Integrated Sample Cleanup−Capillary Electrophoresis Microchip for High-Performance Short Tandem Repeat Genetic Analysis

Abstract

An integrated PCR sample cleanup and preconcentration process is developed for forensic short tandem repeat (STR) analysis using a streptavidin-modified photopolymerized capture gel injector for microchip capillary electrophoresis (microCE). PCR samples generated with one biotinylated primer and one fluorescent primer provide the input to the streptavidin-based affinity capture-microCE device. Monoplex PCR samples processed by the device exhibited approximately 10- to 50-fold increased fluorescence intensities, and DNA profiles generated using 9-plex STR samples displayed approximately 14- to 19-fold higher signal intensities compared to those analyzed using traditional cross injection. Complete STR profiles were obtained with as few as 25 copies of DNA template using the capture-microCE device. Four DNA samples with various degrees of degradation were also tested. Samples analyzed using the capture-microCE device resulted in a significant increase of successful allele detection. The ability of our capture-microCE device and method to remove contaminating ions, to concentrate the sample injection plug, and to eliminate electrokinetic injection bias provides a powerful approach for integrating sample cleanup with DNA separation.

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.…”

“…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. 9,11 In the former case, 14 a proof of concept was achieved with a fluorescently labelled complementary target strand.…”

“…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.…”

“…16 Sample cleanup based on a biotin=streptavidin affinity technique has also been performed. 11,15 Here, streptavidin-modified acrylamide plugs were used to cleanup and preconcentrate products of multiplex amplification of 9 forensically relevant STRs 11 and human respiratory viruses. 15 In each case, the PCR products to be purified were labelled with a biotin tag which later became trapped during electrophoresis through the streptavidin modified hydrogel plugs.…”