How to Measure Gene Activity Using Competitive PCR Kits

A laboratory method and kit for accurately measuring how much a specific gene is being used by cells by comparing it against a known internal standard.

What it covers

This patent describes a kit used to quantify gene expression in small biological samples. It works by performing a multiplex reaction, meaning it amplifies both a target gene and a 'housekeeping' gene (a gene that is always active) at the same time. The kit includes competitive templates for both genes, which are nearly identical to the natural genes but contain a small point mutation. By adding these templates, researchers can compare the amount of product from the natural gene against the known amount of the competitive template, allowing for a precise calculation of how much of the target gene was originally present.

What it doesn't cover

• —Does not cover non-competitive PCR methods where no internal standard template is used.
• —Does not cover gene expression measurement techniques that rely on sequencing or hybridization arrays.
• —Does not cover the use of competitive templates that differ from the target by more than a point mutation.
• —Does not cover methods that amplify the target and housekeeping genes in separate, non-multiplexed reactions.

The clever bit

By using a competitive template that is nearly identical to the target gene, the method ensures that both the target and the competitor compete equally for the same primers and enzymes, effectively canceling out variations in reaction efficiency.

Why it matters

Before this method, quantifying gene expression in tiny tissue samples was notoriously difficult and prone to error. This approach provided a standardized way to normalize data using housekeeping genes, which became a staple technique in molecular biology research for studying diseases like cancer.

Real-world examples

• 1.Oncology research for measuring tumor gene expression
• 2.Molecular diagnostic assays
• 3.Gene expression profiling in clinical biopsies