Integrated sample cleanup-capillary electrophoresis microchip for high-performance short tandem repeat genetic analysis.
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.
Yeung
Stephanie H I
SH
UCSF/UCB Joint Graduate Group in Bioengineering, University of California, Berkeley, California 94720, USA.
Liu
Peng
P
Del Bueno
Nadia
N
Greenspoon
Susan A
SA
Mathies
Richard A
RA
D001710:Biotin / Q000737:chemistry; D004247:DNA / Q000032:analysis / Q000235:genetics; D004277:DNA, Single-Stranded / Q000737:chemistry; D019075:Electrophoresis, Capillary / Q000379:methods*; D018895:Microsatellite Repeats; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods; D017422:Sequence Analysis, DNA / Q000379:methods*; D019809:Streptavidin / Q000737:chemistry
D004277:DNA, Single-Stranded; D001710:Biotin; D004247:DNA; D019809:Streptavidin