Ribonucleotide-Modified Primer Overlapping Blocker Elongation (PROBE) Assay for Highly Specific Mutation Detection

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.
• 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.