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Tools for DNA discovery and innovation

Overview of techniques used to study DNA and genomes.

Ruth MacKinnon

on 1 February 2015

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Transcript of Tools for DNA discovery and innovation

Complementary DNA and RNA
Tools for DNA discovery
- copy DNA
in the test tube

DNA polymerase
makes new DNA

Gel electrophoresis - separates DNA
- allows DNA to be

Restriction enzymes - molecular scissors
Southern blot
- transfers DNA from gel
onto a membrane
DNA vectors
Add a piece of DNA into vector DNA
A host organism will treat it like its own

DNA ligase
- molecular

Plasmids are circular DNA molecules extra to the bacterial chromosome
extract plasmid from bacteria
cut circle open (restriction enzyme)
add foreign DNA - join ends to form a circle again (ligase)
replace in bacteria
-> bacteria treat your DNA like its own - they are the factories making new DNA - and maybe protein - from the inserted gene
there are other types of vectors for different situations and different host organisms like BACs, cosmids in bacteria, YACs in yeast, and viruses can be used as vectors
The DNA strands are separated by heating (heat denature)
Two short single-stranded DNA molecules (= "primers") are added
each is complementary to one end (3') of a strand of the DNA you want to copy
when the single stranded DNA is cooled the primers will pair with the matching DNA
a heat resistant DNA Polymerase copies the DNA strand (Taq polymerase from a heat resistant bacterium)
each new DNA molecule is made of one old strand and one new strand (this is semi-conservative replication)
Heat denature the two new DNA molecules and start again
Several cycles of copying amplifies the DNA exponentially the number of DNA molecules doubles with every cycle
Also known as restriction endonucleases
These are found naturally in different bacteria
bacteria use them to destroy invading viruses (bacteriophages)
cut the DNA at a specific short sequence
each DNA strand is cut a few bases apart
giving a single-stranded overhang on each strand

This is the EcoRI
restriction site
before and after cutting. It comes
from E. coli

At the electrophoresis step the DNA was distributed along the length of the agarose gel
The gel is laid flat and paper towels draw liquid up through a membrane
The DNA is drawn up too but gets stuck to the membrane
On the membrane it can be analysed more easily (a gel's pretty fragile)
Adds nucleotides to the new DNA strand
The nucleotides are complementary to the replicated DNA strand
DNA is added at one end of an agarose gel
An electric current is run through the gel
DNA is negative so it moves towards the positive electrode
Smaller DNA moves faster
-> DNA fragments are ordered by size
DNA fragments of known size are run next door so we'll know how big our DNA is
The DNA is stained so you can see it and work out how long it is
By Minestrone Soup (talk · contribs) (en:File:Recombinant formation of plasmids.svg) [CC BY-SA 3.0 (http://creativecommons.org/licenses/by-sa/3.0)], via Wikimedia Commons
By User Magnus Manske on de.wikipedia [Public domain], via Wikimedia Commons
DNA is made of 2 strands that pair together (A nucleotides pair with T, G with C)
....this has many advantages
DNA can be made from DNA you already have
(RNA is similar but different)
if the 2 strands are separated from each other (denatured) they can then stick (hybridise) to other matching unpaired strands - like VELCRO (R)!
if you add a label when you make the new strand you have a probe that can be used to grab, see or find the matching gene, DNA or RNA sequence
-thousands of short DNA strands on a chip

The short pieces of DNA (oligomers) are made with a known sequence of nucleotides
Labelled nucleic acid is matched up with (hybridised to) the oligonucleotides on the chip
The intensities of the tiny signals are measured
This can produce thousands, even millions of test results at once
Joins cut DNA ends
COMPLEMENTARY single strand overhangs match up and the DNA ends are joined
........back the way it was
DNA ligase sticks it together
and innovation
DNA sequencing

Adds nucleotides to the new DNA strand
The nucleotides are complementary to the replicated DNA strand
DNA sequencing is working out the bases in a stretch of DNA one by one
Traditionally this was done by Sanger sequencing - four separate reactions, one for each nucleotide base, run on a gel and the code read letter by letter
Now we have machines that read sequence - it's getting faster and cheaper
This is a Southern Blot (after the inventor, Ed Southern). There are now also Northerns (for RNA) and Westerns (for protein).
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