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Updated: May 25, 2026

Wild-type Blocking PCR Combined with Direct Sequencing as a Highly Sensitive Method for Detection of Low-Frequency Somatic Mutations
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Ribonucleotide-Modified Primer Overlapping Blocker Elongation (PROBE) Assay for Highly Specific Mutation Detection.

Yongjuan Zhao1, Ziyan Wang1,2,3, Min Zhang1

  • 1Shanghai Public Health Clinical Center, Fudan University, Shanghai 201508, China.

Analytical Chemistry
|May 23, 2026
PubMed
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This summary is machine-generated.

A new ribonucleotide-modified primer (r-primer) overlapping blocker elongation (PROBE) method enhances single-nucleotide variant (SNV) detection. This PROBE method offers high specificity and sensitivity for accurate SNV genotyping, outperforming existing techniques.

Area of Science:

  • Genetics
  • Molecular Biology
  • Biotechnology

Background:

  • Single-nucleotide variants (SNVs) are common genetic variations linked to diseases.
  • Existing SNV detection methods often lack specificity, leading to false positives, particularly for low-frequency variants.
  • Accurate SNV detection is crucial across various scientific and clinical fields.

Purpose of the Study:

  • To introduce and validate a novel mutation detection method, the ribonucleotide-modified primer (r-primer) overlapping blocker elongation (PROBE) method.
  • To demonstrate the PROBE method's capability for highly specific and sensitive SNV detection.
  • To evaluate the clinical performance of PROBE assays for specific genetic polymorphisms.

Main Methods:

  • The PROBE method utilizes RNase H2-mediated cleavage of r-primers and blocker-mediated suppression.

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Last Updated: May 25, 2026

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10:41

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  • It employs allele-specific (AS)- and non-AS-r-primers and a peptide nucleic acid (PNA) or locked nucleic acid (LNA) blocker.
  • Competitive binding of AS-r-primer and blocker to the SNV region enables selective amplification of mutant templates.

    • Main Results:

    • The PROBE method achieved a sensitivity of 200 copies per reaction and a selectivity of 0.1%.
    • It demonstrated complete inhibition of wild-type (WT) template amplification.
    • PROBE assays accurately genotyped *ADH1B* rs1229984 (100% accuracy) and *ALDH2* rs671 (99.1% accuracy), surpassing Sanger sequencing.

    Conclusions:

    • The PROBE method offers a significant advancement in SNV detection, providing high specificity and sensitivity.
    • This novel technique effectively distinguishes between wild-type and mutant alleles, minimizing false positives.
    • PROBE assays show high accuracy for genotyping clinically relevant polymorphisms, with potential for broad application in genetic diagnostics.