PCR (Polymera Chain Reaction)
Polymera Chain Reaction is widely held as one of the most important inventions of the 20th century in molecular biology. Small amounts of the genetic material can now be amplified to be able to a identify, manipulate DNA, detect infectious organisms, including the virus that cau AIDS, hepatitis, tuberculosis, detect genetic variations, including mutations, in human genes and numerous other tasks.
PCR involves the following three steps: Denaturation, Annealing and Extension. First, the genetic material is denatured, converting the double stranded DNA molecules to single strands. The primers are then annealed to the complementary regions of the single stranded molecules. In the third step, they are extended by the action of the DNA polymera. All the steps are temperature nsitive and the common choice of temperatures is 94oC, 60oC and 70oC respectively. Good primer design is esntial for successful reactions. The important design considerations described below are a key to specific amplification with high yield. The preferred values indicated are built into all our products by default.
1. Primer Length: It is generally accepted that the optimal length of PCR primers is 18-22 bp. This length is long enough for adequate specificity and short enough for primers to bind easily to the template at the annealing temperature.
2. Primer Melting Temperature: Primer Melting Temperature (Tm) by definition is the temperature at which one half of the DNA duplex will dissociate to become single stranded and indicates the duplex stability. Primers with melting temperatures in the range of 52-58 oC generally produce the best results. Primers with melting temperatures above 65oC have a tendency for condary annealing. The GC content of the quence gives a fair indication of the primer Tm. All our products calculate it using the nearest neighbor thermodynamic theory, accepted as a much superior method for estimating it, which is considered the most recent and best available.
Formula for primer Tm calculation:
Melting Temperature Tm(oK)=,ΔH/ ΔS + R ln(C)-, Or Melting Temperature Tm(oC) = ,ΔH/ ΔS + R ln(C)} - 273.15 where
ΔH (kcal/mole) : H is the Enthalpy. Enthalpy is the amount of heat energy possd by substances. ΔH is the change in Enthalpy. In the above formula the ΔH is obtained by adding up all the di-nucleotide pairs enthalpy values of each nearest neighbor ba pair.
ΔS (kcal/mole) : S is the amount of disorder a system exhibits is called entropy. ΔS is change in Entropy. Here it is obtained by adding up all the di-nucleotide pairs entropy values of each nearest n
eighbor ba pair. An additional salt correction is added as the Nearest Neighbor parameters were obtained from DNA melting studies conducted in 1M Na+ buffer and this is the
default condition ud for all calculations.
ΔS (salt correction) = ΔS (1M NaCl )+ 0.368 x N x ln(*Na++)
Where
N is the number of nucleotide pairs in the primer ( primer length -1).
[Na+] is salt equivalent in mM.
[Na+] calculation:
[Na+] = Monovalent ion concentration +4 x free Mg2+.
3. Primer annealing temperature: The primer melting temperature is the estimate of the DNA-DNA hybrid stability and critical in determining the annealing temperature. Too high Ta will produce insufficient primer-template hybridization resulting in low PCR product yield. Too low Ta may possibly
lead to non-specific products caud by a high number of ba pair mismatches,. Mismatch tolerance is found to have the strongest influence on PCR specificity.
Ta = 0.3 x Tm(primer) + 0.7 Tm (product) – 14.9
where,
Tm(primer) = Melting Temperature of the primers
Tm(product) = Melting temperature of the product
4. GC Content: The GC content (the number of G's and C's in the primer as a percentage of the total bas) of primer should be 40-60%.
5. GC Clamp: The prence of G or C bas within the last five bas from the 3' end of primers (GC clamp) helps promote specific binding at the 3' end due to the stronger bonding of G and C bas. More than 3 G's or C's should be avoided in the last 5 bas at the 3' end of the primer.
6. Primer Secondary Structures: Prence of the primer condary structures produced by intermolecular or intramolecular interactions can lead to poor or no yield of the product. They advers
ely affect primer template annealing and thus the amplification. They greatly reduce the availability of primers to the reaction.
i) Hairpins : It is formed by intramolecular interaction within the primer and should be avoided. Optimally a 3' end hairpin with a ΔG of -2 kcal/mol and an internal hairpin with a ΔG of -3 kcal/mol is tolerated generally.
ΔG definition : The Gibbs Free Energy G is the measure of the amount of work that can be
extracted from a process operating at a constant pressure. It is the measure of the spontaneity of the reaction. The stability of hairpin is commonly reprented by its ΔG value, the energy required to break the condary structure. Larger negative value for ΔG indicates stable, undesirable hairpins. Prence of hairpins at the 3' end most adverly affects the reaction.
ΔG = ΔH –TΔS
ii) Self Dimer : A primer lf-dimer is formed by intermolecular interactions between the two (same n) primers, where the primer is homologous to itlf. Generally a large amount of primers are ud in PCR compared to the amount of target gene. When primers form intermolecular dimers muc
h more readily than hybridizing to target DNA, they reduce the product yield. Optimally a 3' end lf dimer with a ΔG of -5 kcal/mol and an internal lf dimer with a ΔG of -6 kcal/mol is tolerated generally.
iii) Cross Dimer : Primer cross dimers are formed by intermolecular interaction between n and antin primers, where they are homologous. Optimally a 3' end cross dimer with a ΔG of -5 kcal/mol and an internal cross dimer with a ΔG of -6 kcal/mol is tolerated generally.
7. Repeats: A repeat is a di-nucleotide occurring many times concutively and should be avoided becau they can misprime. For example: ATATATAT. A maximum number of di-nucleotide repeats acceptable in an oligo is 4 di-nucleotides.
8. Runs: Primers with long runs of a single ba should generally be avoided as they can misprime. For example, AGCGGGGGATGGGG has runs of ba 'G' of value 5 and 4. A maximum number of runs accepted is 4bp.
9. 3' End Stability: It is the maximum ΔG value of the five bas from the 3' end. An unstable 3' end (less negative ΔG) will result in less fal priming.
10. Avoid Template condary structure: A single stranded Nucleic acid quences is highly unstable and fold into conformations (condary structures). The stability of the template condary structures depends largely on their free energy and melting temperatures(Tm). Consideration of template condary structures is important in designing primers, especially in qPCR. If primers are designed on a condary structures which is stable even above the annealing temperatures, the primers are unable to bind to the template and the yield of PCR product is significantly affected. Hence, it is important to design primers in the regions of the templates that do not form stable condary structures during the PCR reaction. Our products determine the condary structures of the template and design primers avoiding them.
11. Avoid Cross homology: To improve specificity of the primers it is necessary to avoid regions of homology. Primers designed for a quence must not amplify other genes in the mixture. Commonly, primers are designed and then BLASTed to test the specificity. Our products offer a better alternative. You can avoid regions of cross homology while designing primers. You can BLAST the templates against the appropriate non-redundant databa and the software will
interpret the results. It will identify regions significant cross homologies in each template and avoid them during primer arch.
Parameters for Primer Pair Design
1. Amplicon Length: The amplicon length is dictated by the experimental goals. For qPCR, the target length is clor to 100 bp and for standard PCR, it is near 500 bp. If you know the positions of each primer with respect to the template, the product is calculated as: Product length = (Position of antin primer-Position of n primer) + 1.
2. Product position: Primer can be located near the 5' end, the 3' end or any where within specified length. Generally, the quence clo to the 3' end is known with greater confidence and hence preferred most frequently.
3. Tm of Product: Melting Temperature (Tm) is the temperature at which one half of the DNA duplex will dissociate and become single stranded. The stability of the primer-template DNA duplex can be measured by the melting temperature (Tm).
4. Optimum Annealing temperature (Ta Opt): The formula of Rychlik is most respected. Our products u this formula to calculate it and thousands of our customers have reported good results using it for the annealing step of the PCR cycle. It usually results in good PCR product yield with minimum fal product production.
Ta Opt = 0.3 x(Tm of primer) + 0.7 x(Tm of product) - 14.9
where
Tm of primer is the melting temperature of the less stable primer-template pair
Tm of product is the melting temperature of the PCR product.
5. Primer Pair Tm Mismatch Calculation: The two primers of a primer pair should have cloly matched melting temperatures for maximizing PCR product yield. The difference of 5oC or more can lead no amplification.
