Effect of amplicon length - Effects of amplicon length-qpcr (Sep/09/2008 )
Iam trying to find out the effect of amplicon length in a qpcr reaction. can you please tell me the optimal amplicon size or the size you prefer or the sizes which gave you best results
amplicon size has an influence in the overall efficiency of a qpcr reaction...most of the companies state that amplicon size play a role...how can we decide the size of the amplicon
effect of Amplicon size according to QIAGEN
The smaller the amplicon size used for PCR analysis of FFPE DNA, the greater the chance to detect a specific locus. Real-time PCR of intact DNA compared with DNA from paraffin-embedded samples shows that increasing amplicon sizes dramatically reduces the number of detectable genome equivalents. In the example described in the figure “Real-time PCR analysis using amplicons of different length”, 100% of the genome fragments were detectable using an amplicon size of 100 bp for both intact and paraffin-embedded DNA. With an amplicon of 160 bp, only 1% of the genome fragments could be detected using DNA from paraffin-embedded samples. 99% of fragments were smaller than 160 bp or contained cross-links and could not be detected by real-time PCR. When the amplicon size was further increased to 360 bp, only 0.03% of the copies can be detected with DNA from paraffin-embedded tissue samples.
ACCORDING TO APPLIED BIOSYSTEMS
Amplicon was too long.
Designing primers that generate a very long amplicon may lead to poor amplification efficiency. Ideally, amplicon length should be 50 to 150 bases for optimal PCR efficiency. In cases in which longer amplicons are necessary, optimization of the thermal cycling protocol and reaction components may be necessary.
CAN ANYONE PLEASE STATE THE REASON WHY WE NEED AN OPTIMAL LENGTH OR WHY CANT WE GO FOR LESS OR LARGE AMPLICONS
My guess is incomplete dissociation of the amplicon during the melt cycle with higher amplicon sizes. Taq is pretty fast, so a 100 bp difference shouldn't really be an issue with extension.
Thanks... when i work with amplicon size of 50bp, 80bp,100,bp and so on untill 300bp neither the least nor the higest is working.
can you pls help me reagrding this....... the probable reasons......
You're assuming that because it happens to be the smallest and largest amplicons that aren't working, that amplicon size is the problem, and not some other issue (like primers, for example). What other information do you have to allow more confidence in your conclusion that your difficulties are related to amplicon size?
In my opinion, if your PCR didn't work, this should not be the problem of the amplification size. The amplification size affect the PCR efficiency, the longer the amplification size, the lower the PCR efficiency, this will make the qPCR not so accurate. If the PCR didn't work (no peak in the melting curve), first of all, you should check the primers.
my project is to find the effect of amplicon size in qpcr reaction.... so iam trying to work with different sizes of amplicons.....can you please tell me the sizes you prefer.....and why