Researchers at Vanderbilt University have developed a new way of performing PCR (polymerase chain reaction), or amplifying DNA so there’s enough of it to perform genetic analysis. The technique is called adaptive PCR and it relies on using only left-handed DNA (L-DNA), which is the mirror of normal DNA, to help regulate and monitor PCR. PCR is currently a fragile process that can be impeded by inexact sample preparation and environmental conditions. Having a way of continuously monitoring and guiding the process can lead to faster and cheaper results from genetic analysis and reduce the size of the machines used.
Though molecularly identical to DNA, L-DNA stays out of most biological processes. It can therefore be tagged with fluorescent markers, added to a PCR sample, and tracked as copies of DNA is made.
Changing the temperature of a sample during the tedious PCR process is both necessary and difficult to achieve. It has to be done during a number of steps, including when the DNA is split into strands and after primers are added to begin the PCR reaction. Today, timing of this process is done through estimation rather than in response to seeing when a certain step has actually been completed. Adaptive PCR instead relies on seeing changes in the fluorescence of L-DNA molecules as they undergo the same steps as the DNA in the same sample. In response to increases and decreases in fluorescence, the researchers know when a particular step has completed and so immediately move the process to the next one.
Here’s a captioned diagram explaining the adaptive PCR process, according to the researchers:
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)
Here’s a Vanderbilt video talking more about the new PCR technique:
Study in Analytical Chemistry: Adaptive PCR Based on Hybridization Sensing of Mirror-Image l-DNA…
Via: Vanderbilt…