OCT 05, 2016 12:00 PM PDT

Sensitive and Specific, Multiplex, Real-Time PCR Assays for Assessing the Abundance of Extremely Rare Mutations Associated with Cancer Diagnosis, Prognosis, and Therapy

  • Professor, Public Health Research Institute, New Jersey Medical School, Rutgers University
      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.


    Real-time multiplex PCR assays are potentially the most rapid, most sensitive,
    and least expensive way to assess the abundance of mutant DNA fragments present in liquid biopsies; provided that a way is found to selectively amplify rare mutant fragments without amplifying abundant related wild-type fragments; and provided that the amplicons generated from different mutants that occur in the same or an adjacent codon are prevented from forming heteroduplexes that interfere with exponential amplification, obscuring the threshold values of the rarer mutants.  “SuperSelective” PCR primers, due to their unique design, are extraordinarily specific, able to selectively initiate the synthesis of amplicons
    on ten mutant DNA fragments in the presence of 1,000,000 wild-type DNA fragments,
    even though the only difference between the mutant and the wild-type is a single-nucleotide polymorphism.  Moreover, each SuperSelective primer that is specific for a particular mutation possesses a unique 5' tag sequence that is incorporated into the resulting amplicons and detected in real-time by differently colored molecular beacon probes. 
    Each SuperSelective primer specific for a particular mutation also possesses a unique “bridge” sequence that assures that each primer only copies its intended amplicon,
    and that creates a single-stranded bubble in heteroduplexes that enables the rarest amplicons to be independently exponentially amplified.  And finally, the inclusion of primers for a wild-type reference gene fragment, enables the abundance of each type of mutant
    DNA fragment to be assessed (without measuring the amount of DNA in the sample)
    by determining the difference between its threshold value and the threshold value of
    the reference gene.

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