Quantitative method for specific nucleic acid sequences using competitive polymerase chain reaction with an...

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• Accurate quantification of specific nucleic acid sequences is crucial in molecular biology.
• Existing methods like real-time PCR can be complex and require expensive equipment.
• The presence of PCR inhibitors in biological samples poses a significant challenge for DNA quantification.

Purpose of the Study:

• To develop a simple, cost-effective, and accurate method for nucleic acid quantification.
• To overcome limitations of existing methods, particularly in the presence of PCR inhibitors.
• To introduce alternately binding probe competitive PCR (ABC-PCR) as a viable alternative.

Main Methods:

• Developed alternately binding probe competitive PCR (ABC-PCR).
• Coamplified target and competitor nucleic acid sequences with a sequence-specific fluorescent probe (ABProbe).
• Measured fluorescence intensity at the end-point to calculate the target-to-competitor ratio using fluorescence quenching and FRET.

Main Results:

• ABC-PCR demonstrated sensitivity, accuracy, and precision comparable to real-time PCR.
• The method successfully quantified DNA even in the presence of PCR inhibitors like humic acid.
• End-point fluorescence measurement enabled direct calculation of the target quantity, eliminating complex post-PCR steps.

Conclusions:

• ABC-PCR is a simple, cost-effective, and accurate method for nucleic acid quantification.
• This technique is robust and performs well even with challenging biological samples containing PCR inhibitors.
• ABC-PCR offers a valuable alternative to real-time PCR for DNA quantification applications.