A proportional analysis method using non-kinetic real-time PCR.

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• Increasing interest in proportional analysis of genetic types necessitates efficient quantification methods.
• Existing techniques may require multiple steps or separate assays for target and total gene quantification.

Purpose of the Study:

• To develop a simple, single-tube assay for determining the ratio of a specific target gene within a total gene pool.
• To enable proportional analysis of genetic types with enhanced efficiency.

Main Methods:

• A single-tube real-time PCR system was designed.
• Task 1: Amplification of total genes (including target) using a labeled PCR primer, with real-time monitoring of product copy number.
• Task 2: Detection of the target gene signal using a labeled nucleotide probe during each amplification cycle.

Main Results:

• The assay successfully integrates total gene amplification monitoring and target gene detection in one tube.
• The ratio of target gene to total genes is determined by comparing signals from Task 2 to a prescribed product threshold from Task 1.
• This method provides a straightforward approach for proportional genetic analysis.

Conclusions:

• The developed assay offers a simple and efficient method for determining target gene ratios within total gene populations.
• This technique facilitates proportional genetic analysis using a single-tube real-time PCR system.
• The method combines PCR amplification and target detection for streamlined genetic quantification.