The efficient way to minimize primer dimmers isredesign primers. However, in this case, most of the time it was limited onlyto one specific primer pair as using a universal region of the barcode to identify internal variations. Inthis application, species specific primers were designed by using DNA barcoderegions.
Therefore, redesign of primers is impossible due to this limitation. “Hotstart” is another technique which avoids nonspecific amplifications includingprimer-dimers. The idea here is first heated up the samples around 80–850Cand then add missing critical components such as dNTPS, MgCl2, whichenables high annealing temperature to avoid primer dimmers.1 2If DNA contains more GC bases with more hydrogenbonds, then more energy should be required to break bonds. In this case, thecomposition of the primers directly depends on the melting temperature ofprimers. Primer design guideline3further states that the recommended range for Tm is 52-650C.All theresults of Tm are in the optimal range.
Melting temperature difference of thetwo primers should be close enough to each other. If melting temperaturedifference is less, choosing an annealing temperature becomes easier. Otherwisesetting an annealing temperature is difficult for both primers to anneal duringPCR. All the Tm differences of the primer pairs in this table are less than 20C. If any contradiction occurred in the melting temperature difference, itcould be adjusted for a suitable Tm difference by changing the length of theprimer within 18-30.4The amplicon length5 isdictated by experimental goals. Usually, for real PCR (qPCR), the productlength is closer to 100 while it is 500 in standard PCR. Product length dependson the specificity of the primer pair.
Long amplicons could dissociate forseveral times due to the formation of the secondary structure with variousmelting temperatures. Therefore the specificity of the primer should be highenough.Through a nested PCR, the binding of the primer pairto the other locations in the genome except for the intended gene or DNAfragment can be avoided.
To obtain unique amplification with the speciesspecific primers, first, amplify the barcode region with universal DNA barcodeprimers. If the annealing temperature is much lower than the meltingtemperature this may lead the primers to anneal with sequences other than theintended target and lead nonspecific PCR. Developedsoftware tool for species specific primer designing by using DNA barcode regionsInthis project, the automated application and its configurations worked asplanned. The Primer Designer application was accurate and efficient. Althoughthe use of the automation would not entirely replace human abilities, it wouldreduce the workload considerably. When the process was done manually, thedissimilar regions have to be identified one by one after aligning twosequences and then with those dissimilar bases, primers were designed. Althoughthe forward primer was designed directly, the reverse primer has to be complementaryreversed as both the primers were chosen from the same strand.
Hence, thisprocess was time consuming. With this automated software tool designing of primersfrom the internal variation of DNA barcode sequences could be done within fewseconds. 1http://www.sciencedirect.com/topics/neuroscience/primer-dimer2https://www.nucleics.com/DNA_sequencing_support/DNA-sequencing-primer-dimer.html3https://dnacore.mgh.harvard.edu/cgi-bin/sequencing/PCR Primer DesignGuidelines.htm4https://openwetwear.org/wiki/Design_Primers 5 http://www.premierbiosoft.com/tech_notes/PCR_Primer_Design.html
