Polymerase chain reaction and southern hybridisation Flashcards
What can PCR enable?
Identification, amplification and isolation of DNA of interest from a minute amount of a mixed DNA population.
What does PCR require?
A minimum of sequence information on the DNA molecule of interest.
What polymerase is used in PCR?
Taq polymerase, a thermostable type of polymerase isolated from Thermus aquaticus.
What temperatures can Taq withstand?
Around 95 degrees celsius, but works best at around 75 degrees celsius.
What does Taq polymerase require?
Single stranded DNA template, short DNA primers and supply of dNTPs.
What is PCR carried out in?
A thermocycler.
What are the first stages of PCR?
Mix the DNA, primers, dNTPs, Taq, buffer and water in the thermocycler and heat to 94 to denature the DNA. Cool to 55 so the primers can anneal and heat to 74 to synthesise the first long products - extension.
What happens in the next stages of PCR?
Repeat the cycles and the short products will accumulate exponentially. The DNA is doubled with each cycle.
What are some of the problems with PCR?
There is a risk of contamination, the primers must be highly specific to the gene of interest and PCR often needs optimisation.
How do short primers differ from long primers?
Short primers may hybridise frequently due to chance (resulting in non-specific amplification) whereas longer primers have a higher probability of being specific to the gene or region of interest.
What is the equation for the Tm?
(4x [g+c] + (2x[a+t]) degrees.
What does Tm control?
The annealing temperature of the primers to the template.
How can the products of PCR be analysed?
Electrophoresis, restriction digest and sequencing.
What is T/A cloning of PCR products?
PCR products frequently have three extra adenines (A) due to Taq. The sequence can therefore only be cloned into a vector with an extra 3 T’s. These vectors can be made or bought.
How can restriction sites be used to clone PCR products instead of T/A cloning?
A restriction site can be incorporated into the 5’ end of PCR primers. After a few cycles it will be added into the products and the amplified product can be cut with RE to give a fragment with sticky ends. This fragment can be cloned in a normal plasmid vector.
What is a problem with Taq?
It doesn’t have proof reading and has a larger error rate and can be a problem if individual fragments are cloned.
What does Southern Hybridisation provide information on?
Differences between genes in the same or different organisms and detecting the position of a gene on a DNA molecule.
What is Southern Hybridisation based on?
Restriction digestion, agarose gel electrophoresis, radio-laelling of DNA and nucleic acid hybridisation.
What are the principles of Southern Blotting?
The plasmid is digested with EcoRI and the fragments are separated by electrophoresis. The gel is Southern Blotted onto a nitrocellulose or nylon membrane and capillary movement of the buffer into the paper towels moves the DNA out of the gel and onto the membrane.
What is done after the DNA has been transferred onto the membrane?
The DNA fragments are immobilised so they retain the same position on the membrane as the gel, they are denatured and hybridised with a labelled probe for the gene of interest and exposed onto an x-ray film.
What else can Southern Hybridisation be used for?
Detecting differences via restriction fragment length polymorphisms (RFLPs).
What can restriction fragment length polymorphisms be used to detect?
It can give information differences and changes in genes or pieces of DNA between individuals and within whole populations. These changes may be due to mutations of different kinds.
What application in the world does RFLPs have?
Gene families, mutations in diagnostics and genetics, genetic diversity within a species and between species, population genetics, molecular ecology and criminals.
What are RFLPs?
Changes in the DNA sequence that change the length of restriction fragments due to a change in restriction site and insertion or deletion between restriction sites.
