A new method for controlling the DNA duplication process makes it possible to shrink the current desktop machines to handheld devices that could be used in the field to provide early diagnosis of a wide range of infectious diseases.

 

Prototype adaptive PCR device. The small sample in the center is illuminated by an ultraviolet laser on the right. Varying levels of fluorescence are detected by the spectrophotometer on the left and are used to control the DNA duplication process. [Anne Rayner/Vanderbilt University]
Prototype adaptive PCR device. The small sample in the center is illuminated by an ultraviolet laser on the right. Varying levels of fluorescence are detected by the spectrophotometer on the left and are used to control the DNA duplication process. [Anne Rayner/Vanderbilt University]

The polymerase chain reaction (PCR) is generally considered one of the most significant biomedical advances of the 20th century and a fundamentally integral part of every molecular biology laboratory today. While technological improvements over the years have made PCR more precise and created machines with small footprints, 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.

Now, biomedical engineers from Vanderbilt University have developed an out-of-the-box idea, which they call adaptive PCR. The new method utilizes left-handed DNA (L-DNA) to monitor and control the molecular reactions that take place in the PCR process.

L-DNA is the mirror image of the biologically active form of DNA found in all living things. It has the same physical properties as natural, right-handed DNA but it does not participate in most biological reactions. Thus, when fluorescently tagged L-DNA is added to a PCR sample, it behaves in an identical way to the normal DNA and provides a fluorescent light signal that reports information about the molecular reactions taking place and can be used to control them. 

To test their idea, the scientists created a working prototype of an adaptive PCR machine that they tested extensively. The findings from their study were published recently in Analytical Chemistry in an article entitled “Adaptive PCR Based on Hybridization Sensing of Mirror-Image L-DNA.” The Vanderbilt scientists are confident that they will be able to develop a machine the size of a large smartphone that can be used as a point-of-care device that could be used to screen for multiple diseases within one sample.

Though the technology is generally considered to be mature, PCR machines have proven to be complicated to operate and hypersensitive to tiny variations in the chemical composition of samples and environmental conditions. This is largely due to the fact that there has been no direct way to monitor what is taking place at the molecular level.

Thus, the adaptive approach for controlling the PCR process promises to make the process simpler to operate, improve its reliability, reduce its sensitivity to environmental conditions, and shrink it from desktop to handheld size. Therefore, 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.

Unlike standard PCR, adaptive PCR automatically controls the duplication process by monitoring it at the molecular level. The reaction is controlled during the three stages of the duplication cycle using red and yellow fluorescent labels attached to synthetic left-handed DNA (L-DNA) shown in blue. The L-DNA is added to a sample and mirrors the interactions of the natural DNA (D-DNA) shown in green: (1) In the denaturation stage (top right), the sample is heated enough to cause the DNA strands to separate. This causes the red and yellow fluorescent labels on the L-DNA to light up. (2) In the annealing stage (bottom right), the sample is cooled to cause left-handed PCR primers to bind to the L-DNA. This is detected by quenching of the red fluorescence. (3) In the elongation stage (bottom left), the D-DNA strands are copied by polymerase enzymes. The L-DNAs are not copied during this stage but are transitioning to the denaturation stage as indicated by brightening of the red label on the L-DNA. The total number of D-DNA molecules in the sample doubles each time the cycle repeats: forty cycles produces more than one trillion copies. [Nicholas Adams/Vanderbilt]
Unlike standard PCR, adaptive PCR automatically controls the duplication process by monitoring it at the molecular level. The reaction is controlled during the three stages of the duplication cycle using red and yellow fluorescent labels attached to synthetic left-handed DNA (L-DNA) shown in blue. The L-DNA is added to a sample and mirrors the interactions of the natural DNA (D-DNA) shown in green: (1) In the denaturation stage (top right), the sample is heated enough to cause the DNA strands to separate. This causes the red and yellow fluorescent labels on the L-DNA to light up. (2) In the annealing stage (bottom right), the sample is cooled to cause left-handed PCR primers to bind to the L-DNA. This is detected by quenching of the red fluorescence. (3) In the elongation stage (bottom left), the D-DNA strands are copied by polymerase enzymes. The L-DNAs are not copied during this stage but are transitioning to the denaturation stage as indicated by brightening of the red label on the L-DNA. The total number of D-DNA molecules in the sample doubles each time the cycle repeats: forty cycles produces more than one trillion copies. [Nicholas Adams/Vanderbilt]

“PCR machines are pretty finicky,” noted lead study investigator Nicholas Adams, Ph.D., research assistant professor at Vanderbilt University. Dr. Adams provided an interesting example of the technical problems associated with PCR: “We have three commercial PCR machines in our lab. For a while 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!”

While laboratories have found methods to compensate for many of these problems, simplifying the process, while adding internal controls, would be of huge benefit to molecular biologists.

Adaptive PCR sidesteps all the variables of normal PCR, by relying on the fluorescent L-DNA to determine the ideal cycle temperatures for annealing and denaturing. Since L-DNA sequences are commercially available, the first step is to order L-DNA with the same sequence as the right-handed DNA that is going to be amplified along with left-handed DNA 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, causing 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 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,” the authors concluded.






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