F215 - Revision thread 13th June 2011

Allrighty, let's start with the first bit then:

First, you've gotta get the section of the genome you want - that is the gene you want from which part of the chromosome.
To do this you can, extract the part of the genome you want by breaking it off the chromosome, this is known as a shotgun approach.

Then, you've got a lot of genome present, so you put it into BAC (bacterial artificial chromosome) and you insert that BAC into E.coli

E.coli then reproduces, and you end up with loads of copies of the genome.

You then use restriction enzymes to cut it into fragments, this gives overlapping fragments because you've got loads of sections of the genome which have been cut at different places.

You then mix the fragment types with DNA polymerase and free DNA nucleotides and primers(which are short strands of DNA that bind to the 3' end of DNA)
AND ALSO Terminator bases, which have one of four colours (As there's only 4 possible bases A T C G)

The primer binds/anneals to the 3' End of DNA - this allows DNA polymerase to get working and adds the free nucleotides to the fragment.

So, when the terminator base is added and binds to the DNA fragment, it throws off the enzyme DNA polymerase, so DNA replication is stopped.

Therefore, different fragments will be thrown off at different points, and will have one of four colors ( A could be red, T could be green, C could be yellow and G could be black) - These are fluorescent labeled and you'll see why they're useful later on.

- So now you've got different fragment lengths of your genome, that has been stopped at different points for each fragment.

you then separate using electrophoresis, it involved cutting a well on a slab of agar, electric current is passed through the DNA fragments that you place in the well.
DNA is negatively charged cus of the phosphoryl groups, so it'll move to the positive end.

Shorter DNA fragments will pass through quicker through the gel, so you'll end up with fragments on this gel that are separated by size.

They'll pass through an automated sequencer at the end of the gel, and the flourcent label will be used by this automated sequencer to give the colour of that fragment, therefore the bases to.

can someone explain a part of the polymerase chain reaction to me please?

in the book it says that the double stranded section of DNA only grows from the 3' end
What does 3' and 5' end mean?
please quote, thanks

Hai Iby! hows it goin (if you remember me :P)

Also for you guys...

Module 4 Notes that I made last year (hopefully not much/nothings changed in that content).

http://www.mediafire.com/file/qit5izu1zyt/bio%20notes.doc

good luck, hope you nail these exams

Allrighty, let's start with the first bit then:

First, you've gotta get the section of the genome you want - that is the gene you want from which part of the chromosome.
To do this you can, extract the part of the genome you want by breaking it off the chromosome, this is known as a shotgun approach.

Then, you've got a lot of genome present, so you put it into BAC (bacterial artificial chromosome) and you insert that BAC into E.coli

E.coli then reproduces, and you end up with loads of copies of the genome.

You then use restriction enzymes to cut it into fragments, this gives overlapping fragments because you've got loads of sections of the genome which have been cut at different places.

You then mix the fragment types with DNA polymerase and free DNA nucleotides and primers(which are short strands of DNA that bind to the 3' end of DNA)
AND ALSO Terminator bases, which have one of four colours (As there's only 4 possible bases A T C G)

The primer binds/anneals to the 3' End of DNA - this allows DNA polymerase to get working and adds the free nucleotides to the fragment.

So, when the terminator base is added and binds to the DNA fragment, it throws off the enzyme DNA polymerase, so DNA replication is stopped.

Therefore, different fragments will be thrown off at different points, and will have one of four colors ( A could be red, T could be green, C could be yellow and G could be black) - These are fluorescent labeled and you'll see why they're useful later on.

- So now you've got different fragment lengths of your genome, that has been stopped at different points for each fragment.

you then separate using electrophoresis, it involved cutting a well on a slab of agar, electric current is passed through the DNA fragments that you place in the well.
DNA is negatively charged cus of the phosphoryl groups, so it'll move to the positive end.

Shorter DNA fragments will pass through quicker through the gel, so you'll end up with fragments on this gel that are separated by size.

They'll pass through an automated sequencer at the end of the gel, and the flourcent label will be used by this automated sequencer to give the colour of that fragment, therefore the bases to.

PCR is a way of amplify DNA (increase the sample size)


1. Heat DNA to 95oC to separate strands as the hydrogen bonds break between the complementary bases
2. Add primers (short bits of DNA for DNA polymerase to bind to)
3. Reduce temp to 55oC; primers anneal (bind to complementary bases)
4. Raise temp to 72oC and add DNA polymerase
5. DNA polymerase binds and makes new complementary DNA using free nucleotides
6. Repeat the process to increase DNA exponentially

Hope this helps! Do you remember from AS that the bases bond with condensation reactions with each other? 3' means that the bond is on carbon 3 of a single base molecule, and 5' at on carbon 5

Hay, whats with the overlapping bit in genome sequencing, that really confuses me????????

How far are you guys into revision?
I am moving onto Ecosystems, and should go straight to module 4 in the next 1-2 weeks...

try and do eco systems within a week

What so your on Ecoysystems also?
I need to do that/ finish module 4, and then revrevise everything:/ Hopefully there is enough time.

What so your on Ecoysystems also?
I need to do that/ finish module 4, and then revrevise everything:/ Hopefully there is enough time.