Amplifying gLUL115 (555bp) from clinical samples have resulted for the second time in 2 non-specific band appearing on the top of expected PCR amplicon (size is larger than amplicon )
the primers used are very known and I have tested them on laboratory strains and show specific band (one band of 555bp)
some colleague have suggested that this might be bacterial contamination as they have been collected from immunocompromised patients and possibly human genome as well.
looking for possible explanation to these results
thanks
you might consider that the larger band is caused by a sequence change in the amplified band, If the patient is heterozygous for this change then once you have plenty of pcr product then on the last denaturation and annealing cycle a heteroduplex will form.Because of the bubble in the heteroduplex this band will run apparently larger (slower) than the NN or the MM bands which are both the same size, You can test this by mixing NN and MM amplified product denaturing and reannealing to see if the larger band appears or by cutting out the larger band ,heating it and re annealing to see if this produces the smaller NN band as well as itself again
Dear Mohammed,
Some years ago, I worked about the HCMV glycoprotein B (gB) gene.
It is known, that HCMV laboratory strains contain less and altered genes in their genome.
Perhaps, in your clinical samples you amplify some 'wild' HCMV gL gene variants.
HCMV (HHV-5) is part of the Human Herpes Virus (HHV) family. All of these family members have similar genes and proteins. Perhaps you should look for the gL sequences of the other HHV's (particular HHV-6 and HHV-7), too?
Try to isolate and sequence your big amplicons!
After this, make a BLAST search with your big amplicon sequences against the sequence data bank.
Best regards,
Thomas
Dear Mohammed,
I remember, we had a similar problem, when we amplfied parts of the HCMV gB gene. We even had an unwanted bigger amplicon in some amplification reactions. For the first moment we were very happy, because we thought, that we have found a gene of new unknown HHV.
After isolation and sequencing this big amplicon, we could identify it as a part of the human endogenous retrovirus HERV-K envelope protein gene.
In this case only one primer produced the big amplicon. This primer worked in both directions: 5' -> 3' and 3' -> 5'.
Best regards,
Thomas
Dear Paul Rutland and Thomas M.C. Binder
Thanks for your thoughts and the answers you have provided is absolutely valuable.
Paul Rutland , I really did not get your idea. Do u mean that those bands might be of human DNA origin ??
Thomas M.C. Binder, your answer makes more sense to me and in fact it is the same answer I got from a professor in virology who once saw the bands he told me it is possible that those bands are a modified and inserted version of the same gene. the virus is trying to adopt to the environment by expressing and genetically change genes necessary for entry such as gL.
Also, as u mentioned, that these extra bands of gL sequences can be of other HHV's (particular HHV-6 and HHV-7).
I believe that cutting those band and sequencing them may give us the answer, but unfortunately the thesis practical time does not allow me to do so.
Thanks again for both of you
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