Molecular diagnostics: basic terms and principles

Dec. 19, 2012

Laboratory medicine in the developed world relies more and more on a collection of analytical tools collectively referred to as ‘”Molecular Diagnostics.” While the term is undoubtedly familiar to the reader, the relative newness of the field (compared to standard chemical, biochemical, and microbiological laboratory testing methods) and its continuing rapid evolution leaves many laboratorians uncertain of what some molecular methods may be, their underlying science, applications, strengths, weaknesses, and most importantly—their utilities. Why is molecular diagnostics useful? What is it used for?

Beginning with this installment, “The Primer” will be a 13-part series aimed at helping de-mystify the terms, underlying science, methods, applications, and issues surrounding many of the core and emerging molecular technologies available in today’s clinical laboratory. Starting from the basics and progressing through the most common methods, the series aims to be of interest and a source of information for readers at all levels of prior familiarity with the topic. While every branch of medicine and science has its own particular terms and conventions, an effort will be made to minimize jargon and focus on explaining underlying concepts and applications. Doing so will provide the reader with a basis for not only understanding the covered methods, but being comfortable in assessing other approaches.

Each monthly section will be short enough to be read in a single sitting. Where simple illustrations are of use in explaining concepts and methods, they will be included. Monthly sections will cross-reference one another when relevant. I hope that readers will be able to use the complete set of articles as a miniature textbook on the subject for future reference.

A look at what’s ahead

The topics and order of the 13 monthly sections will be as follows:

December 2012: Review of Basic Terms and Principles
January 2013: DNA Structure
February 2013: DNA Replication
March 2013: Sample Extraction Methods
April 2013: PCR
May 2013: Endpoint PCR Detection
June 2013: Real-Time PCR (1)
July 2013: Real-Time PCR (2)
August 2013: Quantitative Methods
September 2013: Multiplexing and Array Methods
October 2013: High Throughput Sequencing
November 2013: Process Controls
December 2013: Future Expansions of Molecular Diagnostics

While topics such as thermodynamics and kinetics will of necessity be touched on, the reader is assured that the treatment of these often intimidating subjects can be satisfactorily achieved for our purposes using relatively simple concepts. Best of all, there’s no final exam to worry about!

With those aims and reassurances in hand, the author invites readers of MLO to delve into what will hopefully be both an entertaining and educational journey through the world of molecular diagnostics.

Defining molecular diagnostics

A good starting point is to define what “Molecular Diagnostics” is. The term is itself somewhat misleading; a great deal of laboratory medicine, from blood gas chemistry to ELISA assays, involves the detection and measurement of specific molecules. Rather than adhere to a strict literal meaning, the term “molecular diagnostics” (MDx for short) can be used to mean the detection and/or analysis of nucleic acid molecules (DNA or RNA) to provide clinical information.

If it just came down to a definition, we’d be done and wouldn’t need another 12 sections to examine the topic. In fact, however the definition above really raises more questions than it answers. For example, “How do we detect DNA or RNA?” “What sorts of analysis can we do to nucleic acids?” “What sorts of clinical information can this provide?”

For now let’s examine the last of these. In general, MDx is applied in addressing two distinct types of clinical questions: pathogen detection (searching for exogenous, non-human nucleic acids) and genetic testing (searching for endogenous, host-derived nucleic acids). In the case of pathogen detection, MDx is used to search for (and possibly quantify) specific, pathogen-derived nucleic acid sequences in a patient sample. It makes the supposition that if these are detected, the patient is infected with this agent. Note that the word “specific” here can have more than one meaning; it can in different contexts mean specific to level of species, to level of strain, or possibly even to level of single isolate, and this ability to widen or narrow the focus of test specificity is one of the strengths of MDx techniques.

From the detection and possible analysis of host (patient) nucleic acids, a number of types of clinical information can be gathered which can be informative on topics including the presence, absence, or carriership, of hereditary disorders or projections of responsiveness to particular drug therapies; or hereditary and relationship information (which naturally leads toward forensic applications, which albeit fascinating will be outside the scope of our examination). The MDx detection and analysis of cancer cells, inasmuch as they arise from host cells, is a particular subset of this second general class of MDx applications and affords opportunities for still other types of clinical information to be gathered, including therapy response, progression, and relapse. As this series progresses, examples of the different methods available and how they can apply in one or both of these two general classes of MDx will be considered in detail.

As for the other two questions arising from our definition of MDx, “methods of detection” will be the direct focus of later sections, and “types of analysis” will be a recurring theme across the series.

Before proceeding further, there are a couple of basic chemistry topics we should review. Those topics are pH and hydrogen bonds, which although deceptively simple will lie at the very heart of much of our topic. One other point, however, before we tackle these. Remember my earlier reassurance that the math and thermodynamics will be kept to a minimum? Well, in order to do that, we’re going to have to rely on using some analogies which make the concepts easy to grasp in our applications, but are inherently, by virtue of being analogies, not the complete, correct, or full story. That’s okay—science abounds with models and analogies to explain and understand things—but it’s also important to recognize when we’re using these mental tools as stand-ins for the real deal, and I’ll point them out as we apply them.

pH is something pretty much everyone is familiar with, at least as a term to describe the acidity (or its opposite, alkalinity) of a solution. Acidity in turn is just a measure of the concentration of free hydrogen ions (H+) in solution; acidic solutions, those with pH values below 7.0, contain more H+ ions than do basic solutions (those with pH values above 7.0). Where do these H+ ions come from? In molecules which contain functional groups such as –OH, or –NH or –SH, the covalent (electron-sharing) bond between the –O (or –N or –S) and the H is not tremendously strong, and there’s a certain tendency for the H to essentially “fall off,” leaving its electron behind on what is now –O- (–N-, –S-) and entering the surrounding environment as H+. This is a reversible process resulting in a state of “dynamic equilibrium,” where a certain fraction of molecules are in this state at any given time. Water, with a structure of H–O–H, is an example of a molecule that undergoes this, and in pure water at room temperature this works out to be 1x10e-7 moles/litre of H+ as the steady state. Since the actual mathematical definition of pH is “the negative log10 of the H+ ion concentration,” we just take the 7 as the value we see. Since we live in a world of water-based life, this pH 7 is what we consider to be “neutral” pH, neither acidic nor basic. (Don’t worry if you didn’t follow that math there; the key points are the idea of H+ ions being able to come off these sorts of functional groups, and that pH values below 7 are acidic, and above 7 are basic.) This simple concept is something we’ll come back to in future sections.

The second topic we will need to have in mind for understanding MDx is that of hydrogen (H) bonds, which are in many ways interrelated with the idea of pH. If we consider one of the functional groups referenced earlier (–OH, –NH, –SH), it turns out that even when the H is still attached, it’s electrostatically polarized; its electron is closer to the –O, –N, or –S side, leaving the H slightly positive. This faint charge has a directional nature, extending out from the H directly away from the –O, –N, or –S, and if it can line up within a short distance with a similar size negative charge (usually again from an O, N, or S), an H bond is formed between the H (the bond donor) and the O, N, or S (the bond acceptor). Now here’s where we’re going to apply one of our analogies. Think of H bonds as being like weak magnets; just like two magnets, a bond donor and acceptor have to be paired (like an N and S pole) to attract each other; there’s a directionality; the attraction is only across a short distance; and if you pull on the two sides, you can pull them apart. This analogy is completely inaccurate in a physical sense, but as a mental model of what we’re going to deal with at the crux of MDx, it’s simple and effective.

With those points to think about, we’ll close off this first section of The Primer. Next month we’ll review what nucleic acids are and see how this month’s two simple ideas apply to nucleic acid structure as a basis for all MDx methods.