1, primer design

Careful primer design is the most important step in PCR. An ideal primer pair anneals only to a single sequence flanking the sequence of interest, but not to other sequences. Poorly designed primers may amplify other non-target sequences. The following guidelines describe the satisfactory characteristics of a primer that can increase specificity:

1. Typical primers 18 to 24 nucleosides long. Primers need to be long enough to ensure sequence uniqueness and reduce the likelihood that the sequence will be present at a non-target sequence site. However, primers longer than 24 nucleosides do not imply higher specificity. Longer sequences may hybridize to the mismatched sequence, reducing specificity and slower hybridization than short sequences, thereby reducing yield.

2. Select primers with GC content of 40% to 60% or GC content image template GC content.

3. Design primers with a 5' end and a middle region of G or C. This increases the stability of the primer and the stability of the primer hybridization to the sequence of interest.

4. Avoid the presence of complementary sequences at the 3' end of the primer pair, which would result in primer dimers that inhibit amplification.

5. Avoid GC enrichment at the 3' end. When designing the primers, it is guaranteed to contain 3 A or T in the last 5 nucleosides.

6. Avoid mismatching at the 3' end. The 3' terminal nucleoside needs to be annealed to the template for catalytic extension by the polymerase.

7. Avoid the presence of sequences that may create internal secondary structures, which can impair primer annealing stability.

Additional sequences that are not present on the sequence of interest, such as restriction sites and promoter sequences, can be added to the 5' end of the primer without affecting specificity. These sequences are not included when calculating the Tm value of the primer, but should be tested for complementarity and internal secondary structure.

Sometimes, only limited sequence information is known for primer design. For example, if only the amino acid sequence is known, the degenerate primer can be designed. A degenerate primer refers to a mixture of different sequences that represent the possibility of encoding all of the different bases of a single amino acid. In order to increase the specificity, reference can be made to the codon usage table to reduce the degree of merger according to the base usage preference of different organisms. Hypoxanthine can be paired with all bases to reduce the annealing temperature of the primers. Do not use a merging base at the 3' end of the primer because the annealing of the last 3 bases at the 3' end is sufficient to initiate PCR at the wrong site. Higher primer concentrations (1 μM to 3 μM) were used because the primers in many of the pooled mixtures were not specific to the template of interest.

2. Primer annealing temperature

Another important parameter of the primer is the melting temperature (Tm). This is the temperature at which 50% of the primers and complementary sequences behave as double-stranded DNA molecules. Tm is necessary to set the PCR annealing temperature. Ideally, the annealing temperature is low enough to ensure efficient annealing of the primers to the desired sequence while still being high enough to reduce non-specific binding. Reasonable annealing temperatures range from 55 ° C to 70 ° C. The annealing temperature is generally set to be 5 ° C lower than the Tm of the primer.

There are several formulas for setting Tm. Table 4 lists the two most common methods for determining the primer Tm. The first formula is derived from hybridization in a high salt solution and is suitable for primers of less than 18 bases. The second formula estimates Tm based on GC content. The most reliable method for determining the primer Tm is the proximity analysis method. This method predicts the hybridization stability of the primers from the sequence primary structure and the properties of adjacent bases. Most computer programs use neighborhood analysis.

Depending on the formula used and the sequence of the primers, the Tm will vary greatly. Since most of the formulas provide an estimated Tm value, all annealing temperatures are only a starting point. The specificity can be improved by analyzing several reactions that gradually increase the annealing temperature. Start below the estimated Tm 5 ° C and gradually increase the annealing temperature in 2 ° C increments. Higher annealing temperatures reduce the formation of primer dimers and non-specific products. For best results, the two primers should have an approximate Tm value. If the Tm difference of the primer pair exceeds 5 ° C, the primer will exhibit a significant erroneous onset using a lower annealing temperature in the cycle. If the two primers Tm are different, the annealing temperature is set to be 5 ° C lower than the lowest Tm. Alternatively, to increase specificity, 5 cycles can be performed first at an annealing temperature designed according to a higher Tm, and then the remaining cycle is performed at an annealing temperature designed according to a lower Tm. This allows partial copies of the target template to be obtained under more stringent conditions.

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