Colorimetric quantification of in vitro-amplified template DNA to be used for solid phase sequencing

Area of Science:

• Molecular Biology
• Biotechnology
• Analytical Chemistry

Background:

• Accurate quantification of DNA is essential for molecular biology applications.
• Existing methods for DNA determination can be time-consuming and complex.
• Solid-phase immobilization offers advantages for DNA manipulation and analysis.

Purpose of the Study:

• To describe a novel colorimetric protocol for determining DNA amplified by polymerase chain reaction (PCR).
• To evaluate the suitability of the protocol for both manual and automated workflows.
• To assess the impact of template concentration on DNA sequencing quality.

Main Methods:

• DNA amplification using PCR.
• Immobilization of PCR products onto magnetic beads via a lac operator-DNA sequence.
• Binding of a Lac repressor-beta-galactosidase fusion protein to the immobilized DNA.
• Colorimetric detection of the bound beta-galactosidase enzyme.

Main Results:

• A three-step protocol was developed for colorimetric DNA determination.
• Steps for DNA binding and protein complex formation can be performed concurrently.
• The immobilized DNA template is suitable for direct use in solid-phase sequencing after melting.
• Template concentration was demonstrated to be a critical factor affecting automated DNA sequencer data quality.

Conclusions:

• The described colorimetric assay provides an efficient method for quantifying PCR-amplified DNA.
• The protocol is adaptable to both manual and automated systems.
• Optimizing template concentration is vital for achieving high-quality DNA sequence data.