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Advantages of LATE-PCR Technology

BioQuick News recently had the opportunity to speak with Dr. Lawrence Wangh about the novel technology of LATE-PCR (LATE stands for Linear After The Exponential) (see references 1-3 below). Dr. Wangh is professor of biology and director of the Laboratory of Molecular Diagnostics and Global Health at Brandeis University near Boston. Over the past ten years, he and his laboratory colleagues have invented LATE-PCR and a suite of allied technologies. Dr. Wangh said that LATE-PCR is an advanced form of non-symmetric PCR that overcomes many of the limitations of conventional (symmetric and asymmetric) PCR. These limitations are particularly evident in samples containing low numbers of initial targets. LATE-PCR makes efficient use of a limiting primer and an excess primer that together cause the reaction to abruptly switch from efficient production of double-stranded to single-stranded DNA, when the limiting primer is used up. This is of great utility because single-stranded amplicons can be readily probed at end-point over a broad range of temperatures. This, in turn, allows for the use of probes that hybridize to target sequence variants at different temperatures. LATE-PCR assays are further enhanced by use of PrimeSafeTM, a proprietary reagent that suppresses all forms of mis-priming throughout amplification and makes it far easier to build highly multiplexed reactions. In addition, the lab has recently invented ThermalightTM Lights-On/Lights-Off probes that allow several different long target sequences to be scanned for sequence variants, each in a different fluorescent color. Dr. Wangh said that LATE-PCR makes it possible to build highly informative cost-effective, closed-tube assays for detection of mutations in hundreds of nucleotides. These assays are sensitive down to the single cell and even single molecule level, realms where DNA sequencing is not practical. Dr. Wangh pointed out that LATE-PCR is readily carried out on all commercially available qPCR instruments and is particularly applicable to clinical diagnostic situations in which the quantity of available sample is often low. The Wangh group is currently developing LATE-PCR assays in fields as diverse as infectious diseases, forensics, and cancer diagnostics. The researchers are also exploring ways to further exploit LATE-PCR, including placing it on inexpensive portable point-of-care instruments that take full advantage of the entire 75⁰C of useful temperature space.

1. Rice, J.E., Sanchez, J.A., Pierce, K.E., Reis, A.H., Osborne, A., and Wangh, L.J. (2007) Monoplex/multiplex linear-after-the-exponential-PCR assays combined with PrimeSafe and Dilute-'N'-Go sequencing. Nature Protocols 2(10): 2429-2438.
(http://www.nature.com/nprot/journal/v2/n10/abs/nprot.2007.362.html)

2. Pierce, K.E., Sanchez, J.A., Rice, J.E., and Wangh, L.J. (2005) Linear-After-The-Exponential (LATE)-PCR: Primer design criteria for high yields of specific single-stranded DNA and improved real-time detection. PNAS 102:8609-8614.
(http://www.pnas.org/content/102/24/8609.abstract)

3. Sanchez, J.A., Pierce, K.E., Rice, J.E., and Wangh, L.J. (2004) Linear-After-The-Exponential (LATE)-PCR: An advanced method of asymmetric PCR and its uses in quantitative real-time analysis. PNAS 101(7):1933-1938.
(http://www.pnas.org/content/101/7/1933.abstract)