关于PCR引物设计原则
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 secondary annealing. The GC content of the sequence 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(K)={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 possessed 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 base 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 neighbor base 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 used 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 caused by a high number of base 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 bases) of primer should be 40-60%.
5. GC Clamp: The presence of G or C bases within the last five bases 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 bases. More than 3 G's or C's should be avoided in the last 5 bases at the 3' end of the primer.
6. Primer Secondary Structures: Presence of the primer secondary structures produced by intermolecular or intramolecular interactions can lead to poor or no yield of the product. They adversely 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 represented by its ΔG value, the energy required to break the secondary structure. Larger negative value for ΔG indicates stable, undesirable hairpins. Presence of hairpins at the 3' end most adversely affects the reaction.
ΔG = ΔH – TΔS
ii) Self Dimer: A primer self-dimer is formed by intermolecular interactions between the two (same sense) primers, where the primer is homologous to itself. Generally a large amount of primers are used in PCR compared to the amount of target gene. When primers form intermolecular dimers much more readily than hybridizing to target DNA, they reduce the product yield. Optimally a 3' end self dimer with a ΔG of -5 kcal/mol and an internal self dimer with a ΔG of -6 kcal/mol is tolerated generally. iii) Cross Dimer: Primer cross dimers are formed by intermolecular interaction between sense and antisense 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 consecutively and should be avoided because 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 base should generally be avoided as they can misprime. For example, AGCGGGGGATGGGG has runs of base '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 bases from the 3' end. An unstable 3' end (less negative ΔG) will result in less false priming.
10. Avoid Template Secondary Structure: A single stranded Nucleic acid sequences is highly unstable and fold into conformations (secondary structures). The stability of these template secondary structures depends largely on their free energy and melting temperatures(Tm). Consideration of template secondary structures is important in designing primers, especially in qPCR. If primers are designed on a secondary 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 secondary structures during the PCR reaction. Our products determine the secondary 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 sequence 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 database and the software will interpret the results. It will identify regions significant cross homologies in each template and avoid them during primer search.
Parameters for Primer Pair Design
1. Amplicon Length: The amplicon length is dictated by the experimental goals. For qPCR, the target length is closer 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 antisense primer-Position of sense primer) + 1.
2. Product Position: Primer can be located near the 5' end, the 3' end or any where within specified length. Generally, the sequence close 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 use 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 false 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 closely matched melting temperatures for maximizing PCR product yield. The difference of 5oC or more can lead no amplification.
Primer Design Parameters for Multiplex PCR
Design of specific primer sets is essential for a successful multiplex reaction. The important primer design considerations described below are a key to specific amplification with high yield.
1. Primer Length Multiplex PCR assays involve designing of large number of primers, hence it is required that the designed primer should be of appropriate length. Usually, primers of short length, in the range of 18-22 bases are used.
2. Melting Temperature Primers with similar Tm, preferably between 55°C-60°C are used. For sequences with high GC content, primers with a higher Tm (preferably 75°C-80°C) are recommended. A Tm variation of between 3°-5° C is acceptable for primers used in a pool.
3. Specificity It is important to consider the specificity of designed primers to the target sequences, while preparing a multiplex assay, especially since competition exists when multiple target sequences are in a single reaction vessel.
4. Avoid Primer Dimer Formation The designed primers should be checked for formation of primer dimers, with all the primers present in the reaction mixture. Dimerization leads to unspecific amplification.
All other parameters are similar to standard PCR primer design guidelines.
Advantages of Multiplex PCR
1. Internal Controls Potential problems in a simple PCR include false negatives due to reaction failure or false positives due to contamination. False negatives are often revealed in multiplex assays because each amplicon provides an internal control for the other amplified fragments.
2. Efficiency The expense of reagents and preparation time is less in multiplex PCR than in systems where several tubes of uniplex PCRs are used. A multiplex reaction is ideal for conserving costly polymerase and templates in short supply.
3. Indication of Template Quality The quality of the template may be determined more effectively in multiplex than in a simple PCR reaction.
4. Indication of Template Quantity The exponential amplification and internal standards of multiplex PCR can be used to assess the amount of a particular template in a sample. To quantitate templates accurately by multiplex PCR, the amount of reference template, the number of reaction cycles, and the minimum inhibition of the theoretical doubling of product for each cycle must be accounted.
Applications of Multiplex PCR
- Pathogen Identification
- High Throughput SNP Genotyping
- Mutation Analysis
- Gene Deletion Analysis
- Template Quantitation
- Linkage Analysis
- RNA Detection
- Forensic Studies