Contributed by Ning Chin
Polymerase Chain Reaction (PCR) is one of the most important technique in the field of molecular biology. The idea of PCR came to Kary Mullis in 1983 when he was driving from Berkeley to Mendocino. 34 years later, PCR technique is used heavily and taught to students as a basic molecular biology technique. The invention of PCR catalyzed the advancement in the field of molecular biology, which also benefited PCR the medical field and the forensic field, to name a few.
The theory of PCR is simple yet effective: you increase temperature to denature double-stranded DNA, lower temperature to anneal primers and then increase temperature again to allow polymerase to make the DNA based on your DNA template. However, as simple as it sounds, there are many details that we can tweak to improve the PCR technique. The details in PCR condition are especially important when dealing with low template DNA. My instructor from the forensic science program once jokingly said that PCR is the short-form of Pure Chance Reaction. How true is this when dealing with low template DNA? When you have very low amount of template DNA, the PCR reaction result become a statistical problem: what is the probability that you will pipette the amount of DNA needed for your PCR reaction?
In forensic science, a stochastic effect is often solved by increasing the cycle number—the strictly controlled nature of forensic science does not allow for a lot of personal creativity to ensure the quality and standards of the analysis. In research settings, there are many other factors that you can change to increase your PCR efficiency. Many of these were explored by other researchers, such as primer design, salt concentration, polymerase type, and others. However, I haven’t seen any research on how secondary structure of template DNA, more specifically hairpin structures, can affect PCR. Theoretically, double-stranded DNA denatures completely at 95C – but when the temperature cools down to allow primer annealing, would single-stranded DNA form a hairpin and block primer from binding? Is this the reason why I couldn’t amplify my low template PCR? A simple experiment will solve this mystery – adding dithiothreitol into the PCR master mix to denature hairpin formation.
And no. My PCR is still unsuccessful.