We can't amplify the first exon -out of twelve- of a gene. All the other were easily amplified. A set of primers designed in the central region of this exon could amplify a piece of the expected size, suggesting that the exon is present. We changed several sets of primers and annealing temperature, however a lot of background was amplified but not a band of the correct expected size. Could you suggest us other possible things to do in order to succeed in the amplification? Thank you.
Dear Gesicca,
what template are you using? If it is DNA from a cell line, you could have some mutations or indels in that reagion...
Have you tried amplifying it with one of the primers in the preceeding intron? If that amplifies, I'd sequence it... cloning might make it easier to then amplify your exon from the insert.
Have you check if this exon has a conserved region from a gene family. It is likely that you are amplifying a region that is present multiple times in the genome. For this to work, you should first align the exon with the genomic sequence of the same species and see what you get. You will likely find some sequences that are specific for the exon you want and use those for your primers.
Another possibility if the presence of high GC content in the region that you are amplifying, which will make it difficult for standard PCR condition to work. If that is the case, there are things you can do to improve the results.
Please, let me know which is the case so I can provide more info.
The initial exons of genes (you don't mention organism) are often close to or within CpG islands, so you may be having problems with amplifying a segment with high GC content.
If so, you may find this thread useful:
https://www.researchgate.net/post/How_can_I_sequence_human_COQ2_DNA_exon_1
Thank you all, actually I found this first exon is in a large GC-rich region, therefore we will try the suggestions in the link by David and the related paper. Marcos, if you have additional suggestions please let me know!
Suggestion for these type of templates are:
5-10 % DMSO with or without glycerol
Betaine
Qiagen Q solution
even 2-10% Formamide with DMSO
What is key is to design longer primers with high Tm so you can perform annealing at high temperatures, even trying two-step PCR. Make sure you optimize by trying at different annealing temperatures. Some recommend to reduce the annealing time to 10-15 s.
In my experience, most times, just using high Tm primers and DMSO have worked fine, but some templates may require other tricks.
Good Luck!
if it is large and gc rich I would ampolify in presence of 1M betaine final concentartion which make the template melt easier so helps amplification and reduces the structural complexity of the target. If this fails you could try nested primers where 1 or both second amplification primers are just inside the first roung primers and ampify 1/10th ul of first product for 20 cycles or fewer as it is very efficient. As exon1 of many genes is larger then it will reanneal to itself easily before the primers anneal so use primers that anneal at a high temperature if possible
Dear all, this is to thank you all: we could successfully amplify our first exon! Thank you again for your help !
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