Why us? why we were the lucky ones that developed the new multiplex PCR strategies?
The Polymerase Chain Reaction was invented more than 20 years ago, the inventor, Kary Mullis, won Nobel Price in 1993. I believe that the reason why so many people failed to make multiplexing work is because we all trusted the TM (melting temperature) too much.
During early years of PCR, some cleaver engineers developed software programs to aid the primer design. And at the core of many of these software, is the prediction of TM. It makes sense, for a pair of primers (or many pairs) to work with each other, they both need to anneal to the target DNA at the same efficiency.
What many people do not aware are the following facts:
1) There are many formulas to estimate TM. The same sequence may have different TM values based on the different formulas. Which formula do you trust?
2) TM calculations usually make many assumptions, because many factors may influence the final TM. For example, the concentration of the substrates (dNTPs, primers, templates), enzymes, and pH, iron concentration (Mg++) etc should be considered when using these equations. However, when we use these formulas, we rarely considered these.
3) Research shown that the estimated TM and measured TM value could be quite different.
4) Even a perfect TM was identified, it only means that the primers have equal opportunity to BIND to the templates, it does not means the primers will be used to PRIMING from the templates at equal efficiency.
The reason people use TM calculating software is not because they are good, but because they are not bad enough to be abandoned!
Our multiplex PCR methods worked because we largely ignored TM and let biology dictate how we should design our PCR primers. If there is only a section of the sequences were conserved, we may have to pick it disregards of its TM and CG content.
More than often, what we trusted the most (such as TM) is the one thing that limiting us from inventing new. So, when things are not working, do not forget to question our basic believes.
* Disclaimer*
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