Hello, Norah Hur If you have performed sequencing I suggest comparing the two sequences by Multiple Sequence Alignment. In my opinion, there might be a deletion.

This is a loading amount effect. When you have a large amount of DNA, the front-running molecules can damage the agarose gel structure as they pass through. Imagine it like they're digging little trenches, that make it easier for subsequent molecules to follow. That's why generally heavily loaded wells seem to run faster than lightly loaded wells.

Annealing temperature and other PCR reaction conditions also sometimes change the specificity of primers.

Did you find any differences in the two sequenced products? I'd also let that gel run for longer to get better separation in your bands. It might actually be a smaller product (Indel polymorphism, homologous gene, homopolymer alleles, etc.).

Thank you so much for your insights ...

John Schloendorn , Vinay Kumar Katie A Burnette

PCR was done with 4 ul DNA , load 10 ul all wells are the same.

I repeat the PCR and run for 2 h its still the same and there were no differences in sequencing

I used these primes (same Annealing temperature and PCR conditions) before this is the first time this happened

am really puzzled

Hello Norah,

You mentioned sequencing gave same results. Have you sequenced the bands completely or partially? As Dr. Katie A Burnette mentioned, the two bands may have indel polymorphisms or they may be splice variants (if generated from RNA). Partial sequence may match, complete sequence may give you some information. The other reason I can think of is the variation in ionic strength of the two PCR reactions, that may also show differential migration of bands. kindly check this aspect. also

Loading amount variations as suggested by Dr. John Schloendorn seems less likely here.

Hello, Norah Hur If you have performed sequencing I suggest comparing the two sequences by Multiple Sequence Alignment. In my opinion, there might be a deletion.

Ajay Saini

It was DNA, no splicing and we sequence the whole gene

Navonil Gogoi

we did Multiple Sequence Alignment there the same. no deletion.

Thank you for your insight . I changed every things repeat the whole prosedure and even tried the master mix with other genes and it was ok.

Norah Hur Try performing MSA of the normal and the abnormal sequence with a reference sequence. You might be amplifying a pseudogene. Look for nonsense and frameshift mutations in the abnormal sequence. Hope it helps.

May have just been gel band shifting, a phenomenon discovered in the forensic sciences. Suggest you run the gel under different conditions e.g. voltage, current and allow adequate time for the bands to migrate.