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Restriction Digest Assignment Difficulty


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#1 Cist

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Posted 30 October 2010 - 04:33 PM

I am having trouble with one of the questions in my restriction digest assignment, a classmate and I have been thinking about it for days but we can't figure it out. Can anyone help?

A large number of 10kbp plasmids are digested:

Endonuclease / Fragments
BamHI / 10
EcoRI / 10
PstI / 2.7 , 7.3
NotI / 10
SstI / 4.8 , 5.2
TaqI / 6 , 10 , 17 , 23

It then goes on to do double digests with varying combinations and I drew the circular restriction map. The 2nd part of the question asks to identify anomalies and say why they could happen.

Obviously the anomaly is that somehow a 10kbp fragment is digested into 6, 10, 17, and 23 fragments. Also, for example, if you mix TaqI with PstI you get 2.7 and 7.3 fragments. This leads me to believe that the TaqI does not cut at all. But I have no idea whats going on with the 6, 10, 17, and 23.

#2 perneseblue

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Posted 31 October 2010 - 06:04 AM

This leads me to believe that the TaqI does not cut at all.

Good job

I shall offer you a clue... Topology.

A plasmid isn't simply a circle, it is a circle which has been twisted... supercoiled. Imagine putting a twist into a rubber band and you will get idea. Supercoiling alters the effective size of the plasmid. You should also note that circular DNA (supercoiled or not) do not move at the same speed as linear DNA.

Lastly, if a plasmid is a circular loop of DNA, what happens when 2 plasmids are linked together like two rings in a chain?
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#3 Cist

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Posted 31 October 2010 - 06:23 AM

So...

The gel electrophoresis is more or less designed for linear DNA molecules? So the specific size, shape, and charge density of the plasmid moves faster through the gel giving a false impression that it is bigger than it actually is? And the cross-linking of plasmids gives the impression that there are multiple fragment? (i.e. 1 plasmid looks like a 23 fragment, 2 cross-linked plasmids look like 17 fragment, 3 cross-linked looks like 10, etc.) and therefore there is nothing specific about the numbers 6, 10, 17, 23, they just happen to be what the false results look like.

I'm really racking my brain here, that's the best I've got.

and thanks for your time!

#4 perneseblue

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Posted 02 November 2010 - 09:18 PM

Um not quite.

From the restriction digests, TaqI/Pst , Pst and TaqI, you have established that TaqI isn't cutting the plasmid.

Plasmid DNA is normally supercoiled... imagine a rubber band with several twist in it... resulting in a molecule with a compact rod shape. Being more compact, a supercoiled plasmid moves faster through the agarose gel matrix (a sieve) than a linear plasmid of equivalent length. This supercoiled plasmid is probably represented as the 6kb band.

The process of preparing plasmid DNA is a physical process that may damage the DNA.

If a double stand break is made in the plasmid, you get linear molecule. The 10kb band is probably represents this.

If only one DNA stand is broken, the twist in the plasmid is allowed to unwind and the plasmid relaxes. What you get is a plasmid in an open circle configuration which takes up alot more space. This molecule will move through the agarose gel slower.. and is probably represented by the 17kb band.

As for the 23kb... if you have two plasmids interlinked together like two rings in a chain.. it was cause the two molecules to migrates as one.. at a slow pace.

And yes, in a sense when we do gel electrophoresis we are thinking linear DNA. Commercial DNA ladders are made of linear DNA fragments. Thus to make any meaning full comparison with these ladders, all DNA samples must be linearised by restriction enzymes. Linearisation also has the added benefit of converting all topological forms of a plasmids into one.
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