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4.1 Explain how the PCR reaction amplifies a target nucleic acid so that it can be readily detected using electrophoresis.
Refer the reader to the figure 4.1. DNA is amplified following a three step reaction. DNA is primarily denatured at high temperature (usually 94 or 95 degrees). Specific primers to the region being amplified in the second step will anneal (meaning they will be complementary to the sequence to amplify and therefore will hybridise to the nucleic acid). This step will be done at a specific temperature specific to the sequence of the primers. Following the annealing of the primers, the taq polymerase (enzyme) will use the primer to start to extend the DNA fragment using complementary nucleotide to the sequence to be amplified. This step is usually performed at a temperature of 72 degrees. The succession of this three step constitute a cycle. A standard PCR will be made of a certain number of cycles starting from 30 cycles to 40 cycles. optimisation might need to be done and number of cycles should be tested.
4.2 What are the main sources of contamination in a PCR and how can these be avoided?
The main sources of contamination are genomic DNA and / or amplicons from previous reactions.
The best way to avoid this is to have a unidirectional workflow. For instance to have a pre- and a post-amplification room separated from each other and, if possible, with different air pressures in each. For instance the pre-PCR room, with no DNA and only PCR reagents, would be positively pressurised so that nothing can get in. The post-amplification room, which would have amplicons, would be negatively pressurised so that nothing can get out. It is always best to start work in the pre-amplification room then finish in post-amplification room, but not the other way around. If separate rooms cannot be provided then having separate laboratory areas is good practice.
Make sure to decontaminate equipment such as work surface and pipettes on a regular basis with 5 or 10% bleach solution that will degrade any DNA that might be present. Also try to have different sets of pipettes for setting up PCR and to check PCR products. Furthermore, use filter tips for reaction setup and when pipetting to avoid creating aerosols.
4.3 What are the different chemistries used in real time PCR to detect the result of the amplification?’
There are two main chemistries that can be used. One is the use of a fluorescent dye which is a DNA intercalating agent such as SYBR green or Eva green. These are not specific to the product and will intercalate any double stranded DNA. Therefore, when used, they must be carefully optimised. The second is the use of a oligonucleotide DNA probe that is designed to be specific to the amplified DNA target. This DNA probe is labelled with fluorescent molecule to permit the detection of the PCR product. Examples of DNA probes for real time PCR are TaqMan probes, Molecular Beacon probes and Scorpion probes.
4.4 What are the different types of microarrays and what applications can they be used for?
There are various types of microarrays that can be used for the assessment of:
- level of gene expression (mRNA and miRNA level)
- identification of SNPs (polymorphisms at the genomic DNA level)
- gain or losses of genetic material (aCGH, SNPs array, gDNA level)
- copy number variations
- methylation levels.
Gene expression microarrays can be used to stratified patients as ‘good’ or ‘bad’ responders to certain drugs; to identify pathways in a particular disease that can be targeted for drug therapy and to distinguish sub-types of tumours i.e., for tumour classification or tissue of origin.
DNA microarrays such as SNP arrays, for example Affymetrix SNP 6.0, can be used to screen for polymorphisms associated with a certain population or group of people in relation to a particular disease / phenotype.
4.5 What are the key advantages of using Western blotting?’
Western blotting remains the simplest semi qualitative method of protein identification and assessment of changes due, for example, to degradation or post-translational modification.
4.6 What are the key differences between ELISA and multi analyte assays?’
Both assays rely on a sandwich antibody format. However, the multi-analyte assay can measure multiple targets at the same time. Luminex xMAP, MSD and SIMOA are all immunoassay platforms that allow for multi analyte measurement.
4.7 How does 2-DGE differ from 2D-DIGE?
DIGE uses fluorescent protein labels to simultaneously measure several independent samples at the same time.
4.8 All complete TMT tags are identical in mass and structure. Using Fig 4.9, highlight which section(s) of a TMT tag is unique in mass.
The Reporter is different for each TMT tag as this is the unqiue identifier. After the cleavable linker is broken by MS fragmentation, the unique reporter will be measured.
4.9 Why is sample enrichment needed for phosphoproteomics and what are the most common amino acid sites where phosphorylation occurs?
Although phosphorylation is a common event, the ratio between non-phosphorylated and phosphorylated peptides is extremely imbalanced and therefore non-phosphorylated peptides need to remove from the matrix for an accurate measurement can be made. Tyrosine, serine and threonine are the most common “phosphosites”.