Good qPCR Starts with Good Primers
One of the most frustrating problems in the development of a qPCR assay is complications due to off-target amplicon formation. Undesired amplicon formation can happen from extension when one or more primers anneal to each other (forming primer dimers) or when primers anneal to undesired regions of the template with lesser complementarity (forming mispriming products). Off-target amplicon formation can significantly affect PCR performance by reducing PCR efficiency and by increasing the propensity for false-positive and false-negative results.
TriLink BioTechnologies has developed a strategy for chemically protecting the 3´ end of the dNTP. In her presentation at the meeting, senior staff scientist Natasha Paul, Ph.D., described this method including a proof-of-principle experiment. TriLink’s new hot-start PCR makes use of a tetrahydrofuranyl protecting group on the 3´ end of the dNTP. The use of 3´-protected dNTPs blocks the primer from being extended in the 3´ direction, with the protecting group coming off at the elevated temperatures of PCR. Thus, nonspecific amplification is prevented during the set-up stages, but PCR proceeds normally once the temperature is raised to 95ºC.
In a proof-of-concept experiment, Dr. Paul verified this mechanism by showing that 3´-THF dNTPs were not incorporated by DNA polymerase prior to hot-start activation. Furthermore, the utility of 3´-THF dNTPs was verified for a number of different targets and in real-time PCR, where amplicon detection was linear from five to 5,000 copies. “This technology provides a way to block any DNA polymerase of interest from being active until your PCR begins,” Dr. Paul said. “The introduction of these dNTPs into your reaction set-up will allow for conversion of your polymerase of choice into a hot-start polymerase.”
qPCR methods excel at detecting small amounts of nucleic acids, but by its nature the technique is vulnerable to contamination and amplification bias. Particularly when using complex samples such as clinical blood samples, there is a risk that contamination will introduce an amplification bias or that the wrong template entirely will be amplified.
Reginald Beer, Ph.D., a principal investigator at Lawrence Livermore National Laboratory (LLNL), presented his work on monodisperse picoliter PCR. LLNL studies viral and bacterial pathogens that threaten the population and food supply. “Viruses mutate quickly, constantly probing the immune defenses,” said Dr. Beer. “It’s hard to find causes of agents in new pandemics. When people first present sick, it’s hard for people in public health organizations to know what they’re sick with.” The goal is to reduce the time to treatment in a disease outbreak by optimizing diagnostic methods using qPCR. To this end, LLNL has harnessed microfluidic technologies to create chip qPCR.
Running qPCR in sample volumes of 10 picoliters not only reduces sample volumes and reagent volumes, it isolates the nucleic acids in the droplets and reduces PCR amplification bias. Standard or bulk PCR works quite well for oligonucleotides at high concentrations, but not so well when the concentration of target DNA or RNA is low and the background is complex.
A 10 picoliter droplet is 1,000,000 times smaller than a standard PCR sample volume. Poisson statistics predicts that this droplet will only contain one copy of viral DNA (or none) at typical concentrations, Dr. Beer said. That means that there is only one possible template in each reaction. Not every droplet will amplify, because not every droplet will contain DNA.
In their first publication, using qPCR with vaccinia, Dr. Beer and his team were able to come within 6% of the actual titer of the sample on the bench qPCR system. “Generally, when you talk to people in the field they are happy to get 40%,” he explained.