A laboratory must quantify a rare mutant allele present at approximately 0.1 percent among wild-type copies in a liquid biopsy sample. Which PCR-based method partitions the sample into thousands of individual nanolitre reactions so that mutant molecules can be counted absolutely without a standard curve?

• A Conventional endpoint PCR followed by gel electrophoresis
• B Sanger sequencing of the bulk amplicon
• C Allele-specific oligonucleotide hybridisation on a dot blot
• D Digital droplet PCR ✓

Explanation

Digital droplet PCR splits the sample into tens of thousands of water-in-oil droplets, most containing zero or one target molecule. Each droplet is scored positive or negative by endpoint fluorescence, and Poisson statistics convert the fraction of positive droplets into absolute copy numbers. This partitioning gives the sensitivity needed to detect a 0.1 percent allele. Bulk Sanger sequencing cannot resolve variants below roughly 15 to 20 percent, and endpoint PCR is only semi-quantitative.

Reference: Harper's Illustrated Biochemistry, 32nd ed.

High-yield for: NEET PGINI-CETNExTFMGEUSMLEPLABMRCP

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