Calibration-curve-free quantitative PCR: a quantitative method for specific nucleic acid sequences without using...

Area of Science:

• Molecular Biology
• Biotechnology
• Analytical Chemistry

Background:

• Accurate quantification of specific nucleic acid sequences is crucial for various applications, including genetic analysis and quality control.
• Traditional methods often require complex calibration curves, limiting their speed and applicability in field settings.
• Developing rapid, sensitive, and calibration-free methods for nucleic acid quantification remains a significant challenge.

Purpose of the Study:

• To develop a simple, quantitative method for specific nucleic acid sequences that eliminates the need for calibration curves.
• To combine competitive polymerase chain reaction (PCR) with fluorescence quenching for enhanced detection sensitivity and accuracy.
• To validate the method's efficacy by quantifying recombinant DNA in genetically modified (GM) soybean samples.

Main Methods:

• Utilized competitive polymerase chain reaction (PCR) to amplify both a target gene and an internal standard (competitor).
• Employed a sequence-specific fluorescent probe whose fluorescence is quenched by guanine bases upon hybridization with the target, but not with the competitor.
• Measured fluorescence intensities before and after PCR to determine the quench rate, which is proportional to the target-to-competitor ratio.

Main Results:

• Demonstrated that the quench rate directly correlates with the ratio of target to competitor DNA.
• Successfully calculated the target-to-competitor ratio from the quench rate without requiring a calibration curve.
• Quantified the copy number of recombinant DNA in GM soybean samples, accurately estimating GM soybean content.

Conclusions:

• The developed method provides a simple and accurate way to quantify specific nucleic acid sequences without calibration curves.
• The combination of competitive PCR and fluorescence quenching offers a powerful tool for nucleic acid quantification.
• This technique is particularly suitable for rapid field tests, such as detecting specific gene contamination in food products like GM soybean.