Adaptive PCR Technique Offers Simpler Alternative
Prototype adaptive PCR device. The small sample in the center is illuminated by an ultraviolet laser on the right and varying levels of fluorescence are detected by the spectrophotometer on the left and are used to control the DNA duplication process. Credit: Anne Rayner / Vanderbilt
Imagine a “DNA photocopier” small enough to hold in your hand that could identify the bacteria or virus causing an infection even before the symptoms appear.
This possibility is raised by a fundamentally new method for controlling polymerase chain reaction (PCR).
Vanderbilt University biomedical engineers Nicholas Adams and Frederick Haselton came up with an out-of-the-box idea, which they call adaptive PCR. It uses left-handed DNA (L-DNA) to monitor and control the molecular reactions that take place in the PCR process.
Left-handed DNA is the mirror image of the DNA found in all living things. It has the same physical properties as regular, right-handed DNA but it does not participate in most biological reactions. As a result, when fluorescently tagged L-DNA is added to a PCR sample, it behaves in an identical way to the regular DNA and provides a fluorescent light signal that reports information about the molecular reactions taking place and can be used to control them.
In order to test their idea, Adams and Haselton recruited Research Assistant Professor of Physics William Gabella to create a working prototype of an adaptive PCR machine and then they tested it extensively with the assistance of biomedical engineering undergraduate Austin Hardcastle.
A description of the technique has been published in the journal Analytical Chemistry.
Although the technology is generally considered to be mature, PCR machines have proven to be complicated to operate and hypersensitive to small variations in the chemical composition of samples and environmental conditions. That is largely because there has been no direct way to monitor what is taking place at the molecular level.
As a result, the adaptive approach for controlling the PCR process promises to make it simpler to operate, improve its reliability, reduce its sensitivity to environmental conditions and shrink it from desktop to handheld size. As a consequence, it could free PCR from the laboratory setting and allow it to work in the field or at the bedside where it could be used to identify different diseases by their DNA signatures.
“PCR machines are pretty finicky,” said Adams, giving an example: “We have three commercial PCR machines in our lab. For awhile one of them wasn’t working. When we put identically prepared samples in all three machines, two of them worked and one didn’t. As I was discussing this problem on the phone with one of the company’s technicians, she asked me if the problem machine was within eight inches of a wall. It turned out it was. According to the technician the wall was interfering with the air flow to the machine. She was right because when I moved it out from the wall it began working properly!”
Laboratory technicians have found methods to compensate for these problems. The machines are kept in temperature-controlled rooms. They purify the DNA samples so they have a uniform chemical composition. Even so, it can take operators several weeks to optimize the machines to run samples from new sources. And, even when optimized, they run samples in triplicate, just in case one of them fails.
Adaptive PCR sidesteps many of these classical PCR problems by relying on the fluorescent L-DNA to determine the ideal cycle temperatures for annealing and denaturing. L-DNA sequences are commercially available. So the first step is to order L-DNA with the same sequence as the right-handed DNA that you want to amplify along with left-handed primers. The L-DNAs are ordered with a fluorescent dye on one strand and a “quencher” on the other strand. The quencher suppresses the fluorescence of the dye. So, as the L-DNA strands separate in the denaturing step, the quencher and dye also separate which causes the fluorescence level in the sample to increase. By analyzing the rate of change of the fluorescent level, a microprocessor can determine when virtually all of the DNA has separated.
Similarly, a dye quencher is attached to the left-handed primers. So as the process moves into the annealing step and the primers attach to the L-DNA strands, the quenchers they carry begin suppressing the fluorescent dye on the L-DNA. This provides a dimming signal that can be analyzed to identify the point when the primers are attached to virtually all the DNA strands. The amount of L-DNA in the sample remains constant from cycle to cycle because it does not participate in the amplification step.
The researchers report that experiments with the prototype system have demonstrated that the technique duplicates the results of conventional PCR machines in controlled conditions and can efficiently amplify DNA under conditions that cause standard PCR to fail. “These advantages have the potential to make PCR-based diagnostics more accessible outside of well-controlled laboratories, such as point-of-care and field settings that lack the resources to accurately control the reaction temperature or perform high quality sample preparation.”
This article has been republished from materials provided by Vanderbilt University. Note: material may have been edited for length and content. For further information, please contact the cited source.