Multiplex PCR
Multiple primer pairs can be added in the same tube to do the PCR
Good for amplifying multiple sites
Application example: genome identification
Design difficulty
Melting temperatures should be similar
No dimer formulation
Universal Primers
Primers can be designed to amplify only one product.
Primers can also be designed to amplify multiple products. We call such primers “universal primers”. For example, design primers to amplify all HPV genes.
Strategy:
1.Align groups of sequences you want to amplify.
2.Find the most conservative regions at 5’ end and at 3’ end.
3.Design forward primer at the 5’ conservative region.
4.Design reverse primer at the 3’ conservative regions.
5.Matching forward and reverse primers to find the best pair.
6.Ensure uniqueness in all template sequences.
7.Ensure uniqueness in possible contaminant sources.
Semi-Universal Primers
Primers can be designed to amplify only a subset of template sequences from a large group of similar sequences. For example, design primer to amplify HPV type 1 and type 6 gene, but not other types.
Strategy:
1.Align all types of HPV genes.
2.Identify a subset of genes that are more similar to each other than to other subsets. In this case, type 1 and type 6.
3.Find the 5’ and 3’ regions that are conserved between type 1 and type 6, but are variable in other types.
4.Design forward primers from the 5’ region and reverse primers from the 3’ region.
5.Matching forward and reverse primers to find the best pair.
6.Ensure uniqueness in all template sequences.
7.Ensure uniqueness in possible contaminant sources.
Guessmer
In some cases, DNA sequences are either unavailable or difficult to align. Then, a single/group of related proteins can be back translated into nucleotide sequences that will be used as template to design primers/probes. We call such primers “guessmer”.
Back translation is both problematic and feasible. While the genetic codes are degenerate, different organisms do show preferential biases in codon usage, which can be used to limit the possible back-translated nucleotide sequences.
Strategy:
Back translate the protein sequence using corresponding codon usage table. Identify 5’ and 3’ regions where there is the least ambiguity.
Design and match forward and reverse primers as before. But the primers shall be about 30 bases long in order to offset the decreased hybridization specificity caused by mismatched bases.
Set higher annealing temperature to increase the primer annealing stringency.
Summary ~ Advanced Primer Design
Primers can be designed to serve various purposes. Universal primer, semi-universal primer, guessmers are some of them. There are many more fields where primer design skills are required, such as real-time PCR, population polymorphism study (microsatellite, AFLP, SNP …), internal probe design, and so on.
However, the basic rules always apply –
achieve the appropriate hybridization specificity and stability
