OCT 16, 2013 11:00 AM PDT

SuperSelective PCR Primers for the Detection of Rare Mutant Cancer Cells in Clinical Samples Containing Abundant Normal Cells

Speaker
  • Professor, Public Health Research Institute, New Jersey Medical School, Rutgers University
    Biography
      Fred Russell Kramer is Professor of Microbiology and Molecular Genetics at the New Jersey Medical School, and has been a Principal Investigator at the Public Health Research Institute for the past 25 years. He graduated from the University of Michigan in 1964 and received his doctorate from the Rockefeller University in 1969. He was on the faculty of the Department of Genetics and Development at Columbia University College of Physicians and Surgeons for 17 years and has been a Research Professor and Adjunct Professor in the Department of Microbiology at New York University School of Medicine for the past 24 years.

    Abstract

    "SuperSelective" primers, by virtue of their unique design, enable only a few molecules of a mutant sequence to generate amplicons in conventional, real-time PCR assays without interference from extremely abundant wild-type molecules, even if the only difference between the mutant sequence and the wild-type sequence is a single-nucleotide polymorphism. The basis of this extraordinary selectivity is two-fold: (1) short, perfectly complementary mutant hybrids formed by the primers are thermodynamically more likely to be present at any given moment than the even shorter, mismatched wild-type hybrids; and (2) because the mean persistence time of mismatched wild-type hybrids (measured in milliseconds) is significantly shorter than the mean persistence time of perfectly complementary mutant hybrids, DNA polymerase molecules are kinetically more likely to encounter mutant hybrids before those hybrids dissociate, compared to the probability of their encountering the shorter-lived wild-type hybrids before they dissociate. Consequently, as few as 10 mutant molecules can routinely be distinguished and quantitated in samples containing 1,000,000 wild-type molecules. Learner Objectives: After completing this activity, the learner will be able to design polymerase chain reaction assays for the detection of rare mutant cells for the sensitive detection of circulating tumor cell DNA, for the quantitation of minimal residual disease, and for the early detection of mutations associated with resistance to therapeutic drugs.


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