Looking at your DNA sequences, I'd say the different band positions you're seeing are most likely down to the sequences behaving differently during electrophoresis, even though they're roughly the same length.

The main culprit is probably secondary structure formation. Your Group 3 sequence particularly caught my eye - it's got those repetitive TATATATA regions that love to form hairpins and loops. When DNA folds up on itself like this, it migrates differently through the gel compared to a more linear piece of the same size.

There's also the GC content to consider. Groups 1 and 2 are quite GC-rich, whilst Group 3 is much more AT-heavy. This affects how the DNA moves through the polyacrylamide matrix - GC-rich sequences tend to be a bit more rigid.

Another possibility is that your annealing hasn't worked equally well across all four groups. The primer return reaction might have been more efficient for some sequences than others, giving you different proportions of annealed versus single-stranded material.

If I were in your shoes, I'd try running a denaturing gel (chuck some urea in there) to see if that evens things out. That would tell you whether it's secondary structure causing the trouble. You could also try running at a higher temperature to keep everything nicely denatured.

It's one of those frustrating things about working with DNA - even when everything looks the same on paper, the actual molecules can behave quite differently!

I'm eye to eye with Richard Monaghan . The most likely explanation is the secondary structure, especially with such long single-chain DNA ends. Running a denaturing gel as he said should solve the problem.

when dealing with small DNA fragments (and even more if they are not perfectly double stranded, base composition and secondary stuctures can modify the migration