restriction enzyme question.

[biology_06er]

Hi there,

Soo I have a this question which I don't seem to be able to find the answer to in my supposedly what should be "helpful" textbook..

Ok, so I get how if I wish to isolate a gene, I need to use restriction enzymes to cut it out from say the chromosome...and I must use those same restriction enzymes within the MCS of a plasmid to make complementary ends (right?)...Now what I don't get is the "finding" of the cutting site within the donor DNA...how come textbooks/online information assume their will be a cutting site nearby the gene..what happens if the RE recognizes the sequence within the said gene..I assume you only choose RE's that won't cut the acutal gene of interest in bits..or what happens if the RE site is located so far away that the insert actually codes for two genes...(I'm not even sure if that is possible..but I'm really hoping someone understands what I'm trying to get at here)..
how is it possible that for every gene one wishes to study there happens to be a cutting site
another thing..Say for example as the RE EcoR1 recognizes the sequence GAATTC, isn't there a extremely high chance that that same sequence will be somewhere present in the gene?
..im really hoping someone understands where I am coming from...ive graduated in bio and im freaking out that this is like first year stuff..i didn't do much genetics so im really sorry if the question seems stupid but at the same time i want to know the answer..so someone please help

thanks heaps
b_06er

[JackBean]

Well, you usually don't get your gene by restriction analysis, because all gDNA would be cut and you would get only smear on gel. Instead you amplify your gene of interest by PCR and by that you nicely introduce the restriction sites as parts of the primers.

[JackBean]

You can calculate how often your RE will cut. Of course, the longer recognized sequence, the lower probability. But still, that's just math, in real, you can get small pieces, which you won't even see on gel or large pieces of several kbp.
Usually you know the sequence first if you want to do anything like that, so you check, whether is your gene cut by particular RE

[canalon]

To expand on jackbean's answer there are 2 situations:
- there is a restriction site of one of the enzyme in your freezer that is there and you are very lucky and can clone it that way.
- But as you have alreadyfigured out, it is usually not the case. There are many things that can be done in that case. The simplest one being to add some with PCR : you add restriction sites as overhangs at the beginning of the primers and run a PCR then digest the poduct and clone. Or simply do away without the RE and do a simple PCR followed by TA cloning, and eventually digestion of the resulting plasmid.

[biology_06er]

Hi there..

hmm ok--well I've looked at clips on online and other online sources and basically they say purify DNA from host and then cut it up and low and behold one of those fragments always seem to contain the gene...but ok that means you have to know before hand which RE to use right..so by my understanding of what you said below about adding to primers is that when I'm PCRing my gene...attach the restriction sites at the start of the primer and therefore when you insert it into the plasmid it has complementary ends because you know what the ends will be like in your MCS...have I understood this properly?..eep

Another thing..PCR makes many copies of my desired gene....so what is the difference in making copies by PCRing and my bacteria dividing and making lots of copies within the plasmid..is it just more time consuming and yay for PCR type thing..also another thing..why would I have to take of the hydrophobic regions of my pre-cursor protein..I hear it's because e.coli doesn't like hydrophobic ...but the actual reasoning as to why it does not like..

hoping once again you guys get meee

thanks
b_06er

[JackBean]

yes

basically yes, but as I said before, the bacteria will multiply all the DNA, while with PCR you get only your piece of DNA (don't forget, that we are talking about cloning from gDNA now!). Futhermore, with PCR you get much more DNA. For such amount you had to use a LOT of bacteria.
Regarding hydrophobic proteins, hydrophobic molecules (in general) don't like contact with water (thus hydro-phobic) and tend to aggregate in water (or in general in polar) solutions. Thus the hydrophobic proteins would aggregate and form inclusion bodies.

[biology_06er]

Thank you for your help so far ...but one last thing (hopefully) regarding attachment of restriction sites to the end of the primers...so say this site is at the 5 end of primer...when the first cycle takes place...how will the complementary stand be made...the Taq polymerase will only extend from the 3 end...on one website I read:

During the first round of PCR the complimentary strand of the overhang will be added, and in subsequent rounds of PCR this site will be amplified along with the rest of your PCR product. In the end, you will have made your DNA of interest plus restriction sites on both ends that you can now cut and ligate into an appropriate site.

Ok, so how will the complementary strand of the overhang be added exactly?...also in subsequent rounds how will "this PCR site be amplified" if the primers will always bind to the start of the gene..do I that make sense..?

Thanks
b_06er

[canalon]

Watch in awe the mighty PCR in action:

http://www.youtube.com/watch?v=_YgXcJ4n-kQ

No really, this movie is quite good at explaining the different step. Worth the 5 minutes of your life that it will take to watch it.

[biology_06er]

It was a video of how PCR works--one which I had already viewed and didn't really answer my question below..nevertheless..thanks for your help.

b_06er

[JackBean]

the RE place is on one end of the primer, thus it is incorporated into the amplicon with PCR