SuperSelective PCR Primers

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Structure of a SuperSelective primer for the detection and quantitation of rare mutant sequences in the presence of 1,000,000 wild-type sequences; and a demonstration that the number of amplification cycles required to generate a signal above background is inversely linearly proportional to the logarithm of the number of mutant molecules in the original sample.

Invention Summary:

“SuperSelective” primers enable the detection and quantitation of somatic mutations whose presence relates to cancer diagnosis, prognosis, and therapy in real-time PCR assays that can analyze rare DNA fragments present in blood samples (or in other biological tissues).

Rutgers researchers have created a design of these deoxyribonucleotide primers which includes both a “5' anchor sequence” that hybridizes strongly to target DNA fragments and a short, physically and functionally separate, “3' foot sequence” that is perfectly complementary to the mutant target but mismatches the wild type. As a consequence, amplification mainly occurs on the mutant target sequences.  As shown in the figure above, as few as ten mutant fragments can 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 can utilize differently colored molecular beacon probes or sloppy molecular beacon probes, each specific for the amplicons generated from a different target mutation, enabling a comprehensive assessment of the relevant somatic mutations present in a patient's clinical sample.

Market Applications:

• Identification and quantification of somatic mutations indicative of cancer diagnosis, choice of therapy, and early detection of recurrence, utilizing non-invasive liquid biopsies (or tissue samples)
• Identification of rare antibiotic resistance mutations in bacterial and fungal samples
• Potential to detect cancer in a routine blood sample taken during an annual medical examination before any symptoms have occurred

Advantages:

• Assays are carried out on widely available spectrofluorometric thermal cyclers
  utilizing commercially available buffers and take only a few hours to complete
• Closely related sequences are much less likely to be amplified
• Sensitivity is greater than analyses utilizing more expensive next-generation sequencing
• SuperSelective PCR primers enable a large number of somatic mutations to be simultaneously identified

Intellectual Property & Development Status: 

• Highly Selective Nucleic Acid Amplification Primers – US Patents 9,909,159;
  US 10,815,512; 11,111,515 and related foreign patents.
• Multiplex Nucleic Acid Assays Capable of Detecting Closely Related Alleles and Reagents Therefore – US Patent 11,542,547 and related foreign patents.

• Assay Methods and Kits for Detecting Rare Sequence Variants – Pending US Patent Application 17/754,032 and related foreign patents.
• Use of Structure-Specific Amplification Primers for the Detection of Genetic Rearrangements – Nonprovisional US Patent Application will be submitted in 2024.

Additional publications and patent applications:

• Full List of Issued and Pending Patents
• SuperSelective primer pairs for sensitive detection of rare somatic mutations
• Ultrasensitive Detection of Multidrug-Resistant Mycobacterium tuberculosis Using SuperSelective Primer-Based Real-Time PCRAssays
• Highly Selective Nucleic Acid Amplification Primers - US Patent 11,111,515 B2
• Multiple Nucleic Acid Assay Methods Capable of Detecting Closely Related Alleles, and Reagents Therefor - US Patent 11,542,547 B2
• Assay Methods and Kits for Detecting Rare Sequence Variants - US Patent - US Patent 2022/0333183 A1
• Highly Selective Nucleic Acid Amplification Primers - US Patent 9,909,159 B2
• Highly Selective Nucleic Acid Amplification Primers - US Patent 10,815,512 B2
• Multiplex Real-Time PCR Assays that Measure the Abundance of Extremely Rare Mutations Associated with Cancer
• Suppression of Wild-Type Amplification by Selectivity Enhancing Agents in PCR Assays that Utilize SuperSelective Primers for the Detection of Rare Somatic Mutations
• Multiplex SuperSelective PCR Assays for the Detection and Quantitation of Rare Somatic Mutations in Liquid Biopsies
• Super Selective PCR Slide Deck

 For any business development and other collaborative partnerships, contact:  marketingbd@research.rutgers.edu