Gene technology Watch

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Ellie4
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#1
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Can anyone give me a nice definition of what 'sticky ends' means, if it come out in the exam? I know in general terms, but don't know if I could define it.

Also, if anyone has the time or patience to explain DNA fingerprinting with probes and all that shibang, I would be externally grateful!
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perkyDani
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sticky ends are unpaired bases which readily can join up with complementary bases.
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Ellie4
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(Original post by perkyDani)
sticky ends are unpaired bases which readily can join up with complementary bases.
So one end of the DNA would be cut at, say C and T, and that would readily pair up with another sticky-ended piece of DNA which was G and A? :confused:
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idiopathic
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Yep! In an applied sense, if the mRNA has those sticky ends, and is then mixed with open plasmids (say from bacterial cells), the complementary bases join up and the mRNA becomes incorporated into the plasmid and this join is made permanent by DNA ligase.
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Ellie4
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(Original post by endeavour)
Yep! In an applied sense, if the mRNA has those sticky ends, and is then mixed with open plasmids (say from bacterial cells), the complementary bases join up and the mRNA becomes incorporated into the plasmid and this join is made permanent by DNA ligase.
So, the plasmid is also cut with sticky ends? As well as the piece of mRNA which is being inserted into the plasmid? (Sorry for being thick )
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Twiglet
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Yep! In an applied sense, if the mRNA has those sticky ends, and is then mixed with open plasmids (say from bacterial cells), the complementary bases join up and the mRNA becomes incorporated into the plasmid and this join is made permanent by DNA ligase.
woooo, why would you want to put mRNA into a plasmid? Don't they normally just put DNA in ?
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Twiglet
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So, the plasmid is also cut with sticky ends? As well as the piece of mRNA which is being inserted into the plasmid? (Sorry for being thick )
Yup the plasmid and whatever you want to insert are both cut by the same restriction endonuclease, since it cuts at recognition sites, complementary sticky ends will be produced so they can join together. There would be no point in the plasmid and the gene having different sticky ends - the ligase would not be able to anneal them as the bases are not complementary.
franks x
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Ellie4
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(Original post by franks)
Yup the plasmid and whatever you want to insert are both cut by the same restriction endonuclease, since it cuts at recognition sites, complementary sticky ends will be produced so they can join together. There would be no point in the plasmid and the gene having different sticky ends - the ligase would not be able to anneal them as the bases are not complementary.
franks x
So the plasmid is cut at say, A and C
and the DNA is cut at T and G?

That what you mean, yeah?
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idiopathic
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(Original post by Ellie4)
So, the plasmid is also cut with sticky ends? As well as the piece of mRNA which is being inserted into the plasmid? (Sorry for being thick )
Yep, with "restriction endonucleases" enzymes.

(Original post by franks)
woooo, why would you want to put mRNA into a plasmid? Don't they normally just put DNA in ?
*whips out dusty biology book*

Yep you're right. The mRNA they get which carries the code for whatever (eg insulin) is treated with reverse transcriptase to convert mRNA -> cDNA (complementary DNA) which is then put into the plasmid, after the sticky ends are put in.
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Twiglet
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So the plasmid is cut at say, A and C
and the DNA is cut at T and G?
Well, remember DNA has 2 strands, the cut made is staggered;
ATCGATCG
TAGCTAGC
if this is the DNA sequence then then it may be cut so that one sticky end it:
AT
TAGCTAGC
and so the complementary one will be:
--CGATCG
-------GC

Both the plasmid and the gene will have the same sticky ends, its way easier to understand if you can find a pic on the internet or in a text book, what I drew above might be a bit confusing...
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Ellie4
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(Original post by franks)
Well, remember DNA has 2 strands, the cut made is staggered;
ATCGATCG
TAGCTAGC
if this is the DNA sequence then then it may be cut so that one sticky end it:
AT
TAGCTAGC
and so the complementary one will be:
--CGATCG
-------GC

Both the plasmid and the gene will have the same sticky ends, its way easier to understand if you can find a pic on the internet or in a text book, what I drew above might be a bit confusing...
Ok, thanks! Sorry, but I really hate this topic, so it's making my brain hurt! It's worse than respiration and photosynthesis put together! :p:

Don't suppose you'd like to explin DNA fingerprinting to me
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idiopathic
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Try here: http://biologymad.com/master.html?ht.../ASBiology.htm
then click on "Genetic Engineering".

Then you should find info on the website, and also links on the right hand side to external interactive fun such as this (under Additional Support Materials).
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Twiglet
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Don't suppose you'd like to explin DNA fingerprinting to me
lol, thats a bit tricky isnt it ! hehe
basically you get a piece of DNA which you want to find out the sequence of bases, get 4 test tubes, in each you have got the normal nuclotides A T C G and some DNA polymerase. If this is all you had then the strand of DNA would be copied again and again until the nuclotides ran out - all the strands of DNA would be the same length.

BUT in each of the 4 test tubes you add some altered nucleotides, in the first test tube you would have lots of altered A's, in the secton lots of altered T's etc etc. When the DNA polymerase is busy copying the DNA strand, sometimes one of these altered bases will be added , when an altered base is added the replication stops. therefore when replication of the DNA strand has finished you will be left with lots of chains of nucleotides, all of different lengths. All the strands in the tube with the altered A nucleotide will end in A , all the strands in the tube with alterd t will end in T etc etc.

woo this is a long explanation - sorry ! Now you need to separate the strands using electrophoresis - this separates the strands according to length, the shortest strands diffuse the furthest. The electrophoresis tray is set up with 4 columns - each column contains the contents of one test tube. BY taking an autoradiograph of the positions of the bands on the final electrophoresis tray the sequence of DNA bases can be determined.

I think you could also do the whole of this by adding restriction enzyme to the original sample...

I hope that made some kind of sense, i kinda ran out of steam at the end !
franks x
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Ellie4
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(Original post by franks)
lol, thats a bit tricky isnt it ! hehe
basically you get a piece of DNA which you want to find out the sequence of bases, get 4 test tubes, in each you have got the normal nuclotides A T C G and some DNA polymerase. If this is all you had then the strand of DNA would be copied again and again until the nuclotides ran out - all the strands of DNA would be the same length.

BUT in each of the 4 test tubes you add some altered nucleotides, in the first test tube you would have lots of altered A's, in the secton lots of altered T's etc etc. When the DNA polymerase is busy copying the DNA strand, sometimes one of these altered bases will be added , when an altered base is added the replication stops. therefore when replication of the DNA strand has finished you will be left with lots of chains of nucleotides, all of different lengths. All the strands in the tube with the altered A nucleotide will end in A , all the strands in the tube with alterd t will end in T etc etc.

woo this is a long explanation - sorry ! Now you need to separate the strands using electrophoresis - this separates the strands according to length, the shortest strands diffuse the furthest. The electrophoresis tray is set up with 4 columns - each column contains the contents of one test tube. BY taking an autoradiograph of the positions of the bands on the final electrophoresis tray the sequence of DNA bases can be determined.

I think you could also do the whole of this by adding restriction enzyme to the original sample...

I hope that made some kind of sense, i kinda ran out of steam at the end !
franks x
Thanks for taking the time to write this. I'm having a good look at it tomorrow, so if I have any more q's, I know who to come to! :p: Oh, and I'll rep you after my 24h thing runs out
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Twiglet
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Thanks for taking the time to write this. I'm having a good look at it tomorrow,
pleasure, have a good look at a text book after - and if it disagrees with me , believe what it says ! hehe
franks x
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theunforgiven
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hey guys just wanted to confirm this:
C + G = A + T this is right yes.
then there was this question tht if a DNA sample has 15% of guanine nucleotides then what percentage of addenine would you expect?
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Ellie4
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(Original post by theunforgiven)
hey guys just wanted to confirm this:
C + G = A + T this is right yes.
then there was this question tht if a DNA sample has 15% of guanine nucleotides then what percentage of addenine would you expect?
If you have 15% G, you'd have 15% C = 30%
That leaves 70%, half of which will be T and half will be A

So 35% adenine
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Saffie
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(Original post by franks)
I think you could also do the whole of this by adding restriction enzyme to the original sample...
oh.. so that's why what you'd written was totally beyond me!

Genetic fingerprinting works because we all have a different amount of minisatelites between exons. So when different people's DNA is cut by the same restriction enzymes, the masses of the fragments are different. In electrophoresis, the DNA fragments are put into wells in gel, covered by a buffer solution that conducts electricity. The DNA is negatively charged so moves to the positive electrode. Different masses move faster and therefore further than others.

franks didn't mention southern blotting after the electrophoresis. You put a nylon membrane over and tissue paper to help absorption / or UV light, to bind the DNA fragments. Heat to separate into single strands (breaking H bonds). Radioactive gene probes are then put onto the nylon membrane. The probes are warmed and incubated for a while so they'll attach to any bits of complementary DNA in the DNA fragments.

The nylon membrane is then put on top of an unexposed photo film, the film goes dark where the radioactive gene probes are, this reveals the position of the DNA fragments as 'bands'. The darker the band, the more radioactive probes attached to it. The fewer the bands the less probes used (i believe they're specific so if you only have one type, there will only be one band)

I think you'd remember it better if you'd actually done it :cool: It's not so bad as breathing control or blood pressurey stuff :eek:
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Aired
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(Original post by theunforgiven)
hey guys just wanted to confirm this:
C + G = A + T this is right yes.
then there was this question tht if a DNA sample has 15% of guanine nucleotides then what percentage of addenine would you expect?
If you have 15% guanine you must also have 15% cytosine, as they're complementary. That means 70% (100 - [15x2]) of the DNA is made of adenine and thymine, therefore there is 35% adenine (75/2)

Franks was describing Sanger sequencing by the way, lol.
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Twiglet
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Franks was describing Sanger sequencing by the way,
yeah sorry, should have said!

franks didn't mention southern blotting after the electrophoresis. You put a nylon membrane over and tissue paper to help absorption / or UV light, to bind the DNA fragments. Heat to separate into single strands (breaking H bonds).
I think usually when using the Sanger method, single DNA strands are used (presumably what would be the equivalent of the sense strand), so heat would not be needed to separate into single strands.

Arrrgh, why are there so many ways of doing exactly the same thing!!
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