Integrated affinity capture, purification, and capillary electrophoresis microdevice for quantitative double-stranded DNA analysis.
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.
Toriello
Nicholas M
NM
UCSF/UC Berkeley Joint Graduate Group in Bioengineering, University of California, Berkeley, California 94720, USA.
Liu
Chung N
CN
Blazej
Robert G
RG
Thaitrong
Numrin
N
Mathies
Richard A
RA
D004247:DNA / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification*; D019075:Electrophoresis, Capillary / Q000295:instrumentation* / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics
D004247:DNA