June 21-23, 2004
Cambridge Healthtech Institute's Nucleic Acid-Based Technologies: Applications Amplified
LATE-PCR for Precise Asymmetric Amplification of Single-Stranded DNA
Lawrence J. Wangh
LATE-PCR is an advanced form of asymmetric PCR invented in my laboratory (patent pending)(PNAS in Press). This technology achieves reliable asymmetric amplification by making use of several innovations in the design of primers and utilizes an improved thermal cycle. These changes makes it possible, in turn, to detect accumulating target strands over a wider and lower range of temperatures, with increases in the strength and reliability of signals and decreased scatter among replicate reactions. We are currently constructing a single-tube sample-to-sequences system based on LATE-PCR, for applications in bio-defense, cancer diagnostics, infectious diseases and many other fields.
March 3-6, 2004
1st International qPCR Symposium and Application Workshop
LATE-PCR and Allied Technologies for Amplification and Utilization of Single-stranded DNA
L. Wangh, A. Sanchez, J. Rice, and K. Pierce
Symmetric PCR generates double stranded DNA products via exponential amplification, but the reaction plateaus when analyzed in real-time. Signal strength in such reactions can be low because the fluorescent probe has to compete with the reannealing of the product strands, and the final fluorescence intensity among replicate reactions is highly variable. LATE-PCR starts out as exponential amplification of a double-stranded product and then switches to sustained (non-plateauing) linear amplification of just one strand. Thus, LATE-PCR is an improved form of asymmetric PCR but, in contrast to conventional asymmetric PCR, is just as efficient as symmetric PCR. LATE-PCR achieves reliable asymmetric amplification by making use of several innovations in the design of primers. LATE-PCR also utilizes an improved thermal cycle in which the annealing of the two primers to their respective target strands takes place prior to primer extension, while hybridization of the probe to its accumulating single-stranded target takes place after primer extension. This change makes it possible, in turn, to detect accumulating target strands over a wider and lower range of temperatures using probes that have lower melting temperatures. Probes of this design, such as molecular beacons, are more allele-discriminating and exhibited decreased levels of background fluorescence. Collectively these improvements increase the strength and reliability of signals and minimize scatter among replicate reactions. The wider detection-temperature range of LATE-PCR also makes it possible to use combinations of probes to determine which of many possible variant-target sequences, such as mutant strains of an infectious agent, is actually present in a sample. LATE-PCR also yields single-stranded amplicons in sufficient quantity and purity to be used directly for DNA sequencing. Our progress toward construction of a single-tube “sample-to-sequences” system based on LATE-PCR will be discussed. LATE-PCR is patent pending and available for licensing from Brandeis University.
Pur-Amp - a New Quantitative Method for Preparation, Synthesis, and Amplification of Both cDNA and Genomic DNA in a Single Tube
L. Wangh, C. Hartshorn, and A. Anshelevich
PurAmp is a novel single-tube method for preparing protein-free RNA from samples (even those as small as a single cell or less), reverse transcribing that RNA into cDNA, and then amplifying sequences within the cDNA via real-time PCR. Genomic DNA in the sample is also rendered protein-free and can either remain in the sample for amplification along with the cDNA, or can be removed by DNase treatment prior to reverse transcription. In either case, all of the steps are carried out in a single tube thereby minimizing losses, and each of the steps in the process has been fully optimized to permit quantitative detection over the range of 1-to-10,000 target molecules. In the application illustrated here a single mouse embryo comprised of 2-to~200 cells is placed onto a dried “LysoDot” previously spotted into the lid of a standard PCR tube. The sample is then heated briefly, after which it can be either stored or processed immediately.
Non-transcribed genomic sequences in the sample serve as an internal standard for reaction reliability, as well as a means of counting the numbers of genomes present. In the case of transcribed genes the number of cDNA copies present in the sample is deduced from the CT value of the real-time reaction, after correcting for the number of genomic DNA copies amplified by the same primers.
The utility of the PurAmp method will be illustrated for several genes in intact mouse embryos, or single cells recovered from mouse embryos via laser ablation of the zona pellucida. PurAmp is a patent pending technology that can be licensed from Brandeis University.