Field of Science

And the USS is bent at...

So yesterday I poured and ran my first polyacrylamide gel in many many years.  Actually I ran my second gel, because the first one didn't set at all.  I was quite proud of myself for remembering a lot of little tricks, like flushing the wells before loading them and flushing the bubbles out from under the gel.

And the results told me that the USS is bent at the T-tracts.

Above is the gel photo. I tested seven different versions of the USS sequence, each embedded in an otherwise-identical 200 bp fragment.  The white bands in the gel are the positions that the DNAs migrated to during the electrophoresis (6% acrylamide in 0.5 x Tris-Borate buffer, run at 60-70 volts for about 5 hours).  'S' indicates DNAs that I scored as running slower, and 'F' DNAs that ran faster.

Below is the key to the differences between the DNAs:

The first good result is that the randomized-sequence DNA did run faster than the consensus USS, as it had in the former postdoc's experiment five years ago.  That might not look obvious in the gel photo, where the random fragment (in the leftmost lane) is at about the same position as the USS fragment on its right.  But this end lane ran slower than the others because it stayed cooler - I've tried to indicate this by the shape of the blue tracking-dye bands I've drawn onto the gel.

Most of the other DNA fragments ran with the same mobility as the USS, but DNAs 6 and 7 ran faster, like the randomized-sequence DNA.  These are the only two DNAs whose T-tracts are changed: 6 has one T-G substitution in each T-tract, and 7 has these plus the same two outer-core changes as DNA 4.

You may wonder why I didn't run the DNAs for longer. to better resolve the migration differences.  But I expected the DNA to have run much further; the Molecular Cloning manual I was using as a guide said that xylene cyanol (the upper turquoise dye band, labeled 'xc') would migrate at the same rate as a 260 bp DNA fragment in a 5% polyacrylamide gel, and at the same rate as a 160 bp fragment in an 8% gel, so I expected my DNAs to coincide with the xc band.

The only explanation I can think of is that I put 10 mM MgCl2 into the gel buffer but forgot to also put it in the running buffer in the tanks.  The manual says that even minor differences in ionic strength 'can greatly distort the migration of DNA'.  So I should probably repeat the gel with the correct buffer.  Maybe I'll also try running it in the cold room.

1 comment:

  1. It's probably from the MgCl2 itself. All the sources I've ever seen regarding migration of DNA in PAGE are based on TBE buffer, without Mg. I know from experience that including Mg substantially reduces DNA migration in the gel.

    The other possibility is if your acrylamide:bisacrylamide ratio is a lot different than the one in the cloning manual. That can change the degree of crosslinking in the gel and alter migration, but not to the degree you're showing here (at least, not in my experience).

    You're aware that there's a significant literature on sequence-specific bends in DNA, right? IIRC, having 2-3 consecutive As repeated at ~ 10-11 base intervals (i.e. once per turn) can cause a significant bend.

    One thing I remember people did to help prove bending is to circularly permute the supposedly bent sequence. That is, imagine putting your presumed bent sequence into a 200-300 bp circular DNA. Now imagine cutting the circle at different places so the base sequence stays the same, but the presumed bent part is at different distances from the end. If it's truly bent, you expect the slowest gel migration when the bend is in the middle. When the bend is at either end, migration should be similar to a random sequence.

    Apologies if you're already up on all this!


Markup Key:
- <b>bold</b> = bold
- <i>italic</i> = italic
- <a href="">FoS</a> = FoS