My laboratory is currently devoted to the invention and application of new technology in the field of nucleic acid diagnostics. Over the past five years we have discovered and developed a new method of DNA amplification that overcomes the inherent limitations of conventional, symmetric PCR that has been the dominant method of DNA amplification for the last 20 years. In the case of symmetric PCR, each amplicon is generated by the use of two primers of equal concentration and equal Tm (melting temperature) and is therefore double-stranded. Amplification proceeds exponentially, but then slows down and reaches plateau in a stochastic manner. Thus, although such reactions are well known and are often robust, they nevertheless suffer from limitations which are particularly evident in samples containing low numbers of initial targets. These limitations include:

• Design difficulties, particularly for multiplexing
• Reduced efficiency and sensitivity due to mis-priming
• Variability among replicate reactions, particularly at end-point
• Limited availability of fluorescent colors
• Limitations in probe design
• Requirement for hot-start enzymes
• Costs

Our method, known as Linear-After-The-Exponential-PCR or LATE-PCR, is an advanced form of asymmetric PCR that efficiently generates single-stranded amplicons under predictable defined conditions. LATE-PCR is further enhanced by additional technologies, also invented in my laboratory. These methods and reagents include Quantilyse, PurAmp, PrimeSafe®, and several types of fluorescent probes. When used together these methods and reagents:

• Improve sample preparation
• Suppress mispriming
• Make it possible to routinely amplify single molecules
• Make it possible to routinely construct multiplexed reactions
• Make it possible to rapidly and inexpensively sequence single-stranded amplicons, even multiple products in a multiplex reaction.

Collectively these technologies promise to reduce the cost of individual assays while increasing the amount of information that they generate. In addition, new portable instrumentation (now under development) for use with LATE-PCR will make sophisticated point-of-care or penside assays available for the first time.

We are developing applications of the LATE-PCR technology in fields as diverse as detection of infectious diseases, genetic analysis of forensic samples, cancer detection, and basic research. For example, we are using LATE-PCR to develop a new, experimentally convenient strategy for the rapid detection of chromosomal numerical abnormalities (aneuploidies). Our strategy combines sample preparation, amplification, and quantitative end-point analysis into a clinically-compatible, single-tube assay. This assay is suitable for detection of copy number changes resulting from chromosomal and sub-chromosomal numerical abnormalities. This method is also applicable for detection of deletions or duplications of sub-chromosomal regions beyond the limits of detection of conventional cytogenetic techniques.

We are also currently studying levels of gene expression in single cells of cleavage stage mouse embryos. This research is aimed at generating a more complete understanding of early embryonic development, as well as generating tools needed to manipulate cells of the early embryo. Most specifically we hope to determine which cells and which cell culture conditions have the greatest potential of generating pluripotent embryonic stem cells capable of sustained cell division and development into particular therapeutically useful types of differentiated cells. Currently, we are using the LATE-PCR technology platform to quantify levels of two mRNAs, Oct4 and Cdx2, in individual blastomeres recovered from 8-cell mouse embryos because it is possible that these two mRNA's are regulated reciprocally and may therefore presage cell fate. Oct4 has been well established as a marker for cell pluripotency and by the blastocyst stage is exclusively expressed at high levels in cells of the ICM. In contrast, Cdx2 is known to be involved in the development of the trophectoderm. Our goal is to determine how early in development each of these genes is expressed and whether the cells of the early embryo display consistent patterns of expression. If such patterns exist they are expected to be quantitative rather than qualitative (yes-no), thereby increasing the technical challenge involved in investigating this problem.

In the field of infectious diseases, a LATE-PCR multiplex assay able to detect any of a number of avian viral targets is under development in our laboratory. This assay will be a single-tube assay designed to detect and distinguish between avian flu virus (serotypes H5 or H7 or H9), Newcastle Disease virus and Gumboro Disease virus.

Selected Publications:

Pierce, K.E. and Wangh, L.J. (2007) "LATE-PCR and allied technologies: Real-time detection strategies for rapid, reliable diagnosis from single cells." In: Single Cell Diagnostics, Methods in Molecular Medicine Series (ed. Alan Thornhill), Series Editor John Walker, Humana Press, UK. [full text] (.pdf)

Sanchez, J. A., Abramowitz, J.D., Salk, J.J., Reis, Jr, A.H., Rice, J.E., Pierce, K.E., and Wangh, L.J. (2006) "Two-temperature LATE-PCR endpoint genotyping", BMC Biotechnology, 6:44. [full text] (.pdf)

Salk, J.J., Sanchez, J.A., Pierce, K.E., Rice, J.E., Soares, K.C., and Wangh, L.J. (2006) "Direct amplification of single-stranded DNA for pyrosequencing using Linear-After-The-Exponential (LATE)-PCR", Analytical Biochemistry, 353:124-132. [full text] (.pdf)

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 USA, 102:8609-8614. [full text] (.pdf)

Hartshorn, C., Anshelevich, A., and Wangh, L.J. (2005) "Laser zona-drilling does not induce hsp70i transcription in blastomeres of 8-cell mouse embryos", Fertility and Sterility, 84(5):1547-1550. [full text] (.pdf)

Hartshorn, C., Anshelevich, A., and Wangh, L.J. (2005) "Rapid, single-tube method for quantitative preparation and analysis of RNA and DNA in samples as small as one cell", BMC Biotechnology, 5:2. [full text] (.pdf)

Hartshorn, C., Rice, J.E., and Wangh, L.J. (2004), "Optimized Real-time RT-PCR for Quantitative Measurements of DNA and RNA in Single Embryos and Blastomeres", In:A-Z of quantitative PCR (Editor: SA Bustin), International University Line, La Jolla, Ca., 675-702.

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", Proc Natl Acad Sci USA. 101:1933-8 [abstract] [full text].

Pierce KE, Wangh LJ. (2004) "Effectiveness and limitations of uracil-DNA glycosylases in sensitive real-time PCR assays. Biotechniques", 36:44-6, 48. [full text]

Pierce KE, Rice JE, Sanchez JA, Wangh LJ. (2003) "Detection of cystic fibrosis alleles from single cells using molecular beacons and a novel method of asymmetric real-time PCR", Mol Hum Reprod. 9:815-20 [abstract] [full text]

Hartshorn, C., Rice, J.E., and Wangh, L.J. (2003), "Differential Pattern of Xist RNA Accumulation in Single Blastomeres Isolated from 8-Cell Stage Mouse Embryos Following Laser Zona Drilling", Molecular Reproduction and Development, 64: 41-51 [abstract] [full text].

Rice, J.E., Sanchez, J.A., Pierce, K.E., Wangh, L.J. (2002), " Real-Time PCR with Molecular Beacons Provides a Highly Accurate Assay for Detection of Tay-Sachs Alleles in Single Cells", Prenatal Diagnosis, 22: 1130-1134. [abstract] [full text]

Pierce, K., Rice, J., Sanchez, J.A., and Wangh, L.J. (2002) “QuantiLyseTM: Reliable DNA Amplification from Single Cells”, BioTechniques 32: 1106-1111. [abstract] [full text]

Hartshorn C., Rice J.E., and Wangh, L.J. (2002) "Developmentally-Regulated Changes of Xist RNA Levels in Single Preimplantation Mouse Embryos, as Revealed by Quantitative Real-Time PCR", Molecular Reproduction and Development, 61:425-436. [abstract] [full text]

Karni R.J., Wangh L.J., and Sanchez J.A. (2001) Non-Random Distribution of the Inactive-X Chromosome In Human Neutrophil Nuclei. Chromosoma 110: 267-274. [abstract]

Pierce, K.E., Rice, J.E., Sanchez, J.A., Brenner, C. & Wangh, L.J. (2000). "Real-time PCR using molecular beacons for accurate detection of the Y chromosome in single human blastomeres", Molecular Human Reproduction, 6:1155-1164. [abstract]

Sanchez, J.A., and Wangh, L.J. (1999). New Insights Into the Mechanisms of Nuclear Segmentation in Human Neutrophils, Review article, J. Cellular Biochemistry 73:1-10. [abstract]

Sanchez, J.A., Karni, R.J., and Wangh, L.J. (1997). Fluorescent in situ hybridization (FISH) analysis of the relationship between chromosome location and nuclear morphology in human neutrophils., Chromosoma., 106:168-177. [abstract]

 

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Last reviewed: February 16, 2007.

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