We are working with a comparative functional genomics approach, where we are trying to make primers for a sequenced organism, but using these primers to pick-up genes in a related genus (but same family). When we blast the sequence of the amplicon generated by our primers from this related genus, the results indicate "no match". Moreover, the primers are picking up a smaller fragment, almost half the size expected for the native organism. Any explanations?
Hello Tarun,
I agree with the above and I'd ask you a few things that you have not clarified in your questions in order to be able to help with your puzzle.
1) how big is the fragment you want to amplify?
2) what's your template...DNA or cDNA?
3) when you said "we blast it" you mean using NCBI blast tool or some other software?
4) you blasted your amplicon against what exactly?
5) have you used high fidelity taq? this is important for several reason, HiFi taq will chew up your primers if they don't match perfectly, remember that's the whole point of the HiFi taq, and your actual final primers will be in fact smaller and with higher chance of annealing somewhere else in the genome. My suggestion will be to use common taq first to have a feeling of how things are working, you may not be able to get the right sequence after this because of the high mutation rate of common taq but at least you can play around with it. I'd also suggest to design a few extra set of primers.
Thanks!
Hi Tarun
Although your are using primers from a sequenced related organism..but this does not ensures that they will also amplify the region from other organisms in the family...you can address some of the aspects:
1) Are your primers really specifc to gene....for which they are designed....check them at Primer Blast at NCBI web site...if the primer bind to some other genomic regions also...you'll have some unexpected bands
2) Check the conservation of sequence at the primer site in similar organisms by doing a MSA..you may need to introduce some degenerate sites..
3) Check your PCR conditions..in related organisms ..you may need to lower the stringency
4) are you amplifying from genomic DNA or cDNA ...in genomic DNA...variation in the gene length (intron/exon organisation) may give the the observed results...in case of cDNA an alternative spliced variant may also be observed as a smaller product..
check some of these things..and you may get the answer
bye
Hello Tarun,
I agree with the above and I'd ask you a few things that you have not clarified in your questions in order to be able to help with your puzzle.
1) how big is the fragment you want to amplify?
2) what's your template...DNA or cDNA?
3) when you said "we blast it" you mean using NCBI blast tool or some other software?
4) you blasted your amplicon against what exactly?
5) have you used high fidelity taq? this is important for several reason, HiFi taq will chew up your primers if they don't match perfectly, remember that's the whole point of the HiFi taq, and your actual final primers will be in fact smaller and with higher chance of annealing somewhere else in the genome. My suggestion will be to use common taq first to have a feeling of how things are working, you may not be able to get the right sequence after this because of the high mutation rate of common taq but at least you can play around with it. I'd also suggest to design a few extra set of primers.
Thanks!
@Luciano...You have really raised few more important issue regarding problem Tarun is facing in PCR amplification
bye
Tarun you already have good suggestions from Ajay and Lucinao. What i want to add here is that during comparative studies when you are using information from one genome to identify related genes from another related species, is not very straight forward at most of the times. whenever you make a primer there are chanced that it will bind to some another regions if the conditions will be less stringent and because you are amplifying from a related species, you need to reduce the stringency little bit. Therefore it is very important to have exact match at the 3` end of the primer (at least three baspairs). most of the times mismatch at 5` end or center of the primer sequences is well tolerated but mismatch at 3` end is deleterious. I would suggest that you pick the couple of homologous genes from other plants and look for your primer binding sites across these sequences and select primer with exact match at 3` end in all of them. I am sure that will give you your amplicon. All the best
Thanks a lot Ajay, Luciano and Manoj for the discussion and suggestions. Here are the answers to a few questions asked by Luciano.
1. Size of my fragment: 843 bp while the size of the amplicon being generated is 488 bp
2. The template is cDNA
3. Yes it is NCBI blast that has been done
4. And blast was done against the genome of original sequenced organism (Arabidopsis)
To add further to the details, I would like to tell you that we have designed several primer sets. These were tested with NCBI primer blast, to ascertain specificity before proceeding with the amplification.
What measures are recommended to be take now, to reduce the stringency of the PCR?
in a case where some of the primers do not match, are the mismatched primers included in the amplicon?
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