Lower size band in genomic PCR with 300 bp missing - (Sep/27/2005 )
hihi, i am new in this forum and i got a question which trouble me a lot....
i am now optimising the PCR condition of a new primer which i always get multiple bands on agarose gel... i am using very stringent condition and i am now trying nested PCR.... and now i got generally 2 bands on the agarose gel.... i use a few DNA samples for optimisation and surprisingly some got the lower size band and some got the upper sized band and some got both !!! I picked the one with lower size for sequencing and it shows clean results... which is the sequence i expected but with 300bp missing in the middle of the sequence..... do you think it is a real mutation or is it just the problem of PCR????
Can anyone help me !! Many thanks!!!
Dear Sylvia,
What organism are you dealling with?
Is the region you looking at present in multiple copies in the genome?
Is the same sample giving you reproducible band?
I do observed the same situation in the virus genome that I looking at. The gene present in two copies in the genome. However, one of the copy of certain strain is either inserted or deleted. Hence i result me multiple band although single primer set is applied.
If your result is reproducible, and conferm by sequencing, it should be a mutation.
Best regards
Could be a pseudogene.
If you're using cDNA as your template, it could be a splice variant (depending on what organism you're working with, of course)...
Note that 300 bp (is that what was missing, exactly?) is divisible by three (obviously), and so could remain in frame...
Hi everybody,
I have a similar problem.I am using 2 different primer pairs to clone different region of my template.My forward primer is same in both pairs but only the reverse primers are different(like F1/R2, F1/R3) 
.When I run pcr products of these two primer combination on agarose gel I see same bands on the gel for both two pairs!!! and any of these bands is the desired one.They have lower size.I don't know what it is??
I expect 1200 bp with one of my primer combinations( this is from beginning to the middle of the fragment) and 1970 with the other combination ( this is from beginning to the end).
I attach the gel photo.
first lane after DNA marker is for the smaller fragment pcr, second lane is for the whole fragment, the other lane is not important:)
Any comments?Thank you:(
i am dealling with human genomic DNA and the region i amplify is the promoter region of a gene. i have try on my real samples (>100) and all shows similar results ... (some with lower size and some with larger size and some both).... and the results are reproducible!!! i am wondering if my DNA is degraded but i truely get clean sequencing results!!!
and one thing to add, the larger sized PCR product isn't exactly the sequence i expected, too, with about 70bp missing in the middle...... that's weird!!!
i don't believe that i got so many mutations in my samples..... 
Katanin, have you do sequencing on your samples?? will it be the same case like me or is it just other non-specific binding of the primers???
Thank you very much for all your suggestions!!!
and one thing to add, the larger sized PCR product isn't exactly the sequence i expected, too, with about 70bp missing in the middle...... that's weird!!!
i don't believe that i got so many mutations in my samples.....
Katanin, have you do sequencing on your samples?? will it be the same case like me or is it just other non-specific binding of the primers???
Thank you very much for all your suggestions!!!
Hi sylvia123,
I didn't do sequencing because I am still trying but if it continues like this I will do sequencing and see what they are!!
I think maybe my fragment is a little bit long so I can not see the desired band but only the smaller size bands,but in your situation how long is your template?As I understand it is not too much, so it is very strange that you can't get what you want! and it is also strange that there is such a big mutation in all of your samples!!
I can not find a solution for now:)
See you...
the pcr product i expected is just ~700bp.... i don't know why it is so difficult to amplify..... in fact i have redesign the primers for several times and i can't get clear band.....
do anyone know that are the promoter region more difficult for doing PCR?? because some of my colleagues also face difficulties in doing pcr of the promoter region......
wish everyone good luck!
Promoter regions can be higher G+C, try increasing Mg2 to 5mM and adding DMSO 5% to your PCr reaction.
when you are PCRing promoters you really have to blast your primers to the genome of interest. transcription factor binding sites can be exactly the same sequence for different genes so if your primer contains a TFBS you have an increased probability of having other sequences in the genome with your primer binding site... Be careful when your primers are designed to avoid these conserved TFBS totally if possible but at least at the 3' end of the primer...
Another problem is that many promoters are within a CpG island this extremely GC rich template is very difficult to amplify try to do a longer denaturation time to ensure the GC rich template is denatured for amplification --also what Tap14 says is correct