Naked Science Forum
Life Sciences => Cells, Microbes & Viruses => Topic started by: fibintern on 07/02/2007 20:14:07
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After attepmting to research genetic fingerprinting and gel electrophoresis I was still left with the question about what the white dots visually represent. Are they nucleotides? Or are they the individual pieces of DNA themselves showing up on the gel at varying lengths according to the size of each strand?If this is the case how is that an indication of someones individual identity rather than just the size of each sample of DNA?
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in normal DNA electrophoresis, the bands that appear on the gel are at different distances from the well that the sample was loaded in. this distance is representative of the size of the DNA fragment. The larger a fragment is, the less distance it will travel, as it will be hindered by the actual gel, whereas small fragments will pass through teh gaps in the gel structure easily, and so will travel longer distances. so you second answer is the correct one.
an example is when you have a plasmid, and you cut it with retriction enzyme. if the enzyme cuts in only one place you will only get one band, but if it cuts in two places, you will get 2 bands. running the sample on a gel will show you the sizes of these two bands. [diagram=143_0]
The diagram is a bit rubbish!
You can run markers alongside your sample, which are DNA fragments of known lengths, so you can work out the approximate sizes of your pieces of DNA. This is a useful technique, and i often used it to determine that I had correctly inserted a gene into a plasmid - if it was there the length of the DNA fragment between two 'cut' locations would be bigger.
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oh yes, you can also get sequencing gels, which I have actually ran before, but I can't remember anything about them! I'll have a go:
essentially four separate PCR reactions are run alongside each other for the same piece of DNA, but each uses a different terminator nucleotide. this means that of the nucleotides provided to enable the generation of new DNA strands from the template, some of one type in each mix (A,G,C or T) will be slightly different to normal, and will cause the termination of elongation. Because the incorporation of a terminating nucleotide is random, the result is a mix of lengths of prematurely truncated pieces of the same DNA sequence. These can be run on a gel, as I described in my previous post, with the smallest fragment at the bottom and the largest at the top. in between will be all the possible lengths (hopefully!) where that particular nucleotide is the last one. If you run all four mixes next to each other on the same gel you should get a band appear for every possible length of DNA possible, which basically tells you the order of the nucleotides in that sequence. very clever.
[diagram=145_0]
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Just to add on . . .
If this is the case how is that an indication of someones individual identity rather than just the size of each sample of DNA?
Different individuals have small differences in their DNA. As a simple example: if we cut a sample of your DNA with an enzyme that recognizes the sequence GAATTC and ran those out on a gel you would see fragments of many different sizes depending on where the cuts are. Now if we cut my DNA with the same enzyme, but at one or two places where you have GAATTC, I have GAATTT or AAATTC the enzyme now won't cut at those spots and the fragmentation pattern that you see when you run out on the gel will look different (I will have some longer pieces). Now if we make this more complicated and add in a few other enzymes we can start to cover enough of the genome to tell people apart.
I'm not sure if this is exactly what you are doing, but this is the basic idea.
Colleen
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One more thought . . .
If you ran DNA out on a gel by itself, you probably wouldn't be able to see it- it has some intrinsic flourescence but very little. So to make it more visible we add a dye (there are a few different types that can be used). Commonly, ethidium bromide is used this dye likes the area in between the DNA bases and when it gets into this area will increase its flourescence. So where you have a lot of DNA accumulating (at a band) you will get a lot of flourescence and hence a "white spot" that you can see under UV light.
Since ethidium bromide loves to go into DNA it is considered to be a potential carcinogen and mutagen so other dyes are starting to be used a bit more commonly to avoid having to deal with ethidium.
Colleen
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I liked the diagrams
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Here's a link to an article I wrote with Dalya Rosner about how genetic fingerprinting works:
http://www.thenakedscientists.com/HTML/articles/article/dalyacolumn8.htm
I think this covers most of the story.
Chris
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Glad you liked the diagrams!
I don't know if you read sciencey-type magazines, but there's a new cool gel running piece of equipment out that runs gels in minutes and you can see the bands instantly without having to faff about with UV light boxes and ethidium bromide!
I'm not in the lab anymore, but if I were I'd want one!
only a geeky scientist could think something like this is cool [:-\]
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I have seen those. . .
but agarose and ethidium are sooo cheap, it is hard to justify that.
There are so many other experiments that could be done for the cost of those,
but they sure look cool . .I'd like to try one someday.
I think it's cool (and I'm not geeky at all) snort snort.
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This video we made explains DNA fingerprinting in a visual way:
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DNA electrophoresis is a technique used in sorting DNA molecules by length. DNA pieces are usually floating in a gel’s tray and subjected to an electric field, which make them move around one end of the tray to another which separates them out into bands. This technique is helpful in detecting genes for diagnosing disease and useful in genetic researches too.
[MOD EDIT - THANKS FOR THE SPAMMY LINK, WHICH WE'VE REMOVED FOR YOU SO THAT YOU STAY WITHIN OUR GUIDELINES]