Method of the Month: TaqMan qPCR
During my research internship last summer, I tested for the presence of two parasites, Trypanosoma cruzi and Trypanosoma rangeli, in DNA extracted from several hundred kissing bugs. The laboratory technique I used to accomplish this was TaqMan quantitative polymerase chain reaction (commonly referred to as “TaqMan qPCR”). This technique is a modified version of regular polymerase chain reaction (the procedure used to replicate specific sequences of DNA). TaqMan qPCR differs from regular PCR because its purpose is to detect the presence and amount of one or more specific DNA sequences in a sample.
Before a TaqMan qPCR can be performed, a sample of DNA must be obtained. In my internship, I used extracts that contained both DNA from the kissing bugs’ genome and DNA from any microorganisms that happened to be inside the kissing bug at the time of extraction. Since TaqMan qPCR tests for the presence of specific organisms, the presence of DNA from other microorganisms doesn’t affect the results.
The TaqMan qPCR process consists of the following steps:
1. Each sample of DNA is combined with reagents and pipetted into a single well of a reaction plate. This must be done in cold, low-light conditions to prevent the reagents from prematurely reacting with each other or with the DNA. Positive and negative controls are also added to the plate.
2. The reaction plate is placed in a thermocycler. This machine will heat and cool the sample around 40 times in succession.
3. Heating denatures all DNA in the sample (separates it into single strands).
4. A specially selected primer binds to the specific sequence of DNA that we want to amplify. In my case, two primers were used to bind a region of DNA in the T. cruzi genome and a region of DNA in the T. rangeli genome.
5. A probe binds to a specific sequence of DNA located downstream from where the primer has attached. This probe consists of a specific sequence of nucleotides bound to two other molecules: a fluorescent dye molecule and a quencher molecule. As long as the quencher is bound to the probe, the dye does not glow. In my case, two separate probes were used, one for T. cruzi and one for T. rangeli. The dye molecule attached to the probe for T. cruzi was different from the dye molecule on the probe for T. rangeli.
6. Taq DNA polymerase begins to add new nucleotides to the end of the primer. This process replicates the target sequence.
7. As the primer extends, the probe is cut off the target DNA by Taq polymerase. In the process, the fluorescent dye molecule is separated from the quencher molecule and begins to glow.
8. This process is repeated again and again, through many cycles of heating and cooling. The qPCR machine is programmed to heat and cool the samples repeatedly to allow the target sequence to be replicated many times. The more the target sequence is replicated, the more dye molecules are released. This increases the strength of the fluorescence.
9. The qPCR machine detects and interprets the fluorescence. The presence of any fluorescence indicates that the target sequence is actually present in the sample. The intensity of the fluorescence is correlated with the amount of the target sequence present in the sample.
Interpretation of the TaqMan qPCR results is straightforward. In my research, the thermocycler generated two graphs for each DNA sample. One graph showed the amount of fluorescence from the dye on the T. cruzi probe, and the other showed the amount of fluorescence from the dye on the T. rangeli probe. To determine if a sample was “positive” for each parasite, we looked at the location of the cq value (the point where the fluorescence exceeded the normal background level). For a sample to be considered positive, the cq value needs to appear somewhere between cycles 12 and 38. If it appears outside this range, there is a good chance that something has gone wrong with the sample, and the results cannot be considered reliable.
Because this is a highly sensitive test, it is very easy to accidentally mess it up. During my internship, the biggest problem we encountered with our TaqMan qPCRs was getting positive results for our negative controls. This indicated possible contamination with environmental DNA (airborne fragments of DNA, or DNA on laboratory surfaces) and invalidated our results for that run. We learned the hard way that meticulous sterilization practices and precautions against airborne DNA contamination are absolutely essential to the success of this test.
