11 replies to this topic

[relamberth]

Hi everyone:
I'm new here so I'm hoping I'm doing this right. I am a sophmore at a small univeristy studying cell biology. We finished a plasmid DNA lab and after running a gel on it, were left with two bands. My professor wants to know why there are two bands of PBR322 (I assume the plasmid?) when the DNA is circular.

Here is what I know so far:
The smaller the particle, the faster it moves through the gel.
Supercoiling has an effect. It moves the fastest, so chances are the lower band is supercoiled DNA.

Could anyone help me with the rest of the problem?

Thanks!

[HomeBrew]

Hi relamberth -- Welcome to the BioForums!

I'm going to move this post to the "homework questions" category. We don't just give answers, but will help you get unstuck if you've put a little thought into it...

You're right in your two points -- smaller is faster through a gel, and supercoiling of plasmid DNA does make it effectively smaller, so it moves faster. Knowing these two things, what could have happened to the plasmid to make it effectively larger when compared to the supercoiled form? We know the actual size of the plasmid (pBR322), as measured in number of base pairs, has not changed -- it is the same plasmid DNA in both bands.

[relamberth]

I didn't mean to imply that I wanted a direct answer--my professor would have my head! I just needed a new direction. The only thing I can come up with is that the shape of the plasmid changed--perhaps it was nicked? I know the plasmid will remain in circular form because its a plasmid--it has no chance of going linear...am I any further along?

[HomeBrew]

relamberth, on Oct 16 2009, 11:13 PM, said:

I didn't mean to imply that I wanted a direct answer--my professor would have my head! I just needed a new direction.

Yes, I realize that -- it's just kind of a standard disclaimer we put out there so people won't think we just answer their homework questions for them -- who learns anything that way?

relamberth, on Oct 16 2009, 11:13 PM, said:

The only thing I can come up with is that the shape of the plasmid changed--perhaps it was nicked? I know the plasmid will remain in circular form because its a plasmid--it has no chance of going linear...am I any further along?

Yes, you are on the right track. Since you realize that supercoiling packs the plasmid into a more compact shape, allowing it to progress through an agarose gel matrix more rapidly, and you understand that some portion of the population of plasmids can become nicked (a break in one of the two strands), put the two ideas together -- what effect would breaking one strand of the circular DNA have on the supercoiling?

[relamberth]

Wouldn't a nick in the DNA then cause the supercoiling to loosen, slowing it down through the gel?

[HomeBrew]

relamberth, on Oct 17 2009, 12:41 AM, said:

Wouldn't a nick in the DNA then cause the supercoiling to loosen, slowing it down through the gel?

Correct. Now, if you had a plasmid DNA extract in which say 40% of the plasmids had been nicked and the rest were fully intact, and you loaded a sample of that extract on a gel and ran it, what banding pattern would be produced?

[relamberth]

There would be two bands of plasmids on the gel? One higher up--the nicked plasmid and one further down--the supercoiled plasmid? And the supercoiled plasmid band would be a little thicker because more of the DNA remained intact?

[HomeBrew]

relamberth, on Oct 17 2009, 12:54 AM, said:

There would be two bands of plasmids on the gel? One higher up--the nicked plasmid and one further down--the supercoiled plasmid? And the supercoiled plasmid band would be a little thicker because more of the DNA remained intact?

Exactly right -- and that is what you saw in your lab, isn't it? The bands are two three-dimensional forms of the same plasmid -- the faster migrating form is supercoiled and the less rapidly migrating form is nicked circles in which the supercoiling has been relaxed. The forms are separated into two bands by agarose gel electrophoresis because they differ in the rate at which they can worm their way through the pores of the gel matrix under the influence of an electric field.

Good job!

[relamberth]

Thank you so much! I totally understand now. But, I do have one more question: what caused the DNA to be nicked or slightly uncoiled? Was it the process? Something we did or something that happens naturally?

[HomeBrew]

Single strand nicks can be introduced enzymatically (by DNA topoisomerases, for example) or mechanically by shearing during the plasmid extraction procedure. There are other causes -- for example, radiation and some chemicals can also cause single-stranded breaks in DNA -- but they're likely not in play here.

[relamberth]

Great, I can now not only answer his question but explain it to. Thanks again for your help.

[HomeBrew]

Glad to help, relamberth. You had all the information and understanding, you just needed a little push -- exactly what we do here...

Not to confuse the issue, but you should understand that there are also other forms of plasmid DNA possible -- linear (which can in fact happen without restriction digestion -- what form would the plasmid DNA be in if it by chance it acquired two single-strand breaks on opposite strands but at the same location?), denatured (usually encountered when the DNA is left under alkaline conditions for too long), and various multimeric forms. These forms will also migrate through a gel at different rates -- sometimes running a gel of plasmid DNA (especially if it's improperly prepared) produces more than two bands.

Assuming, however, that your professor prepared the plasmid DNA for your lab with some care, which is probably a safe assumption, the two bands you saw were most likely the supercoiled and nicked forms -- they are the two most common forms seen when running out an untreated sample of plasmid DNA.

The take-home message is that the rate at which plasmid DNA migrates through a gel under set electrophoresis conditions (the voltage, gel concentration, buffer system, pH, etc.) is determined by the plasmid's size (mass) and its three-dimensional conformation. In a preparation containing just a single plasmid, the mass of these plasmids is all the same, so if multiple bands are seen, it indicates multiple forms of the plasmid exist in the preparation.