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DNA capture-probe based separation of double-stranded polymerase chain reaction amplification products in poly(dimethylsiloxane) microfluidic channels
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10.1063/1.4729131
/content/aip/journal/bmf/6/2/10.1063/1.4729131
http://aip.metastore.ingenta.com/content/aip/journal/bmf/6/2/10.1063/1.4729131

Figures

Image of FIG. 1.
FIG. 1.

Schematic of covalent immobilization of ss-oligonucleotide capture probes in a PDMS microchannel. Note the surface modification scheme is not to scale.

Image of FIG. 2.
FIG. 2.

(a) Schematic of ss-capture probes immobilized on a PDMS surface of a Y-junction microfluidic device, (b) combined fluorescent image and fluorescent signal intensity distribution (distance from the Y-junction to the cross-section is around 500 μm), respectively, after the hybridization of CP #1 with CS #1 and CS #2 within the upper inlet channel, (c) combined fluorescent image and fluorescent signal intensity distribution, respectively, after the hybridization of CP #2 with CS #1 and CS#2 within the lower inlet channel, and (d) combined fluorescent image and fluorescent signal intensity distribution of the “empty” zone (the distance from the Y-junction to the cross-section is around 2000 μm) after the hybridization. Arrows indicate the direction at which the fluorescent signal intensity distributions were measured across.

Image of FIG. 3.
FIG. 3.

Schematic of the production of ds-PCR products tailed with a PEGylated ss-oligonucleotide. At the PCR denaturation stage, both the target DNA and PEGylated hairpin primers are melted. Then at the annealing temperature (approximately 65 °C) hybridization of both reverse and forward hairpin primer’s main sequence with the target DNA occurs and elongation starts. The PEG-linker of the hairpin primer prevents the formation of the complementary strands to the “hybridization” sequence of the primer resulting in a ds-PCR product tailed with a PEGylated ss-oligonucleotide. During the following hybridization the ds-PCR products tailed with a PEGylated ss-oligonucleotide hybridize with immobilized ss-oligonucleotide capture probes. All unreacted hairpin-like primers form “closed” secondary structures at room temperature and are unable to hybridize with the ss-capture probe.

Image of FIG. 4.
FIG. 4.

A gel electropherogram of PCR amplification products with PEGylated hairpin modified forward primers. Lane 1—product of amplification of AMEL gene, lane 3—product of amplification of CSF1PO gene, lane 5—product of multiplex amplification of both AMEL and CSF1PO genes, lanes 2,4,6—negative controls, lane 7—DNA ruler.

Image of FIG. 5.
FIG. 5.

(a) fluorescent image (left) and fluorescent intensity distribution (right, the distance from the Y-junction to the cross-section is around 500 μm), respectively, of the hybridization of AMEL-CP and CSF1PO-CP immobilized onto the upper and lower inlet microchannels, with ss-tailed AMEL and CSF1PO amplification products labelled with Cy5 and FAM dyes, respectively. (b) fluorescent image (left) and fluorescent intensity distribution (right), respectively, of the hybridization of AMEL-CP and CSF1PO-CP immobilized onto the upper and lower inlet microchannels with AMEL and CSF1PO amplification products labelled with FAM and Cy5 dyes, respectively. Arrows indicate the direction at which the fluorescent signal intensity distribution was measured across.

Image of FIG. 6.
FIG. 6.

(a1)-(a3) and (b1-b3) refer to the upper and lower microchannels, respectively, within a single microfluidic devices were (a1)–(a3) show the fluorescent images (left) and fluorescent intensity distributions (the distance from the Y-junction to the cross-section is around 500 μm) across the channels (right) of AMEL-CP immobilized onto the upper inlet microchannel, after hybridization (a1) with complementary ss-tailed AMEL amplification products labelled with Cy5 dye, after denaturation (a2) and after rehybridization with the same amplification product. (a3). (b1)–(b3) show the fluorescent images and fluorescent intensity distributions across the channels (right) of CSF1PO-CP immobilized onto the lower inlet microchannel, after hybridization with complementary ss-tailed CSF1PO amplification products labelled with Cy5 dye (b1), after denaturation (b2) and hybridization with non-complementary ss-tailed AMEL amplification products labelled with Cy5 dye (b3). Arrows indicate the direction at which the fluorescent signal intensity distribution was measured across.

Tables

Generic image for table
Table I.

Sequences and melting temperatures of primers and capture probes.

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/content/aip/journal/bmf/6/2/10.1063/1.4729131
2012-06-12
2014-04-18
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752b84549af89a08dbdd7fdb8b9568b5 journal.articlezxybnytfddd
Scitation: DNA capture-probe based separation of double-stranded polymerase chain reaction amplification products in poly(dimethylsiloxane) microfluidic channels
http://aip.metastore.ingenta.com/content/aip/journal/bmf/6/2/10.1063/1.4729131
10.1063/1.4729131
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