title: Multiplex Real-Time PCR Assays that Measure the Abundance of Extremely Rare Mutations Associated with Cancer.
abstract: We describe the use of "SuperSelective" primers that enable the detection and quantitation of somatic mutations whose presence relates to cancer diagnosis, prognosis, and therapy, in real-time PCR assays that can potentially analyze rare DNA fragments present in blood samples (liquid biopsies). The design of these deoxyribonucleotide primers incorporates both a relatively long "5' anchor sequence" that hybridizes strongly to target DNA fragments, and a very short, physically and functionally separate, "3' foot sequence" that is perfectly complementary to the mutant target sequence, but mismatches the wild-type sequence. As few as ten mutant fragments can reliably be detected in the presence of 1,000,000 wild-type fragments, even when the difference between the mutant and the wild type is only a single nucleotide polymorphism. Multiplex PCR assays employing a set of SuperSelective primers, and a corresponding set of differently colored molecular beacon probes, can be used in situations where the different mutations, though occurring in different cells, are located in the same codon. These non-symmetric real-time multiplex PCR assays contain limited concentrations of each SuperSelective primer, thereby enabling the simultaneous determination of each mutation's abundance by comparing its threshold value to the threshold value of a reference gene present in the sample.
authors.affiliation: Public Health Research Institute, Rutgers University, Newark, New Jersey, United States of America.
authors.forename: Diana Y
authors.initials: DY
authors.lastname: Vargas
authors.affiliation: Public Health Research Institute, Rutgers University, Newark, New Jersey, United States of America.|Department of Microbiology, Biochemistry and Molecular Genetics, New Jersey Medical School, Rutgers University, Newark, New Jersey, United States of America.
authors.forename: Fred Russell
authors.initials: FR
authors.lastname: Kramer
authors.affiliation: Public Health Research Institute, Rutgers University, Newark, New Jersey, United States of America.|Department of Medicine, New Jersey Medical School, Rutgers University, Newark, New Jersey, United States of America.
authors.forename: Sanjay
authors.initials: S
authors.lastname: Tyagi
authors.affiliation: Public Health Research Institute, Rutgers University, Newark, New Jersey, United States of America.|Department of Microbiology, Biochemistry and Molecular Genetics, New Jersey Medical School, Rutgers University, Newark, New Jersey, United States of America.
authors.forename: Salvatore A E
authors.initials: SA
authors.lastname: Marras
mesh_terms: D004247:DNA / Q000097:blood* / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000379:methods; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D009154:Mutation / Q000235:genetics; D009369:Neoplasms / Q000175:diagnosis* / Q000235:genetics*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods; D009693:Nucleic Acid Hybridization; D020641:Polymorphism, Single Nucleotide / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*
chemical_list: D017931:DNA Primers; D004247:DNA