Multiplex SuperSelective PCR Assays for the Detection of Rare Somatic Mutations


SuperSelective primers can enable the simultaneous identification and quantitation of inherited reference genes and rare somatic mutations in routine multiplex PCR assays, while virtually eliminating signals from abundant wild-type sequences closely related to the target mutations. These assays are ultrasensitive, specific, rapid, and low cost, and can be performed in widely available spectrofluorometric thermal cyclers.

Using a routine blood sample, investigators have developed an extremely sensitive assay to detect and quantify DNA fragments containing rare genetic mutations in a patient’s cancer cells. This assay uses “Super Selective” PCR primers that virtually ignore abundant closely related non-mutant DNA fragments derived from patients’ normal cells. This new assay is faster and considerably cheaper than current methods and can be performed on widely available instruments. The investigators led by Diana Yaneth Vargas and Fred Russell Kramer from the Public Health Research Institute of the New Jersey Medical School of Rutgers University demonstrated the ease of utility of this approach in a new report in the Journal of Molecular Diagnostics.

The research team provide examples of SuperSelective PCR assays that target eight different somatic EGFR mutations, irrespective of whether they occur in the same codon, occur at separate sites within the same exon, or involve deletions. In addition, they demonstrated examples of SuperSelective PCR assays that detect specific EGFR mutations in circulating tumor DNA present in the plasma of liquid biopsies obtained from patients with non–small-cell lung cancer. The findings suggest that multiplex SuperSelective PCR assays may enable the choice, and subsequent modification, of effective targeted therapies for the treatment of an individual’s cancer, utilizing frequent noninvasive liquid biopsies.

The research team objective is to develop highly multiplex assays that enable both the early detection of mutations relevant to cancer before any symptoms have occurred and the application of an effective early treatment. These assays will use noninvasive liquid biopsies (routine blood samples) taken during a person’s annual medical checkup, and the DNA fragments transiently present in the blood sample (originating from cells that have died) will then be analyzed by highly multiplex real-time or digital PCR techniques.

Current techniques for analyzing liquid biopsies involve either next-generation sequence analysis, which is expensive, lengthy, and has limited sensitivity; or use a PCR technique in which both the mutant DNA fragments and the closely related wild-type DNA fragments are amplified, limiting sensitivity when the mutant DNA fragments are rare. The unique design of SuperSelective PCR primers, on the other hand, enables the amplification of the mutant DNA fragments, while effectively preventing the amplification of the abundant closely related wild-type DNA fragments. Consequently, the SuperSelective PCR assays reported in the new study are very sensitive.

Multiplex Super Selective PCR assays, utilizing frequent non-invasive liquid biopsies, enable the choice and subsequent modification of an effective targeted therapy for the treatment of an individual’s cancer. Furthermore, after treatment, these assays may help monitor the patient to detect the presence of minimal residual disease, enabling further intervention if necessary.

To demonstrate the clinical potential of their approach, six DNA samples from the plasma of patients with cancer were assayed. The samples from five of the patients were blinded. Multiplex SuperSelective PCR assays were performed to confirm the presence or absence of particular human epidermal growth factor receptor (EGFR) mutations in each sample. These mutations were chosen because their presence determines which targeted therapy is most likely to be effective against non-small-cell lung cancer. Four samples were positive for different EGFR target mutations and two samples did not give signals for EGFR target mutations. After the blinded samples were unblinded, it was revealed that the two negative samples were from patients who did not have EGFR mutations, and those samples were purposely provided as a control. The assays identified the previously known mutations in the other samples. Significantly, the assay also identified a very rare resistance mutation in one blinded sample that was not previously known to be present, suggesting that the patient’s targeted therapy needed to be changed.

The authors note that this technology might have other medical applications, such as the identification of rare antibiotic-resistant bacteria and the identification of rare mutant genes found in fetal cells circulating in a mother’s plasma. Thus, the availability of sensitive multiplex SuperSelective PCR assays is likely to have wide clinical utility.

Multiplex SuperSelective PCR Assays for the Detection and Quantitation of Rare Somatic Mutations in Liquid Biopsies - Medicine Innovates


About the author

Fred Russell Kramer, Ph.D.
Professor of Microbiology, Biochemistry & Molecular Genetics

Professor Kramer lab is developing sensitive, multiplex polymerase chain reaction assays that utilize the DNA fragments transiently occurring in blood samples to detect and quantitate rare somatic mutations that are indicative of cancer diagnosis, prognosis, and therapy. These assays employ specialized PCR primers that selectively amplify mutant DNA fragments without amplifying the almost identical DNA fragments that come from normal cells. Because these assays are non-invasive and inexpensive, a patient can be followed over the course of treatment, enabling individualized adjustments in therapy to be made as new somatic mutations occur, resulting in longer life with fewer symptoms.  Our long-term goal is to design an assay that can be carried out on a blood sample obtained during a routine annual medical examination that identifies actionable somatic mutations indicative of cancer anywhere in a person’s body, even though no symptoms have yet occurred.


Vargas DY, Tyagi S, Marras SAE, Moerzinger P, Abin-Carriquiry JA, Cuello M, Rodriguez C, Martinez A, Makhnin A, Farina A, Patel C, Chuang TL, Li BT, Kramer FR. Multiplex SuperSelective PCR Assays for the Detection and Quantitation of Rare Somatic Mutations in Liquid Biopsies. J Mol Diagn. 2021 Dec 23:S1525-1578(21)00428-1. doi: 10.1016/j.jmoldx.2021.11.006.