Integrated affinity capture, purification, and capillary electrophoresis microdevice for quantitative double-stranded...

Area of Science:

• Biochemistry
• Analytical Chemistry
• Molecular Biology

Background:

• Quantitative analysis of DNA, particularly double-stranded DNA (dsDNA) like PCR amplicons, is crucial in molecular biology.
• Traditional methods for DNA concentration, purification, and injection in capillary electrophoresis (CE) can be inefficient and introduce bias.
• There is a need for improved methods to handle small sample volumes and achieve high sensitivity in DNA quantification.

Purpose of the Study:

• To develop and validate a novel injection method for quantitative microchip capillary electrophoresis analysis of dsDNA.
• To utilize a thermally switchable oligonucleotide affinity capture gel for simultaneous concentration, purification, and injection of DNA targets.
• To demonstrate the efficiency and sensitivity of this method for analyzing PCR amplicons and other DNA fragments.

Main Methods:

• Development of a thermally switchable affinity capture matrix using an oligonucleotide-modified polyacrylamide gel.
• Integration of the capture matrix directly into the CE separation channel.
• Utilizing helix invasion for capturing dsDNA targets and thermal release for injection.
• Performing dsDNA titration assays and dilution studies to determine limit of detection and capture efficiency.
• Demonstrating multiplex target capture using a mixture of capture probes.

Main Results:

• The oligonucleotide affinity capture gel efficiently captures both single-stranded DNA (ssDNA) and dsDNA, including PCR amplicons.
• Optimal capture efficiency was achieved at 44°C and 100 V/cm with a 20 µM affinity capture probe.
• A limit of detection of 1 pM was achieved for dsDNA from 250 nL of solution.
• Simultaneous capture and injection of amplicons from different bacterial sources (E. coli K12 and M13mp18) demonstrated multiplexing capability.
• The method showed comparable capture efficiency for dsDNA and ssDNA.

Conclusions:

• The novel thermally switchable oligonucleotide affinity capture gel provides an efficient and unbiased method for concentrating, purifying, and injecting dsDNA for quantitative CE.
• This integrated approach significantly enhances the sensitivity and quantitative accuracy of DNA analysis, particularly for nanoliter-scale samples.
• The method's ability to perform multiplex target capture facilitates the analysis of complex DNA samples and products from integrated PCR reactors.