Principles of PCR
Examination of the PCR amplification mechanism reveal its simplicity but also its elegance. Oligonucleotide primers are first designed to be complementary to the ends of the sequence to be amplified, and then mixed in molar excess with the DNA template and deoxyribonucleotides in an appropriate buffer. Following heating to denature the original strands and cooling to promote primer annealing, the oligonucleotides each bind to a different strand of the target fragment. The primers are positioned so that when each is extended by the action of a DNA polymerase, the newly synthesized strands will overlap the binding site of the opposite oligonucleotide. As the process of denaturation, annealing, and polymerase extension is continued the primers repeatedly bind to both the original DNA template and complementary sites in the newly synthesized strands and are extended to produce new copies of DNA. The end result is an exponential increase in the total number of DNA fragments that include the sequences between the PCR primers, which are finally represented at a theoretical abundance of 2n, where n is the number of cycles
Utility of PCR
In addition to the production of double-stranded, blunt-ended DNA fragments which may be formed by PCR, two other features of the PCR scheme contribute greatly to the utility of PCR. First, the position of binding of the primers defines the boundaries of the amplified fragment and therefore the prior molecular cloning requirement of restriction endonuclease recognition sites is not required for PCR. As only a limited number of DNA sequences are restriction sites, PCR greatly increases the flexibility of choice of fragment size and composition. Secondly, it is not necessary for PCR oligonucleotides to be exactly complementary to the template DNA. "Tails" may be added to the 5' end of the primer to introduce sequences within the priming sites which thus may be exploited to introduce restriction endonuclease recognition sites or other useful sequences such as mutations into the amplified DNA. This phenomena allowed the emergence of PCR as a method for rapid DNA cloning
Designing PCR programs
Pipetting and DNA template
• It is best to start pipetting water first, followed by the other ingredients. There was no difference in results when various components of the reaction were pipetted in different orders.
• To minimize the chance of primer binding to the DNA template and to prevent the polymerase from working (even theoretically) prior to the first denaturing step, it is useful to keep the vials on ice while pipetting the ingredients of the reaction.
• Depending on the profile of the laboratory (i.e. current DNA probes in use), pipetting can be done under a laminar flow of sterile air (when plasmids are commonly used in the lab ) or at the bench (when the template DNA is genomic DNA or when a larger amount of DNA is used).
• When plasmids, phages or cosmids are used as templates in PCR, it is very important to use aerosol-resistant pipette tips, otherwise, false positive results are almost always the rule (even trace amounts of these targets provide a sufficient number of copies to allow amplification to work). When using complex templates like genomic DNA (of which, sometimes, tens or hundreds of nano grams are taken in one reaction) such precaution may not be necessary. However, to be on the safe side, it is a good idea to use aerosol resistant tips for every PCR reaction.
• Another problem when pipetting small volumes (1-2 µL) of a complex DNA sample (like genomic DNA) is the likelihood of differences in the amount of DNA actually taken in each PCR vial.
Choosing/designing PCR primers
In designing primers for PCR, the following steps/rules were tested and proven to be useful:
• Length of individual primers between 18-24 bases. Longer primers (30-35 bp) seem to work in more similar cycling conditions compared with shorter primers, and can make multiplexing easier .
• It is desirable (but not absolutely necessary) that the two primers have a close melting temperature or Tm (say, within 5o C or so). If Tm difference between the two primers is high, the lower Tm can be increased by increasing the length of that primer at the 3' end (this can also keep the size of the amplified locus constant) or the 5' end.
• purine:pyrimidine content around 1:1 (maybe 40-60%)
• if possible, primer sequence should start and end with 1-2 GC pairs
• Each primer pair should be tested for primer-primer interactions. For this purpose a useful Macintosh program is "CPrimer", a freeware available at ftp.bio.indiana.edu. This program also provides the melting temperature for the sequences entered, thus helping in designing PCR programs. Very convenient, some web sites offer programs that can be used directly on those sites to do the same functions: (search for optimal primers, melting temperatures).
• Primer sequences should be aligned with all DNA sequences entered in the databases (using BLAST programs) and checked for similarities with repetitive sequences or with other loci, elsewhere in the genome. If two loci are very similar (for example across species) it is useful to design the primers so that at least 1-2 bases at the 3' end are specific for the locus to be amplified
• Cycling conditions and buffer concentrations should be adjusted for each primer pair, so that amplification of the desired locus is specific, with no secondary products (see other pages). If this is not possible, the sequences of the primers should be either elongated with 4-5 bases or simply, changed entirely.
The requirement of an optimal PCR reaction is to amplify a specific locus without any unspecific by-products. Therefore, annealing needs to take place at a sufficiently high temperature to allow only the perfect DNA-DNA matches to occur in the reaction. For any given primer pair, the PCR program can be selected based on the composition (GC content) of the primers and the length of the expected PCR product. In the majority of the cases, products expected to be amplified are relatively small (from 0.1 to 2-3 kb). (For long-range PCR (amplifying products of 10 to 20-30 kb) commercial kits are available). The activity of the Taq polymerase is about 2000 nucleotides/minute at optimal temperature (72-78o C) and the extension time in the reaction can be calculated accordingly.
• As the activity of the enzyme may not be always optimal during the reaction, an easy rule applied successfully by the author was to consider an extension time (in minutes) equal to the number of kb of the product to be amplified (1 min for a 1 kb product, 2 min for a two kb product etc.). Later on, after the product(s) become "known", extension time may be further reduced.
• Many researchers use a 2-5 minutes first denaturing step before the actual cycling starts. This is supposed to help denaturing the target DNA better (especially the hard to denature templates). Also, a final last extension time, of 5-10 minutes, is described in many papers (supposedly to help finish the elongation of many or most PCR products initiated during the last cycle). Both these steps have been tested for a number of different loci, and, based on this experience, neither the first denaturing nor the last extension time changed in any way the outcome of the PCR reaction. Therefore, it is the author's habit not to use these steps (light blue in the table below) anymore.
• The annealing time can be chosen based on the melting temperature of the primers (which can be calculated using the many applications, freely available for molecular biologists). This may work, but sometimes the results may not match the expectations. Therefore, a simple procedure used many times by the author was to use an initial annealing temperature of 54 o C (usually good for most primers with a length around 20 bp or more). If unspecific products result, this temperature should be increased. If the reaction is specific (only the expected product is synthesized) the temperature can be used as is.
• For the seventy or so primers used during this work, a denaturing time of 30-60 seconds was sufficient to achieve good PCR products. To long a denaturing time, will increase the time the Taq polymerase is subjected at high temperatures, and increases the percentage of polymerase molecules that lose their activity.
• Number of cycles. In general, 30 cycles should be sufficient for a usual PCR reaction. An increased number of cycles will not dramatically change the amount of product (see below).
One important observation, coming from experiments with multiplex PCR, is that dNTP stocks are very sensitive to cycles of thawing/freezing. After 3-5 such cycles, multiplex PCR reactions usually did not work well. To avoid such problems, small aliquots (2-5 µl) of dNTP (25 mM each), lasting for only a couple of reactions, can be made and kept frozen at -20o C. However, during long-term freezing, small amounts of water evaporate on the walls of the vial changing the concentration of the dNTP solution. Before using, it is essential to centrifuge these vials at high speed in a microfuge.
This low stability of the dNTP is not so obvious when single loci are amplified.
Another important observation is that, anytime nucleotides are diluted in water, the solution should be buffered (for example with 10mM Tris pH 7.7-8.0, final concentration).Otherwise, an acid pH will promote hydrolysis of dNTP into dNDP and dNMP and will render them useless for enzymatic DNA polymerizing reactions.
An annealing time of 30-45 seconds is commonly used in PCR reactions. Increase in annealing time up o 2-3 minutes did not appreciably influence the outcome of the PCR reactions. However, as the polymerase has some reduced activity between 45 and 65o C (interval in which most annealing temperature are chosen), longer annealing times may increase the likelihood of unspecific amplification products
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