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Centrifugation query

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Centrifugation query

Postby cytochromeP » Fri Aug 19, 2005 12:59 pm

Consider two single stranded DNA molecules. One of them is 1 million bases long. The other is just 20 bases shorter than the first. Can I use centrifugation to separate these two molecules? If not, then is there any technique to separate these molecules ?
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Postby Chris4 » Fri Aug 19, 2005 1:17 pm

Thats only a tiny difference. Seems unlikely, doesn't it?
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Postby MrMistery » Fri Aug 19, 2005 8:42 pm

Electrophoresis with polyacrilamide might work, but the difference is still quite small...
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Postby canalon » Fri Aug 19, 2005 8:54 pm

Hmmm... Probably going to be highly difficult to see such a tiny difference. electrophoresis is not going to work, considering the size of your molecules: they won't move in the gel.

If you do not make any restriction beforehand, I don't see any solution. Have you any possiblity to make any labelling? If yes you could try to mark one with a huge molecule and then be able to differntiate them by centrifugation.

Without further details I can't suggest anything more precise

Good luck

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Postby DevGrp » Mon Aug 22, 2005 9:08 am

You can seperate large DNA molecules with pulse field electrophoresis in agarose (I've seperated whole chromosomes using the Biorad CHEF system), but again the problem is the very small size difference
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Postby Daniel Tillett » Mon Aug 22, 2005 2:49 pm

The simple answer is no. Is this just a hypothetical question? If not there are other techniques that would allow you to separate them.

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Postby cytochromeP » Mon Aug 22, 2005 2:55 pm

Daniel Tillett wrote:The simple answer is no. Is this just a hypothetical question? If not there are other techniques that would allow you to separate them.

Daniel

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Please give me the names of these 'other techniques' u r speaking about.
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Postby Daniel Tillett » Mon Aug 22, 2005 10:26 pm

Can you tell us more about why you need to separate the two DNA- the more details the better? There are a number of ways of doing this and some hard and some are relatively simple. Without knowing what you are trying to accomplish it is impossible to advise what is the best approach to take.

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The Idea

Postby cytochromeP » Tue Aug 23, 2005 7:10 am

Ok. This what and why I intend to use the techniques for.

Problem: To determine the directionality of replication origins i.e. to determine whether the given ori sequence is a uni or a bi-directionally replicating origin. This is to be applied to a circular, double stranded DNA molecule. Its size is approximately 1 million bases (e.g. E.Coli).

Principle: The replication bubble starts from the origin. Replication occurs in a semi-discontinuous fashion (leading and lagging strands). In a bi-directionally replicating ori, a strand which is 'leading' on one side of the ori will be 'lagging' on the other side of ori. But in a uni-directionally replicating ori, the leading strand will be leading throught its replication and the lagging will be lagging.
The replication enzyme being employed is not the holoenzyme E.Coli PolI but its Klenow fragment. A Klenow fragment lacks 'nick-translation' activity. Therefore the RNA primers are not degraded. Hence at the end of the replication, the daughter DNA molecules would be hetero-polymers of DNA and RNA.
The products of a uni-directional replication are subjected to RNAse treatment. The products of a bi-directional replication are also subjected to RNAse treatment. The interveneing RNA segments are destroyed leaving back only the DNA fragments. The kind of DNA fragments resulting would be a function of the directionality of replication.
The set of cleavage products of RNAse, the DNA fragments, would be a characteristic signature of the directionality of ori.

Classical Approaches: The text-books (David Frefielder) have given two techniques for ori's directionality determination: 1) Denaturation Mapping and 2) Radio-isotope pulse chase techinique (tritium, N15). Both these techniques require costly high-tech equipment like a electron microscope and radio-isotopes (which are heavily subjected to liscencing issues).
The technique mentioned above (provided it works !) requires neither of these big toys.

Technical Problem: The problem now is how to separate the long leading DNA fragment, which is the product of RNAse treated uni-directionally replicated DNA, from the two parental DNA strands which remain untouched by RNAse. This large DNA fragment (actually the largest 'fragment') differs in size from the parental DNA fragment by only 20-30bps (the length of an RNA primer). How do I separate these two slightly differing molecules :?:
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Postby Daniel Tillett » Tue Aug 23, 2005 12:19 pm

There are many ways of accomplishing what you want.

1. Do a native Southern (ie don't denature your DNA during transfer to the membrane. RNaseH treat then probe with a labelled oligos that can differentiate bi from unidirectional replication.

2. Capture your DNA using biotin labelled oligos after RNAaseH digestion to free the ssDNA regions.

3. Clone your origin of replication in a plasmid and determine the direction origin from there.

4. Isolated DNA free of other RNA then DNAase digest. This should leave only the RNA primers. Ligate to primers using RNA ligase then RT PCR and clone. The fragments you clone should be the RNA ori primers.

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Postby Jelanen » Tue Aug 23, 2005 10:25 pm

Yes, you CAN separate those DNA strands via centrifugation with the following qualifiers, you need a solution in which allows a large separation and you need alot of time and G. 20bp is alot on a molecular level, but not alot on a human level, so it will take alot of time and alot of high speed spinning. You're better off using something else that has been suggested.

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Postby Daniel Tillett » Tue Aug 23, 2005 11:08 pm

I don't that you would be able to in practice separate two 1,000,000 bp DNA strands that differ by 20 bp as that is only 0.002% difference in length!

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