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I am amplifying 2 fragments of an unique DNA sequence of malaria;
pfmdr1 gene with 2 different sets of primers. All primers are 21
nucleotides length .Set1 primers have Tm of 59 and 61 °C and Set2 have
61.7 and 51.2°C.
PCR is carried out in standard condition and the Tm=40 °C. Using
a same amount of DNA template (5 ul) of purified culture DNA, only Set2
give an amplification. Set1 give an amplification if the template is 20
to 50 times more. How can someone explain this difference in the PCR
efficiency. The gene target is the same only the site of primers
annealing differs. Moreover, Tm primers are quite similar and I used very
low Tm (40 °C) for amplification.
So, when the amount of DNA is not high (naturally infected
sample), Set2 can not amplify the gene however Set1 does. Is there a
problem in my PCR condition? As the PCR conditions are the same for the 2
sets, why this difference?
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