Question regarding Restriction Enzymes

[Methtical]

Hello everybody,

I am currently studying for a Masters degree, its been a few years since I was in education, and genetics was never my strong subject anyway. As such, im rather rusty on the concepts of restriction enzymes. I have an exam coming up in a couple of weeks and expect this topic to come up, so have been researching around these and attempting to answer some questions from past exam papers.

One of the questions presented is shown below:

A restriction fragment produced by Bgl II cleavage of Wulfruna wanderensis DNA
is cloned into the Bam HI site in pATHB1. When attempting to recover this
important piece of DNA from the plasmid why is it neither Bam H1 nor Bgl II will
cleave this insert from the plasmid? Explain your answer diagrammatically.

Enzyme Recognition site
Bam HI G/GATCC
Bgl II A/GATCT


I find this question very confusing, but will decribe some thoughts on it. If the fragment was cloned into the BAm HI site, then surely the using Bam HI should liberate the DNA from the plasmid - unless the insertion of the fragment distrupted the recognition site for Bam HI, therefore meaning the enzyme no longer recognized the site in which the fragment is located.
But even before that, I consider how did they insert the fragment, produced by Bgl II cleavage into the Bam HI site in the first place - I was under the impression that both the desired DNA and the plasmid had to be treated with the same enzyme to produce complementary 'sticky ends' to make insertion possible.

I just figure that there is some fundamental piece of information I am not registering here, my lecturers are not being very helpful either. I asked questions about it, and they just replied with "We don't produce model answers, we're looking for originality". Well, yes I'm sure you are, but surely there's a CORRECT answer and a WRONG answer?

Anyway, if anyone can give me some advice as to how to make sense of the question and come to a reasonable conclusion then that would be great. I also have queries regarding restriction maps but will leave that until a later date.

Thanks,

Methtical

[jonmoulton]

It helps to consider both strands.

Bam H1
gGATCC
CCTAGg

Bgl II
aGATCT
TCTAGa

Bam H1 end
g
CCTAG

Bgl II end
a
TCTAG

Flip the Bgl II end over and anneal it to the Bam H1 site.

Combined site after annealing
gGATCT
CCTAGa

Does this site match either the Bam H1 or the Bgl II recognition sequence?

What if the cut site were at the border of the recognition sequence instead of being one base in from the ends? Could a similar situation arise in that case?

[Methtical]

Yes I think I understand now, I needed to know how the fragment was inserted into the plasmid before I could determine why it would not be possible to cleave out the insert with either enzyme. Thanks.

Seems strange to me though that this would be the method used to insert DNA into the plasmid, i.e using two restriction enzymes instead of just the one. The only reason I can think why is if they have inserted it next to another target sequence and want to use a restriction enzyme to clave out both sequences together.

Thankyou for info, very helpful!

Methtical

[jonmoulton]

What if the fragment of interest is bound by one set of restriction sites, but those restriction sites are not present in the plasmid? Using a different restriction enzyme to cut the plasmid, one the presents sticky ends that are compatible with those on the fragment, at least presents a possible strategy to get the fragment into the plasmid, even if you lose the option of cutting the fragment out at the same positions. If you planned well and inserted the fragment into a polycloning site (with a set of adjacent restriction sites), you can get the fragment back by using a different set of restriction enzymes.

[KMDevi]

so the reason you would use two restriction enzymes instead of one..is to prevent re-ligation of the dna fragments with itself instead of inserting into the plasmid..and also becos..depending on the fragment of interest,u might need two restrictino enzymes to cut the plasmid to present sticky ends suitable for the dna fragment to be inserted in..is my understanding correct?i'm an undergrad student and trying to grasp concepts in molecular genetics as it is pretty new to me..any help in explaining would be gr8ly appreciated..

[jonmoulton]

It's not to prevent re-ligation -- the sticky ends are compatible and would potentially re-ligate anyway. Rather, there might not be one restriction site bordering the sequence you want to clone and the identical restriction site in the plasmid. Since these two restriction enzymes make compatible sticky ends, at least you can get the insert into the plasmid. Unfortunately, you can't get it out, at least not with the same restriction enzymes.