Length of PCR Primers:
Specificity is generally controlled by the length of the primer and the annealing temperature of the PCR. Oligonucleotides between 18 and 24 bases tend to be very sequence-specific if the annealing temperature of the PCR is set within a few degrees of the primer Tm (defined as the dissociation temperature of the Primer:Template duplex). These oligonucleolides work very well for standard PCR of defined targets that do not have any sequence variation. The longer the primer, the smaller the fraction of primed templates there will be in the annealing step of the amplification. Primers of a minimal length that ensures melting temperatures of 55oC or higher provides the best chance for maintenance of specificity and efficiency.
The upper limit on primer length is less critical and has more to do with reaction efficiency. For entropic reasons, the shorter the primer, the more quickly it anneals to target DNA and forms a stable double-stranded template to which Taq DNA polymerase can bind. In general, oligonucleotide primers 28-35 bases long are necessary when amplifying sequences where a degree of heterogeneity is expected.
For primers shorter than 20 bases, Tm = [4 x (G+C)] + [2 x (A+T)].
For longer primers, Tm = 81.5oC + 16.6 x Log[Na+] + 0.41 x (%G+C) - 0.61 (% formamide) - 500/ number of bp in duplex.
The 3'-Terminal Nucleotide in the PCR Primer:
The 3'-terminal position in the primers is essential for controlling mispriming. Care must be taken that the primers are not complementary to each other, particularly at their 3'-ends. Complementarity between primers leads to the undesirable primer-dimer phenomenon, in which the obtained PCR product is the result of the amplification of the primers themselves. In cases where multiple primer pairs are added in the same reaction, it is very important to double check for possible complementarity of all the primers added in the reaction.
GC Content and Tm of PCR Primers:
PCR primers should maintain a reasonable GC content. Oligonucleotides 20 bases long with a 50% [G+C] content generally have Tm values in the range of 56-62oC, which provides a sufficient thermal window for efficient annealing. The GC content and Tm should be well matched within a primer pair.
PCR Product Length and Placement within the Target Sequence:
All of the computer programs provide a place to select a range for the length of the PCR product. In general, the length of the PCR product has an impact on the efficiency of amplification. The length of a PCR product for a specific application is dependent in part on the template material.
Clinical specimens prepared from fixed tissue samples tend to yield DNA that does not support the amplification of large products.
The specifics of the size of the desired products often depend on the application. If the purpose is to develop a clinical assay to detect a specific DNA fragment a small DNA amplification product of 120-300 bp may be optimal.
For the purpose of detecting a DNA sequence, PCR products of 150-1,000 bp are generally produced.
It is relatively straightforward to obtain products greater than 3 kb from pure plasmid or high-molecular-weight DNA.
To monitor gene expression by quantitative RT-PCR, the product must be large enough so that a competitive template can be constructed and both the product and the competitor can be easily resolved on a gel. These products tend to run in the 250-750 bp range. Here the issue is to maximize the efficiency of both the reverse transcriptase step and the PCR.
A Simple Rule for Non-Computer-Based Selection:
Occasionally, PCR primers must be selected from very defined regions at the 5' and 3'- ends of a specific sequence. A simple method of primer design is to choose regions that are deficient in a single nucleotide. Selecting primers in this way reduces the chance of extensive primer-primer homology. Care must be taken to have a balanced primer pair in terms of length and base composition so that the Tm values of the primers are within 2-3oC of each other.
Primers for Nested PCR:
The sample is first amplified for 20-40 cycles using the outer primer set, then a very small aliquot of this reaction is amplified a second time for 15-25 cycles using the inner primer set. The inner set of PCR primers is positioned within the DNA so that the complementary sequence for the inner primer pair is present in the PCR product obtained in the first amplification reaction and available to form a template-primer complex.
In designing primer pair systems to discriminate mutant from wild-type sequences, one needs first to examine the mutations involved. The placement of a mismatch at the 3'-terminus of a primer-template duplex is more detrimental to PCR than internal mismatches.
In addition to 3'-terminal mismatches, other factors should be considered. Shorter primer (< 20 bases, Tm < 55oC), lower dNTP levels, higher annealing temperatures, lower primer concentration, MgCl2 concentration, enzyme concentration, and fewer cycles increase the stringency of the amplifications and can be used to skew amplifications to favor the target sequence.
The use of primers that are at least 25 bases long (Tm > 70oC), high dNTP concentrations (800 uM), and annealing temperatures below the Tm of the primers will better accommodate mismatches.
When designing degenerate primers, the degeneracy of the genetic code for the selected amino acids of the region targeted for amplification must be examined. Obviously, selection of amino acids with the least degeneracy is desirable because it provides the greatest specificity. Several approaches can be considered for increasing the specificity of the amplification using degenerate primers.
The pools may be synthesized as subsets such that one pool may contain either a G or C at a particular position, whereas the other contains either an A or T at the same position.
The degeneracy of the mixed primer may be reduced by using the codon bias for translation.
The size of the degenerate primers can be as short as 4-6 amino acid codons in length. Six-nine base extensions that contain restriction enzyme sites can be added to the 5'-terminus to facilitate cloning.
Degeneracy at the 3'-end of the primer should be avoided, because single-base mismatches may obviate extension.
The inclusion of deoxyinosine at some ambiguous positions may reduce the complexity of the primer pool.
It has been observed that the PCR thermal profile can dramatically alter the success rate of degenerate PCR. The preferred degenerate PCR amplification profile starts with a non- stringent annealing temperature (35-45oC) for 2-5 cycles, followed by 25-40 cycles at a more stringent annealing temperature. The relaxed annealing conditions allow the short complementary primer portion to hybridize to the target. After the second cycle of amplification, the 5'-extension becomes incorporated into the amplified product and serves as the template for subsequent rounds of amplification. By shifting to a more stringent annealing temperature, increased specificity can be achieved.
Primers for Mutagenesis:
The location of the mutated base(s) in the primers depends on the nature of the desired mutation. To introduce single-base substitutions and insertions at a location with a unique restriction enzyme site nearby, only two primers are needed. The mismatches should be placed in the middle of a 24- or 36-base oligonucleotide.
The mutagenesis primers for generating a large deletion should contain sequences flanking the deletion region. Both mutagenesis primers should have a region of more than 12 bases at the 3'-terminus that matches the target sequence and a mismatched tail of more than 7 bases that matches the region flanking the other end of the deletion.
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