Integrated Affinity Capture, Purification, and Capillary Electrophoresis Microdevice for Quantitative Double-Stranded DNA Analysis

Abstract

A novel injection method is developed that utilizes a thermally switchable oligonucleotide affinity capture gel to mediate the concentration, purification, and injection of dsDNA for quantitative microchip capillary electrophoresis analysis. The affinity capture matrix consists of a 20 base acrydite modified oligonucleotide copolymerized into a 6% linear polyacrylamide gel that captures ssDNA or dsDNA analyte including PCR amplicons and synthetic oligonucleotides. Double stranded PCR amplicons with complementarity to the capture probe up to 81 bases from their 5' terminus are reproducibly captured via helix invasion. By integrating the oligo capture matrix directly with the CE separation channel, the electrophoretically mobilized target fragments are quantitatively captured and injected after thermal release for unbiased, efficient, and quantitative analysis. The capture process exhibits optimal efficiency at 44 degrees C and 100 V/cm with a 20 microM affinity capture probe (TM = 57.7 degrees C). A dsDNA titration assay with 20 bp fragments validated that dsDNA is captured at the same efficiency as ssDNA. Dilution studies with a duplex 20mer show that targets can be successfully captured and analyzed with a limit of detection of 1 pM from 250 nL of solution (approximately 150,000 fluorescent molecules). Simultaneous capture and injection of amplicons from E. coli K12 and M13mp18 using a mixture of two different capture probes demonstrates the feasibility of multiplex target capture. Unlike the traditional cross-injector, this method enables efficient capture and injection of dsDNA amplicons which will facilitate the quantitative analysis of products from integrated nanoliter-scale PCR reactors.

Integrated microfluidic bioprocessor for single-cell gene expression analysis

“…In the reactor region, single cells are captured, lysed, and the mRNA of interest is reverse transcribed and amplified by RT-PCR (22). The affinity capture region comprises a hold chamber that acts as a reservoir and a capture chamber where amplicons are immobilized, purified, and concentrated in an affinity capture gel matrix (23). Finally, the system contains a CE separation channel for the size-based separation and quantitation of products.…”

DNA capture-probe based separation of double-stranded polymerase chain reaction amplification products in poly(dimethylsiloxane) microfluidic channels

“…9,11,15 One approach is the biotin-streptavidin affinity system which facilitates the binding of a capture probe and a target probe with very high affinity and strong bonding. 24 However, a drawback of this approach is that it is nearly impossible to release the target probe in a time resolved regime and there is always interference from the unreacted biotin-labelled primers with the strepavidin-modified capture support.…”

“…[8][9][10][11][12] Many strategies are based on a common technique which involves both the concentration of PCR products and separation from unreacted primers, nucleotide triphosphates, and dyes. In these cases, linear or crosslinked acrylamide hydrogel plugs located between the sample and waste channels in a classic double T-junction CE microfluidic system 13 are used.…”

“…In these cases, linear or crosslinked acrylamide hydrogel plugs located between the sample and waste channels in a classic double T-junction CE microfluidic system 13 are used. Typically, the hydrogel plugs are covalently modified with either short oligonucleotide capture probes 9,14 or with streptavidin. 11,15 Oligonucleotide affinity preconcentration=purification has been demonstrated for both synthetic single-stranded (ss) oligonucleotides and ss-products of sequencing reactions, 8,14 as well as double-stranded (ds) PCR products.…”

“…11,15 Oligonucleotide affinity preconcentration=purification has been demonstrated for both synthetic single-stranded (ss) oligonucleotides and ss-products of sequencing reactions, 8,14 as well as double-stranded (ds) PCR products. 9,11 In the former case, 14 a proof of concept was achieved with a fluorescently labelled complementary target strand. The authors 14 also showed separation of a mixture of ss-oligonucleotides after stacking two hydrogel plugs with different capture probes.…”