inPCR

InPCR, commonly referred to as in situ PCR, is a molecular technique that combines polymerase chain reaction with in situ hybridization to detect and amplify nucleic acid sequences directly within fixed cells or tissue sections. The method preserves spatial information, enabling localization of DNA or RNA targets in their native cellular context.

Principle: Samples are fixed to preserve morphology and permeabilized to allow access for enzymes and primers. Specific primers targeting the sequence of interest are applied along with DNA polymerase and nucleotides. PCR amplification occurs within the tissue matrix, producing localized signals that can be detected by fluorescence or chromogenic methods after amplification.

Procedure: Following fixation, specimens undergo permeabilization and often proteolytic treatment to expose nucleic acids. Primers and polymerase are added under conditions suitable for in situ amplification. After cycling, detection is performed using labeled probes or by visualizing amplified products with microscopy, sometimes requiring post-amplification hybridization steps and additional washes.

Applications: InPCR is used to map gene copy number or expression at cellular resolution, detect microdeletions or duplications within tissue, identify pathogens in histological sections, and localize transcripts directly in their tissue context.

Limitations: The technique can be prone to artefacts from nonspecific amplification or diffusion of reagents, and depends heavily on tissue permeability, fixation quality, and primer design. Quantitation is challenging, and results require rigorous controls and optimization.

History and status: InPCR emerged in the late 20th century as a method to combine localization with amplification. It remains useful in certain niche applications and educational settings, though many laboratories prefer separate in situ hybridization and PCR approaches for routine use.