable, an initial aliquot may be added if the interval is up to 3 minutes, longer than this the Taq should be added after the initial denaturation. Taq DNA synthesis is most efficient at temperatures between 70-75ï¿½C and can reach a rate of extension between 2kb and 4kb a minute. After annealing at the required lower temperatures, extension of the primers should be carried out within this range of increased temperatures. For fragments up to 1 kb approximately a 45-second extension is sufficient, for larger fragments of up to 3 kb, an extra 45 seconds to 1 minute per kb should be allowed. PCR is most effective for fragments of about 2kb in length
Number of cycles The number of PCR cycles depends on the amount of template DNA in the reaction mix, smaller amounts of template DNA should be amplified with more cycles to yield enough product, up to around 40 cycles, if the initial quantity of template DNA is higher, between 25 and 35 cycles are sufficient to create enough product in order to get good clear bands when run out during gel electrophoresis.
Concluding remarks There are many factors that affect PCR as discussed, the parameters of which have to be optimised by practical experimentation, but clearly when utilised under the correct conditions, the revolutionary, fast method of amplification has provided an invaluable tool for any molecular research.
Brock, T.D. (1994). Brock Life at High Temperatures; Biotechnology in Yellowstone. Yellowstone Association for Natural Science, History & Education, Inc. Yellowstone National Park, Wyoming 82190. (website; http://www.bact.wisc.edu/bact303/b27) Klug, W.S., and M.R. Cummings (1997) Concepts of genetics. 6th edition. Prentice Hall.