Naked Science Forum
General Science => General Science => Topic started by: mycorrhizae on 15/03/2020 16:41:04
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Hi there!
I'm getting round to completing my biology A levels this year. Currently we're taking a look at all things biotechnology, and my question focuses specifically on DNA sequencing. I've just written about modern Sanger sequencing, which is obviously simplified for the textbook but from what I get-
1. Segment DNA
2.Combine a segment with primers, florescently tagged ddNTPs, dNTPs and DNA polymerase. Place in a thermocycler.
3. Separate the strands of your DNA fragment by heating to 95°c
4. Decrease temperature to 50°c so that primers can anneal
5. At 60°c, DNA polymerase will then build a complementary strand to each template strand; but only so far as the addition of a complementary terminator base/ddNTP will allow.
6. Eventually, you end up with every possible length of complementary strand possible (ie. all complementary strands whether it be to the first base or all bases along the original DNA fragment.
7. The complementary strands are then separated by length through gel or capillary electrophoresis. The lightest fragment is the first base of the complementary strand and the longest complementary strand is tagged at the last base. You look at the florescent tags from the shortest to longest complementary strand and that should reveal the sequential base order of that strand, determined on the colour of each florescent tag in representing a certain base.
These colour signatures and the order that they follow are detected by lasers that feed into a computer, which produces an electropherogram of coloured peaks so that we can observe the results.
8. Using base pairing rules, you can then "invert" the bases of the peaks to find the base order of the original, parent strand.
....thing is, they're not all the same height. It's not required knowledge, but I'd like to know why.
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Why is it also that different bases can overlap in places, or that peaks of the same base can be conjoined? Is it just to do with the sensitivity of your lasers and interface? Is it to do with the fact that, technically, because you separated the original DNA fragment into two, two sets of complementary strands are being made and the machine isn't sensitive enough to differentiate between one of the parent strands being identical to one of your newly constructed DNA strands?
I'm already going off the deep end here, so if anybody could answer any of my question in a way that an A-level student could roundabout get, that would be amazing!
Thank you so much! :) :)
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The vertical axis presumably is intensity of fluorescence and the horizontal, time.
What determines intensity, for a given base? Presumably, the concentration of molecules in the fluoro cell at a given time, and there is no reason why that should be constant.
As you are dealing with complex molecules in a liquid, you can't expect them to be separated as cleanly as simple molecules in a gas carrier, and even in gas chromatography you get a spread of analyte and possible mixing due to viscous drag along the flight tube. Hence some overlap, which is visible on the entire trace but where it occurs between repeats of the same base, it looks like "conjoint" traces.
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I think the separations are good- otherwise the technique simply wouldn't work.
But it's possible that the yield of labeled material is a bit variable.
It's also possible that the intensity of fluorescence is variable because of different quenching by adjacent bases, though I'd expect that effect to be small.
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How times have changed! My A level chromatography involved red, black and blue ink and blotting paper!
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How times have changed! My A level chromatography involved red, black and blue ink and blotting paper!
In my day they improved it considerably- we had a solvent too.