Quantitative polymerase chain reaction using a recombinant DNA internal standard and time-resolved fluorometry

Area of Science:

• Molecular Biology
• Biochemistry
• Analytical Chemistry

Background:

• Quantitative polymerase chain reaction (PCR) is crucial for DNA analysis.
• Reaction-to-reaction variability can affect the accuracy of quantitative PCR (qPCR).
• Internal standards are used to normalize qPCR results and improve precision.

Purpose of the Study:

• To develop and validate a novel internal standard (IS) for quantitative PCR (qPCR).
• To assess the performance of two distinct hybridization-based detection methods for qPCR using the IS.

Main Methods:

• Synthesized a 308 bp DNA fragment as an internal standard (IS) differing by a 26 bp sequence from the target DNA.
• Coamplified target DNA with a constant amount of IS using quantitative PCR (qPCR).
• Assayed PCR products using two hybridization protocols (QPCR-1 and QPCR-2) with digoxigenin-labeled probes and time-resolved fluorometry detection.

Main Results:

• The internal standard (IS) effectively compensated for amplification efficiency variability in qPCR.
• Both QPCR-1 and QPCR-2 demonstrated linear quantification ranges from 1000-200,000 and 2000-200,000 molecules, respectively.
• Coefficient of variations (CVs) ranged from 3.4% to 9.7%, indicating good assay precision.

Conclusions:

• The developed internal standard (IS) allows for accurate and precise DNA quantification using qPCR.
• The two hybridization-based detection methods are suitable for quantifying DNA in the presence of an internal standard.
• This approach enhances the reliability of molecular diagnostic and research applications.