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Discussion question for the nucleic acid section: What are the different variants of PCR and how do they differ from ‘standard’ protocol?
Nested PCR: a nested PCR uses two pairs of primers with one being internal to the first pair of primers to reduce non-specificity. The first PCR need only be a few cycles and the second PCR with internal primers can be up to 30 cycles. Nested PCR is often used to reduce unexpected primer binding sites.
Touch down PCR: the amplification starts with a higher annealing temperature for the primers to avoid non-specific amplification. This temperature is progressively decreased 1°C each cycle to reach the final calculated optimum annealing temperature for the primers in question.
Cold PCR: The principle of COLD-PCRis based on the manipulation of denaturation temperature (Tc) in PCR, which in turns selectively enriches low level of variants. The Tc is strongly dependent on the DNA sequence itself and is lower than Tm. When the denaturation temperature of the PCR is set to Tc (instead of the normally used 94-95ºC) DNA amplicons containing different point mutations will have different amplification efficiency. This property is exploited to selectively enrich low levels of mutations in a mixed pool containing a majority of wild type sequences.
LAMP PCR: Loop-mediated isothermal amplification (LAMP) uses 4-6 primers recognizing 6-8 distinct regions of target DNA. A strand-displacing DNA polymerase initiates synthesis and 2 of the primers form loop structures to facilitate subsequent rounds of amplification.
Multiplex PCR: allows amplification in the same reaction tube of more than one amplicon. Depending on the application, more than a dozen of different pairs of primers can be present.
Discussion question for the protein section: What are the principles and distinguishing features of Western blotting and ELISA methods as used for the identification of proteins.
Western blotting is a qualitative technique that is used to establish the presence and confirm the identity of a protein. The method involves electrophoresis, under denaturing conditions, of a homogenate preparation of the sample to be analysed alongside a series of known molecular weight markers. This is followed by transfer by blotting onto a suitable membrane that is then incubated with an antibody that is specific for the protein of interest. Visualisation of the protein / antibody interaction will reveal if the unmodified protein is present as a single band that can be verified by comparison with the molecular weight markers as being of the correct molecular weight. Modifications to proteins, through the presence of isoforms, glycosylation and phosphorylation, will result in more than one band being demonstrated. As discussed in chapter 3, Box 3.1, Western blotting can also be used to establish the specificity of an antibody as part of the validation process in choosing an antibody for use in diagnostic or research applications.
ELISA (Enzyme-linked immunosorbent assay) also relies on the use of antibodies to detect proteins. In contrast to Western blotting, the method provides a quantitative read out. In its simplest form a test sample is precipitated into a well of a transparent multiwell plate. At the same time other test samples are similarly applied to other wells and their positions are precisely recorded. A series of know quantities of the protein are also precipitated into wells on the same plate and at least one well is left empty. This allows a ‘calibration or standard curve (SC)’ of protein concentration to be constructed at the end of the assay. An enzyme labelled antibody specific to the protein to be detected is incubated with all test and SC samples. When the protein is present it is detected using an enzyme /substrate system that provides a coloured product that is directly proportional to the amount of protein present in each well. The optical density of the coloured product is read using a plate reader and compared with known amounts of the protein applied to make the SC less any background assay reading given by the optical density recorded for the well where no protein was deposited. Analysis of the optical density of each of the test samples against the SC provides a quantitative value of the amount of protein present in each.