August 24-26, 2005
Cambridge Healthtech Institute's Systems Integration in Biodefense
Orthopox Virus Sequence Detection and Identification using LATE-PCR with Mismatch-tolerant Probes
Kenneth Pierce, Aquiles Sanchez, John Rice, and Lawrence Wangh
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
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
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
February 5-8, 2005
ABRF Biomolecular Technologies: Discovery to Hypothesis
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