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11.1
What is the key property of the polymerase chain reaction (PCR)? When we talk about a ‘PCR-based’ test, what variations might we be utilising?
- PCR’s key attribute lies in its capacity to multiply many copies of a target sequence from very small quantities of starting template in a sample. This enables researchers and clinical staff to interrogate nucleic acid sequences with a huge number of analytical methods that simply could not be applied to the low copy number present in the original sample itself.
- Variations of PCR-based tests include restriction-fragment length polymorphism (RFLP) analysis, Sanger sequencing, real-time PCR, pyrosequencing, and various formats of NGS. Any analysis relying on-at some point-enzymatic amplification of NAs is said to be PCR-based.
11.2
When investigating a new disease and/or developing a new test, how would you determine what variations need to be analysed? How could you ensure that a test is fit for purpose in light of the latest relevant findings?
- The biomedical scientist should always be guided foremost by clinical evidence, and the rationale for developing a test must be based upon relevant findings from clinical trials and-where applicable-licensing information. Failing to analyse relevant targets compromises patient management, and testing for extraneous data wastes time and resources, and can confuse clinical decision making processes.
- Existing test pathways must be “sense-checked” periodically. Literature searches and careful monitoring of papers presented to clinical meetings can help in predicting likely future changes to demands on testing pathways, and may reveal where new test pressures are likely to come from as therapeutic agents come to market. Ultimately, however, it is not the job of the Biomedical scientist to dictate patient management and they must ensure tests meet clinical and licensing demands.
11.3
Why might samples submitted for molecular analysis be of limited size and volume? And what might limit the possibility of obtaining further specimens?
- Many molecular diagnostics, especially predictive markers, are applied especially to patients with advanced, late stage disease. They may have widespread disease for which surgical excisions are of little benefit. In order to allow specimens to be obtained from these patient groups, surgeons and radiologists have optimised minimally invasive techniques; so whilst these specimens appear small and more difficult to work with, in the relatively recent past such patients would not have provided specimens of any type. Such patients are usually not fit for more invasive procedures which yield larger volume specimens. At diagnosis they will, if fit enough, immediately proceed to some form of therapeutic intervention and this can further reduce their overall fitness for further procedures at a later stage.
11.4
What do you think the key variables are in deciding on a DNA extraction method to employ, and which sorts of samples do you think might be most suitable for the different methods described?
- The many variables discussed in the chapter at each staged of processing can impact upon suitability of an extraction method. The form of specimen and its pre-analytical processing can both severely limit the overall volume and quality of material available to feed in to the extraction pipeline. Similarly, the downstream applications to be employed should be tailored to, particularly in the instance of NGS methods requiring larger NA quantities. As a rough rule, spin-column and magnetic-rod based methods offer good yields from low volume samples such as very small biopsies.