Abstracts Archive

2006 Abstracts

September 24, 2006
7th International Congress of Veterinary Virology
Lisbon, Portugal

Late-PCR Permits Quantitative End-Point, Closed-Tube Detection and Discrimination of Pathogens and their Sequence Variants

Kenneth E. Pierce, Ph.D., Cristina Hartshorn, Ph.D., John E. Rice, MS, J. Aquiles Sanchez, Ph.D., Arthur H. Reis, Jr., Ph.D., Lawrence J. Wangh, Ph.D., Department of Biology, Brandeis University, Waltham, MA, USA LATE-PCR, Quantitative End Point Assays, Multiprobing, Multiplexing, Dilute-‘N’-Go Sequencing

Introduction and Objective: Conventional PCR and Real-time PCR are well know research technologies, but have serious limitations when used as diagnostic assays for quantitative detection of infectious organisms and viruses in small samples. This is particularly when sequence variants are present in those samples.  LATE-PCR, an advanced form of asymmetric PCR invented in our laboratory overcomes these difficulties and is therefore well suited for detection and analysis of pathogenic organisms in the laboratory and in the field.

Material and Methods: LATE-PCR assays efficiently generate single-stranded DNA amplicons which can be probed for sequence variants over a broad range of temperatures (25-60ºC) using both sequence specific probes, like molecular beacons, and mis-match tolerant probes that detect variant sequences at different temperatures.  PCR-Elixirs, another invention from our laboratory, suppress mis-priming throughout these reactions thereby increasing their sensitivity and fidelity. Construction of multiplex reactions is also made much easier.  In addition, the single-stranded amplicons generated in these reactions can readily be sequenced using an efficient protocol which we call Dilute-‘N’-Go Sequencing.

Results: LATE-PCR assays utilizing two mis-match tolerant probes can readily distinguish the nucleotide sequences of different orthopox viruses Variola, Vaccinia, Monkeypox, Cowpox M-96, and Cowpox B by virtue of small sequence differences.  One probe detects a conserved sequence common to all viruses, while a second probe displays temperature-sensitive hybridization to a site that contains virus-specific nucleotide variations.  The ratio of the two signals comprises a “fluorescent signature” that is unique for each virus.  Unknown viral strains having their own “fluorescent signature” can be readily detected and resolved to the nucleotide level by “Dilute-N’-Go” sequencing.  In addition, LATE-PCR readily permits construction of multiplex assays for simultaneous amplification of five or more products, as well as assays for detecting which single pathogen is present among many possible organisms in a sample.  Assays along these lines are currently being constructed for detection and discrimination between Avian Flu subtypes.

Discussion and Conclusions: The new platform technologies we have developed are broadly applicable to rapid detection and diagnosis of infectious diseases in both animals and humans.  We envision that in the future diagnostic closed-tube LATE-PCR assays will utilize low cost point-of-care devices that are now under development.  These devices will be battery operated and portable for use in the field.

Acknowledgements: The research has been supported in part by a research grant from Smiths Detection, Inc.

References: Jesse J. Salk, J Aquiles Sanchez, Kenneth E. Pierce, John E. Rice, Kevin C. Soares, Lawrence J. Wangh (2006) Direct Amplification of Single-Stranded DNA for Pyrosequencing using Linear-After-The-Exponential (LATE)-PCR. Analytical Biochemistry, Volume 353, Issue 1, 1 June 2006, Pages 124-132.
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, Proc Natl Acad Sci U S A. 102, 8609-8614.
Hartshorn C, Anshelevich A, Wangh LJ. (2005) Rapid, single-tube method for quantitative preparation and analysis of RNA and DNA in samples as small as one cell. BMC Biotechnol, 5:2.
Sanchez,JA., Pierce, KE., Rice, JE., & Wangh. LJ. (2004) Linear-after-the-exponential (LATE)-PCR: An advanced method of asymmetric PCR and its uses in quantitative real-time analysis, PNAS 101:1933-1938.

June 15-18, 2006
12th International Congress on Infectious Diseases
Lisbon, Portugal

Quantitative End-Point Assays of Infectious Agents and Their Sequence Variants using LATE-PCR and Other New Technologies for Multiplexing and Multiprobing
K.E. Pierce, J.E. Rice, J.A. Sanchez, C. Hartshorn, A.H. Reis, Jr., and L.J. Wangh

Background: Conventional PCR and Real-time PCR are well know research technologies, but have serious limitations as clinical diagnostic assays for quantitative detection of infectious organisms and viruses in small samples, particularly when sequence variants are present in those samples. These difficulties are overcome by LATE-PCR, an advanced form of asymmetric PCR invented in our laboratory.

Methods: LATE-PCR assays efficiently generate single-stranded DNA amplicons which can be probed for sequence variants using mis-match tolerant probes over a broad range of temperatures (25-60ºC). Additional improvements greatly reduce amplification errors which otherwise confound detection of small numbers of target molecules.

Results: LATE-PCR assays utilizing two mis-match tolerant probes can readily distinguish the nucleotide sequences of different orthopox viruses Variola, Vaccinia, Monkeypox, Cowpox M-96, and Cowpox B by virtue of small sequence differences. One probe detects a conserved sequence common to all viruses, while a second probe displays temperature-sensitive hybridization to a site that contains virus-specific nucleotide variations. The ratio of the two signals comprises a “fluorescent signature” that is unique for each virus. Unknown viral strains having their own “fluorescent signature” could be readily detected and resolved to the nucleotide level by “Dilute-N’-Go” sequencing, another convenient feature of LATE-PCR. In addition, LATE-PCR readily permits construction of multiplex assays for simultaneous amplification of five or more products, as well as assays for detecting which single pathogen is present among many possible organisms in a sample. Assays along these lines are useful for rapid analysis of blood infections without the need of bacterial culture.

Conclusion: The new platform technologies we have developed are broadly applicable to the field of infectious diseases. We envision that in the future diagnostic closed-tube LATE-PCR assays will utilize low cost point-of-care devices that are now under development. The resulting quantitative end-point information will detect and discriminate between many possible pathogens.

March 29-31, 2006
Cambridge Healthtech Institute's Clinical Biomarker Summit
Coronado, California

Detection of LOH in Barrett's Esophagus using LATE-PCR Quantitative End-point Assays
Lawrence J. Wangh, J. Aquiles Sanchez, Jesse Salk, Patty Galipeau, and Brian J. Reid
Barrett's esophagus (BE) is the only known precursor of esophageal adenocarcinoma (EA). EA represents about 1 percent of the cancers diagnosed in the US and it is one of the cancers whose incidence has been increasing steadily during the past three decades in the Western world. In addition to ploidy abnormalities detected by flow cytometry, loss of heterozygosity, LOH, involving the p16 gene in chromosome 9p, as well as LOH involving the p53 gene in chromosome 17p are among the earliest and most relevant genomic biomarkers of BE. Our two laboratories are employing Quantitative End-Point LATE-PCR, QE LATE-PCR, to detect LOH in these chromosomal regions. 

Each LATE-PCR assay generates both single-stranded and double-stranded DNA and the ratio of these products corrects for variability among replicate samples. In addition, QE LATE-PCR assays make use of single linear probes to distinguish SNP sites that are heterozygous in normal genomes from those that are hemizygous in genomes that have undergone LOH in a single-tube, end-point assay format. We anticipate that clinical assays based on the QE LATE-PCR strategy will be reliable, rapid, and relatively inexpensive compared to current assays for LOH and will thereby make it easier to identify and closely monitor those BE patients at highest risk for developing EA.

March 20-22, 2006
4th Early Detection Research Network (EDRN) Scientific Workshop
Philadelphia, Pennsylvania
Quantitative End-Point LATE-PCR Assays for Detection of LOH as a Biomarker

J. Aquiles Sanchez, Lawrence Wangh, Brandeis University

Jesse Salk, Patricia Galipeau, Brian J. Reid, Divisions of Human Biology and Public Health Services, Fred Hutchinson Cancer Research Center, Seattle, WA

Our two laboratories are employing a new clinically compatible platform for the convenient and reproducible detection of LOH biomarkers in human cancers. Current methods for LOH detection are complex, expensive, and not sufficiently suited for routine clinical use. Our strategy, called Quantitative End-Point LATE-PCR (QE-LATE-PCR) permits robust and reliable LOH detection in small samples comprised of 1-100 premalignant cells. QE LATE-PCR assays are easy to perform in a single-tube format and are sensitive enough to detect chromosome loss in only a fraction of the genomes tested. We demonstrate the use of QE LATE-PCR for detection of LOH biomarkers in Barrett’s Esophagus (BE), a precursor condition for esophageal adenocarcinoma (EA) and a model system for many cancers. A single center prospective (phase IV) study shows that a panel of three specific biomarkers (p16 LOH, p53 LOH, DNA content abnormalities –aneuploidy, tetraploidy) is highly predictive of EA risk in BE patients.

QE LATE-PCR identifies LOH by identifying SNP sites that are heterozygous in normal genomes but become hemizygous in genomes that have undergone LOH. Genotyping is based on the fraction of amplification products detected by a single hybridization probe at the end of the amplification reaction. The probe detects 50% amplification products in samples heterozygous for the probed allele, but either 100% or 0% of amplified products in hemizygous samples depending on whether the probed allele is retained following LOH or not.

Quantitative end-point genotyping is achieved through the combined use of LATE-PCR and mismatch tolerant probes. LATE PCR is a form of asymmetric PCR that generates single-stranded DNA products with high efficiency and specificity. The continued availability of single-stranded DNA products for hybridization at the end of LATE-PCR amplification permits product detection at multiple temperatures using mismatch tolerant linear probes. At a relatively high temperature these probes preferentially hybridize to the fully complementary sequence of a particular SNP, but at lower temperature bind to all variants of that SNP. The ratio of fluorescence signals at the upper and lower temperature corrects for differences in product yield among replicates and reveals with 99.7% accuracy whether the sample in question possesses a normal diploid genome or an LOH genome due to loss of either chromosome.

LOH detection via QE LATE-PCR assays will greatly facilitate early identification of BE patients having the highest risk of developing EA. The same assays are available for rapid and reliable diagnostic of the many cancers and pre-malignant conditions in which LOH is a biomarker of neoplastic progression.