Abstracts Archive

2005 Abstracts

August 24-26, 2005
Cambridge Healthtech Institute's Systems Integration in Biodefense
Washington, D.C.

Orthopox Virus Sequence Detection and Identification using LATE-PCR with Mismatch-tolerant Probes

Kenneth Pierce, Aquiles Sanchez, John Rice, and Lawrence Wangh

The family of orthopox viruses includes smallpox (variola major), monkeypox, and several strains of cowpox. Use of these viruses, particularly variola, as a bioweapon has become a concern of the war on terror. We describe a novel, closed-tube method for the rapid amplification and identification of nucleotide sequences of several orthopox viruses. Amplification is carried out using Linear-After-The-Exponential PCR (LATE-PCR) to generate specific single-stranded DNA. The resulting single-stranded product is free to hybridize to one or more mismatch-tolerant probes over a wide range of temperatures. 

Ratios of the signals from a pair of probes, one to a conserved region and another to a variable region of an orthopox hemaglutinin gene yield "fluorescence signatures" that distinguish all tested sequence variations and virtually eliminate the possibility of a false positive signal for variola. Because mismatch-tolerant probes hybridize to imperfect complementary sequences at low temperatures, the assay is capable of detecting both naturally occurring and genetically engineered variants of orthopox, as well as discriminating between closely related isolates. LATE-PCR also enables target quantification using end-point analysis, since reactions are highly reproducible, unlike standard PCR end-point assays. Internal control primers and targets are included in each assay to verify amplification conditions and guard against false negative results. Assays are compatible with many types of available real-time PCR machines and fluorimeters.  Thus, LATE-PCR assays, with mismatch-tolerant probes, can be used to detect and identify a wide array of infectious agents that could be used as bioweapons.

May 19-21, 2005
Sixth International Symposium on Preimplantation Genetics
London, England

New Technologies for Quantitative Analysis of RNA and DNA in Single Cells Recovered from Cleavage Stage Embryos

C. Hartshorn, J.A. Sanchez, and L.J. Wangh

Introduction: Individual cells of early cleavage stage embryos exhibit distinct patterns of gene expression which likely affects subsequent daughter-cell fates. We have invented several new technologies to investigate this problem in detail.

Materials/Methods: Cell lysis, cDNA synthesis, and DNA amplification are all performed in a recently published single tube method called “PurAmp”.  Amplification is performed using “LATE-PCR”, an advance form of asymmetric PCR. The reliability of such reactions in enhanced using “Elixirs”, a new class of reactions which prevent mis-priming. The fidelity of the amplified single-stranded products is verified via "Dilute-'N-Go" sequencing.

Results: Current experiments are aimed at measuring Xist, Oct4, Sry, and Hsp70i genes and their transcripts in whole mouse embryos, as well as all cells recovered from 3, 4, and 8-cell stage embryos following laser drilling of the zona pellucida. Up-to-date findings using this strategy will be reported.

Conclusions: Our new technologies are convenient and reliable. Our findings should shed light on the origin and fate of pleuripotent stem cells in mammalian embryos.

March 21-22, 2005
Cambridge Healthtech Institute's Quantitative PCR: The Validation Tool of Choice
La Jolla, California

LATE-PCR Multiprobing for Quantitative Detection of Closely Related Sequences

Lawrence J. Wangh, J. Aquiles Sanchez, and Kenneth E. Pierce

We have recently described discovery and use of LATE-PCR, an advanced quantitatively accurate form of asymmetric PCR (Sanchez et al. 2004, Pierce et al. 2003). LATE-PCR also makes it possible to carryout amplicon detection over a larger “temperature space” using “Low-Temperature” probes that are more versatile than those used in conventional symmetric PCR. Our “multiprobing” strategy combines these improvements to achieve quantitative, closed tube detection of many closely related sequences using very few probes. This novel strategy is applicable to the fields of infectious diseases, bio-defense, and forensics.

February 5-8, 2005
ABRF Biomolecular Technologies: Discovery to Hypothesis
Savannah, Georgia

 LATE-PCR and Allied Technologies: Novel Real-time Methods and Reagents for Rapid, Reliable Genetic Analysis Down to the Level of Single Cells

Lawrence J. Wangh and Kenneth E. Pierce

Accurate detection of gene sequences in single cells is the ultimate challenge of PCR sensitivity. Unfortunately, commonly used conventional and real-time PCR techniques are often too unreliable at that level to provide the accuracy needed for clinical diagnosis. In order to circumvent this problem my laboratory has developed Linear-After-The-Exponential-PCR (LATE-PCR), an advanced form of asymmetric PCR incorporating novel criteria for designing and using primers and probes. We have developed an assay procedure in our lab to eliminate primer-dimer formation and mis-priming in both symmetric and LATE-PCR reactions, without need of expensive hot-start forms of Taq polymerase. LATE-PCR with this method is reliable down to the level of single cells and can be used with both allele discriminating and mis-match tolerant probes over a broader range of temperatures. Together these technologies improve the accuracy of genetic diagnosis in a wide variety of settings while reducing the time and cost of designing such reactions.