By Ben Butkus
Life Technologies scientists have developed a technique called asynchronous PCR and demonstrated its ability to produce transient single-stranded DNA targets for subsequent peptide nucleic acid probe or microarray applications.
The method can help increase binding efficiency and amplify signal in any DNA hybridization-based method, Caifu Chen, one of the Life Tech scientists who co-invented the method, told PCR Insider this week.
Despite this, Life Tech will likely not commercialize the asynchronous PCR method on its own, but is further refining the method for use in internal research efforts, and is open to discussions with other companies wishing to apply the technique, Chen said.
Asynchronous PCR, or aPCR, has been around for several years, as Chen and colleagues first filed a patent on the method in 2001, eventually winning a US patent in 2005.
Thus far, aPCR has been a molecular biology technique without a clear killer application. Almost no scientific literature on the method existed until Chen and other Life Tech scientists penned a chapter on it in a recently published PCR Protocols book in the Methods in Molecular Biology series.
According to Chen, in the late 1990s, he and other researchers at Life Technologies and Applied Biosystems predecessor Applera were seeking a way to improve the use of peptide nucleic acid probes, to which Life Tech also owns the rights.
PNAs are DNA analogs in which the phosphate ribose ring of DNA has been replaced with a polyamide backbone, resulting in a probe with superior binding affinity and specificity. They have since been used extensively in endpoint PCR and fluorescence in situ hybridization applications and, more recently, in real-time quantitative PCR methods.
However, because PNA probes cannot be cleaved, they often produce low signal intensity and poor amplification curves, primarily because the probes demonstrate low hybridization efficiency with double-stranded DNA targets, according to Chen.
"And the reason is when you do PCR … you have two primers, and one primer goes one way and the other primer goes the other way simultaneously," Chen said. "The problem is … in standard PCR the PNA probe does not have time, or a chance to bind to the target. Really any other probe, [for example] molecular beacons, [would] have a tough time competing, because PCR is so fast, and priming happens very quickly."
The researchers solved this problem by creating an environment that promoted asymmetric or asynchronous strand-specific amplification. In order to do this they first changed the relative concentrations of the forward and reverse primers such that one primer's concentration was 10 times lower than that of the other primer.
"Indeed we can make the lower concentration primer prime more slowly," Chen said. "But that was not enough. So the other thing we did was to change the [melting temperatures]" such that one primer is around 76° F, and the other is around 55° F, he added. "What that creates is one primer that primes very effectively at the higher temperature, and the other primer doesn’t," Chen said.
The end result was that the researchers were able to more than triple the signal intensity "and greatly improve the success rate" of RT-qPCR experiments using PNA probes, Chen said.
In addition, they found that aPCR could generate more robust single-stranded DNA targets than could standard PCR due to the difference in Tm and concentrations between forward and reverse primers.
Chen explained that in PCR, there are typically two stages: denaturation at around 95° F followed by annealing and extension at around 60° F.
"But we created a unique thermocycling protocol called asynchronous PCR cycling, where we started at 92 or 95 degrees for denaturing, and then cooled down to around 72 degrees, not 60. When you cool down to that temperature, the high Tm primers can prime, no problem. And the low Tm primers are actually delayed … and you generate single-stranded DNA targets during PCR. After you generate the single strand, the PNA probe can bind to that target very effectively."
Chen added that the conditions for generating single-stranded DNA targets are not permanent, and are "only for about a half-cycle;" but that the technique produces "a very significant amount of single-strand target, typically 10 times more. This really creates the perfect target for a lot of applications, like microarrays. And the signal intensity is greatly improved."
Chen said that aPCR can improve the efficiency of any technique that uses a hybridization approach. "For instance, even a competitor of ours like NanoString could use this technology to make hybridization more efficient," Chen said.
As such, Life Tech "is open to having discussions" about potential licensing of the method, but is not specifically interested in commercializing it, Chen said.
"Our company is focused on TaqMan assays, and we don't really focus on any hybridization-based approach, so we cannot spend a lot of time to commercialize this technology," he said. "At the same time, we aren't in the microarray business, so we wouldn't use [aPCR] to make single strands to do hybridization."
Nevertheless, other companies or laboratories employing PNA probes might be able to benefit from the method. Even though Life Tech holds the intellectual property rights to PNA probe technology, it has conferred those rights to Korean biotech firm Panagene, which sells them for applications such as miRNA inhibition, molecular probes, and selectively amplifying mutated target DNA sequences; and AdvanDx, which uses PNAs in molecular diagnostics.
"We are thinking about other applications for internal efforts, as well," Chen said. "The technology is there, we just need to find the right application."
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