Assay Problems

My last post tacitly assumed a certain degree of understanding which could not necessarily be assumed. So here I’ll correct that, at least partially, by addressing the PCR and then use that to discuss briefly what challenges I faced in primer design and how I confronted them.

Early in the senior project, my mentor, Dr. Molina-Cruz, identified to me that it was critical before I could contribute any ‘new’ work that I have some understanding of the polymerase chain reaction. That, beyond a capability of reciting its steps, its components – what is ascertained typically and easily from a textbook – I should know its origin and early history. I have a rather vivid memory of reading one article written by PCR’S discoverer Kary Mullis (which I recommend highly and have linked below) on a rainy afternoon in London. Reveries aside, within these procedures there are a few key points. Critically, PCRs function by repeatedly triggering extant enzymes which elongate and bind strands of DNA, doubling the size of a specified in each passing cycle. The specified region is identified by the presence of two DNA sequences called primers which bond to its beginning and end. These primers need to meet several specifications to be accurate and effective in promoting the PCR, most critically sensitivity to temperature, bonding strength, and likelihood of bonding to incorrect portions.

Thus, significant trouble comes when such primers for regions under analysis cannot be made properly. This is not, one should understand, a limitation of the primers per se. For any arbitrarily long sequence on a DNA strand a set of primers should be easily achievable. The only trouble is that we were and are not generally working with arbitrarily long sequences; we were forced to fix certain areas of the genome and to limit the size of our samples. This challenge was further compounded by the demand that the products of our work be as inexpensive as possible, meaning that most any biotechnology which could potentially change the way our primers or other similar agents which interact with a sequence (CRISPR, notably) are off the table. But, despite this, I – with the benefit of my mentor’s ingenuity and experience – managed to come to a functional, cheap test. On paper, at least.

I can’t really expand on what it was I did to accomplish this despite significant challenge. This is a shame, as it was the critical innovation of the entire project. That being said. With this out of the way, eliding considerably, I can describe my experience in the lab, experience which is more easily fruitfully communicated.

PCR Article:
Mullis, Kary B. “The Unusual Origin of the Polymerase Chain Reaction.” Scientific American, vol. 262, no. 4, 1990, pp. 56–65. JSTOR, http://www.jstor.org/stable/24996713. Accessed 24 May 2023.