plasmid DNA lab question - (Oct/16/2009 )
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!
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.
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?
relamberth on Oct 16 2009, 11:13 PM said:
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:
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?
Wouldn't a nick in the DNA then cause the supercoiling to loosen, slowing it down through the gel?
relamberth on Oct 17 2009, 12:41 AM said:
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?
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?
relamberth on Oct 17 2009, 12:54 AM said:
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!
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?
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.