The General Problem Of Assay Design
The next phase of my research was engineering a test. I needed something which could identify with a high degree of certainty one strand from another based on small (single nucleotide) variations. To accomplish this in another strain of malaria, my mentor previously applied a method called high resolution melting (HRM). Though I can’t speak with much specificity (by obligation, not ignorance) on the method or methods I employed, I can describe HRM and what generally the task of assaying for single nucleotide polymorphisms (SNPs) entails.
The basic problem the assay attempts to address is two-fold: how can we make our DNA visible and how can we make variations in DNA in turn visible. The first problem is addressed generally using PCR, but this option hardly ‘solves’ the fundamental problem, at least not past the point of necessary consideration. Given it is the variation in DNA we need to reveal, the promotion of strands to visibility should be narrowly targeted with respect to the method which is being used to reveal those variations. This is especially critical with HRMs where the factor used to distinguish strands, the derived variability, is the temperature at which DNA strands have their internal structure, a row of hydrogen bonds, broken down – the temperature at which this occurs is dependent upon the composition of base pairings within a single sample, variations contributing continuously to the breakdown of an entire sample. Thus, whenever possible the number of potentials variations beyond the one being tested for, and the number of nucleotides present generally should be watched closely.
Though this is not necessarily directly descriptive of my project, I hope the idea is conveyed. With this basis, my next post, describing some of the challenges one can encounter in designing these tests can make more sense.