The the primers directly depends on the melting temperature

The efficient way to minimize primer dimmers is
redesign primers. However, in this case, most of the time it was limited only
to one specific primer pair as using a universal region of the  barcode to identify internal variations. In
this application, species specific primers were designed by using DNA barcode
regions. Therefore, redesign of primers is impossible due to this limitation. “Hot
start” is another technique which avoids nonspecific amplifications including
primer-dimers. The idea here is first heated up the samples around 80–850C
and then add missing critical components such as dNTPS, MgCl2, which
enables high annealing temperature to avoid primer dimmers.1 2

If DNA contains more GC bases with more hydrogen
bonds, then more energy should be required to break bonds. In this case, the
composition of the primers directly depends on the melting temperature of
primers. Primer design guideline3
further states that the recommended range for Tm is 52-650C.All the
results of Tm are in the optimal range. Melting temperature difference of the
two primers should be close enough to each other. If melting temperature
difference is less, choosing an annealing temperature becomes easier. Otherwise
setting an annealing temperature is difficult for both primers to anneal during
PCR. All the Tm differences of the primer pairs in this table are less than 20
C. If any contradiction occurred in the melting temperature difference, it
could be adjusted for a suitable Tm difference by changing the length of the
primer within 18-30.4

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The amplicon length5 is
dictated by experimental goals. Usually, for real PCR (qPCR), the product
length is closer to 100 while it is 500 in standard PCR. Product length depends
on the specificity of the primer pair. Long amplicons could dissociate for
several times due to the formation of the secondary structure with various
melting temperatures. Therefore the specificity of the primer should be high

Through a nested PCR, the binding of the primer pair
to the other locations in the genome except for the intended gene or DNA
fragment can be avoided. To obtain unique amplification with the species
specific primers, first, amplify the barcode region with universal DNA barcode
primers. If the annealing temperature is much lower than the melting
temperature this may lead the primers to anneal with sequences other than the
intended target and lead nonspecific PCR.





software tool for species specific primer designing by using DNA barcode regions

this project, the automated application and its configurations worked as
planned. The Primer Designer application was accurate and efficient. Although
the use of the automation would not entirely replace human abilities, it would
reduce the workload considerably. When the process was done manually, the
dissimilar regions have to be identified one by one after aligning two
sequences and then with those dissimilar bases, primers were designed. Although
the forward primer was designed directly, the reverse primer has to be complementary
reversed as both the primers were chosen from the same strand. Hence, this
process was time consuming. With this automated software tool designing of primers
from the internal variation of DNA barcode sequences could be done within few



3 Primer Design