Lecture 2 Flashcards
How can we denature DNA?
Heat
Extreme pH
Hydrogen bond breaking agents e.g. concentrated Urea
Under similar conditions the temp at which a (long) DNA double strand will denature depends on
its base composition
DNA with a higher G+C content will denature at a …..
higher temperature
Why would DNA with higher G+C content denature at a higher temperature than DNA with a lower G+C content?
G+C held together more strongly (3 hydrogen bonds) than A+T is (2 hydrogen bonds)
What conditions favour double helix formation?
Altered temperature
High salt conc (ionic strength)
Removal of denaturants
Hybridisation can only occur between DNA strands which are
complementary to each other
What happens in membrane-hybridisation assay?
Melt ds DNA to ss DNA, DNA binds to filter, incubate filter with labelled DNA, wash away labelled DNA that did not hybridise to DNA bound to filter.
What can you use southern blotting for?
Determine how many genes correspond to cDNA probe are present in organisms genome
What happens in southern blotting?
1) DNA cleaved with restriction enzymes
2) Gel electrophoresis
3) Place filter paper over nitrocellulose and gel in alkaline solution
4) Alkaline solution denatures DNA, capillary action transfers DNA from gel to nitrocellulose
5) Hybridise with labelled DNA probe and examine e.g. using autoradiogram
What are northern blots used for?
Standard method of analysing mRNA, gives size and amount of RNA.
What happens in northern blotting?
1) unlabelled RNA, undergoes agarose gel electrophoresis where nucleic acids are separated according to size
2) separated nucleic acids are blotted onto nitrocellulose paper by suction of buffer through gel and paper
3) nitrocellulose paper has labelled probe hybridised to its complementary bands
4) visualised by autoradiography
What is PCR used for?
Amplifying DNA sequences
- cloning
- probes
- forensics
- PCR can be used to amplify rare DNA from a mix (when end sequences known)
- amplification of mutant alleles allows detection of human disease
Requirements of PCR
Taq DNA polymerase
dNTPS
Oligonucleotide primers
Mg2+ ions
What does Taq DNA polymerase do?
Copies original and generates new strands
why are dNTPS needed in PCR?
they are nucleotide bases that form the new DNA strands
Why are oligonucleotide primers needed in PCR?
forms initiation site for Taq polymerase
Why are Mg2+ ions needed in PCR?
stabilise the primer
What are the requirements for the design of a PCR primer?
- primers should be 20 bases long
- The G/C content should be 45-55%
- The annealing temps should be within 1 degree of each other
- The 3’- most base should be a G or C (as 3 hydrogen bonds will clamp tight)
- Primers must not base pair with self or other primers to form hairpins
- Primers must avoid repetitive DNA regions (you’d get multiple binding of primers)
How can you optimise the annealing temp of PCR?
-Primers have a concentrated annealing temp, this temp must be confirmed practically. You can test temps of 2 above and below using gradient cycler.
How can you optimise the Mg2+ conc of PCR?
The fidelity of PCR depends on Mg2+
- Vary Mg2+ in steps of 0.5mM
- Sometimes have to compromise between yield and specificty
How can you optimise the PCR reaction?
CONSIDER
- annealing temp of primers
- Conc of Mg2+ in reaction
- The extension time
- The amount of template
Why is it difficult to find an optimum annealing temp for primers?
At low temperatures you get non-specific binding and whilst this would improve with higher temps, high temps can reduce yield
How do primers form hairpins?
If a primer is self-complementary it can fold into a hairpin, the 3’ end of the primer is therefore base-paired so it can’t anneal to target DNA
How do primers form dimers?
A primer can form a dimer with itself or other primers. Primer dimers are used as a substrate for Taq polymerase (amplifies this and not target DNA)
