h2g2 The Hitchhiker's Guide to the Galaxy: Earth Edition

'these are then arranged in order of length and tagged with radioactive probes. These emit x-rays, so when the sample is photographed they show up'

No, radioactive nuclides do not emit X-rays. Unstable (radioactive) nucle emit three different types of radioactivity. alpha emitters radiate alpha particles which are helium nuclei, beta radiation is high energy electrons (or positrons) and gamma radiation is gamma rays.
It doesn't really matter which type of emitter is present, they will all irradiate photgraphic film. Usually phosphorus-32 is used as the radioactive source in this technique - this is a high energy beta-emitter.
I would say it's a good idea to change the above sentence to something like 'The radiation emitted by the tags will show up on the film when the sample is exposed to a photographic plate.'

'Once the DNA sequences are ordered, chemical probes are added to the sample; like the restriction enzymes, these each select a particular sequence, and bind to it. As they contain radioactive atoms, when the sample is exposed to photographic film, these sequences will show up. An alternative method uses chemiluminescent labelling, where enzymes emit light by a chemical process.'

Ouch. It's difficult to know where to start here (and its' partly my fault as I think I encouraged this paragraph in Peer Review and then didn't follow it up - sorry).
restriction enzymes are used *before* you run the gel -they are what chops the DNA up into managable lengths for sequencing. You do not add anything to the gel to bind to the DNA strands once the gel has run (at least not in this technique). The radioactivity is contained in small amounts of modified nucleotides (DNA is a polymer of nucleotides - they are base-sugar-phosphate monomers) that contain a radioactive phosphorus atom and are modified so that once incorporated into the DNA chain by DNA polymerase (in PCR) the chain will not extend further. There are four bases A,C,T and G in DNA so you add small amounts of modified nucleotides for all of these. The final gel contains four lanes for each chain terminating base. SO you know which lane ends in A, which ends in T etc. You can then simply read off the A,C,T G sequence from the plate. This is how DNA sequencing works.
Lately to avoid the use of radiation, some people came up with some fluorescent tags instead. In this case, instead of the DNA chain terminating with a radioactive nucleotide, it terminates with a nucleotide that contains a fluorescent group. Thus instead of photgraphing the plate, you simple shine a light on it and the flourescent groups show up (like reflectors on your trainers ). This is *not* the same as chemiluminescence (which makes fireflies light up in the dark for example), nor is it phosphorescence which is what makes your clock face remain lit after you have turned out the light). Fluorescence goes away as soon as you've switched off the light source. Also becareful of the use of the word 'enzyme' it's pretty clear from this that the author isn't too sure what an enzyme is (no offence meant ). It is a protein that is capabable of catalyising a specific chemical reaction and will not (except under highly exceptional circumstances) emit light.

'Chemiluminescent labelling is being used increasingly instead of radioactive tags due to the practical problems of using radioactive materials; and the process is now largely automated.'

Again, it is fluorsecent labelling, not chemiluminescent.

Hope that's clear Get back to me if that's all gobbledegook

Orcus

"PCR is a powerful technique which effectively 'amplifies' DNA. It can produce many copies of the DNA from a single cell."

Further to the previous post, PCR only amplifies very small, specific regions of DNA, not the entire genetic complement of the cell.

"Initially the DNA is removed from the sample cells by chemical methods, and the two strands of the double helix are separated."

I don't think the strands are purposefully separated, the enzyme cuts through double-stranded DNA anyway.

I don't think the strands are purposefully separated, the enzyme cuts through double-stranded DNA anyway.

Restriction enzymes do cut double stranded DNA but not I'm not so sure about that. The DNA polymerase used in PCR is from an archaebacterium found in the volcanic vents at the bottom of the oceans and does work at high tempreatures. I'm pretty sure the high temperature does go above the 'melting temperature' of DNA. Certainly the polymerase works on single stranded DNA and the purpose of the elevated temp is to unravel the DNA (otherwise you'd need a topoisomerase in there too {an enzyme that 'unwinds' double stranded DNA}).

The "I don't think the strands are purposefully separated, the enzyme cuts through double-stranded DNA anyway." bit came under the general heading of "DNA fingerprinting", and was described as a stage before the restriction digest. Which is unnecesary.

Taq polymerase (Thermus aquius is the bacteria isn't it?) denatures around 90-something degrees celcius (pretty amazing really). With PCR, the strands dissasociate at 74 degrees. In vivo this strands are separated with the enzymes in the replicon, helicases and as you mentioned, toposiomerases.

Are you a molecular biologist too then?

Just one thing...
About the above posts: I think you might be getting confused between DNA sequencing and DNA fingerprinting. We were taught that to do a DNA fingerprint you just take a section of DNA which has VNTRs in it (Variable number tandem repeats, eg atatatatat for a variable number of times) and then run that on the gel. That way, if you take 2 different people, they may have different numbers of repeats, and therefore have different restriction fragment lengths. If you do this for a large number of different sites, particularly sites which have a large variability between individuals, you get a "unique" fingerprint.

Correct me if I'm wrong (I've got an exam on Sunday!)

=)

I hope I haven't really upset either of you by making a mess of an entry you wanted to write.

Orcus, I understand the technique you explain is the modern method (or one of the modern methods) of DNA sequencing, but I couldn't any information on fingerprinting refering to this method. All the articles I consulted refered to the method I explained, but this may be just because they're out of date (or erroneous). I wanted to research this further, but the scouts and sub-editors were quicker than I was.

I think different sequencing methods are used depending on whether PCR is used or not, it may be the method I describe is only used without PCR.

Chemiluminescent labelling is probably the wrong term, I took the name from a posting someone left on peer review, I should have checked that.

But I do know about radioactive nuclei, as I use them a lot in my research. They certainly can emit X-rays. I don't know which materials are used in DNA sequencing, but I think X-ray emitters would be the most useful.

You're quite right about separating DNA strands.

As for "PCR .. can produce many copies of the DNA from a single cell."
I never said it copied the entire genome, I just meant that you only need a single cell as a sample.

Can I write any more entries on genetics? Or should I stick to particle physics?

MP

Sorry to butt in but...Although it is theoretically possible to do PCR from a single ceel, it is highly unlikely as the processes required to extract the DNA from the cell will most likely lose such a small amount.
There are diferent methods of sequencing...these days it is more likely to use fluorescence (I have been for the last year, even though I can never remember how to spell the fl-word) Radiation is still used for awkward sequences where you have a high percentage of CG pairs.
But sequencing ALWAYS requires PCR...'tis the nature of the beast.

You can use both chemiluminescence as a probing method, as you can radioactivity (usually p32...I think) and fluorescence, with a chemiluminescent tag bound to your probe...which can be an oligonucleotide (DNA) or an oligopeptide (protein)

I'm sure there is more, but I can't remember, and I got to go play in the lab inna bit..

Write on whatever subject you want to Primigenius...... The problems we is having may just be us reading things the wrong way..
And if nuffing else, I quite like finding out what people think we is up to in our hidey-holes (you gotta dashed good grasp of it anyhow) But I'd be interested to read what you do on a day to day basis..

Zantic...that the pompousness over for the day!!!

OK, I do know some radioactive nuclei emit X-rays and one can get X-ray Brehmstrahlung but the nucleus used virtually exclusively in DNA sequencing is Phosphorus 32 and is most definitely a high energy beta-emitter. If you take a look through the Amersham catalogue for radioactive biomolecules there is no mention of X-ray emission except for the mention of brehmstrahlung (and the avoidance of). If you're a particle physicist I'm guessing you use rather more esoteric nuclei than we do. Currently for plate development they actually prefer to use P-33 rather than P-32 as it is a lower energy beta-emitter than P-32 and hence gives nice sharp lines on photographic plates rather than
the huge blobs that P-32 gives - ie. better resolution - the only drawback is that P-33 is considerably more expensive than P-32.

I'm not a molecular biologist but I do do chemistry/biochemistry research so have learned and read up quite a lot on molecular biology as I do use recombinant enzymes quite a bit.

What concerned me most in this article was the worrying remarks about enzymes.

Don't worry though, no treading on toes has been done. There will be a blue moon before I would have got around to writing this Most of the article is fine it was just one or two pragraph in that section that concerned me.

Hi folks,

I'm the Sub who edited this article, and I dropped in to offer a bit of help for the corrections. First of all, I'm no molecular biologist or chemist either, and English is not my first language. So most of this thread is utterly incomprehensible to me. I gathered from the PR thread that there were a few questionable points, which I thought were addressed by Mammuthus Primigenius (I delayed the subbing for two weeks to give him the possibility to do some changes), and left the article more or less as it was.

So, if there are any factual errors left in the article, or if there is some important information missing, could you please post here? And, please, could you write your corrections in such a way that they could easily be pasted into the article? I'm thinking of 'paragraph #2 in section xyzzy should read like this "Yada yada yada."' I would then alert the in-house editorial team to fix it.

Thanks a lot,
SchrEck Inc.