Digital PCR on a SlipChip
Abstract
This paper describes a SlipChip to perform digital PCR in a very simple and inexpensive format. The fluidic path for introducing the sample combined with the PCR mixture was formed using elongated wells in the two plates of the SlipChip designed to overlap during sample loading. This fluidic path was broken up by simple slipping of the two plates that removed the overlap among wells and brought each well in contact with a reservoir preloaded with oil to generate 1280 reaction compartments (2.6 nL each) simultaneously. After thermal cycling, end-point fluorescence intensity was used to detect the presence of nucleic acid. Digital PCR on the SlipChip was tested quantitatively by using Staphylococcus aureus genomic DNA. As the concentration of the template DNA in the reaction mixture was diluted, the fraction of positive wells decreased as expected from the statistical analysis. No cross-contamination was observed during the experiments. At the extremes of the dynamic range of digital PCR the standard confidence interval determined using a normal approximation of the binomial distribution is not satisfactory. Therefore, statistical analysis based on the score method was used to establish these confidence intervals. The SlipChip provides a simple strategy to count nucleic acids by using PCR. It may find applications in research applications such as single cell analysis, prenatal diagnostics, and point-of-care diagnostics. SlipChip would become valuable for diagnostics, including applications in resource-limited areas after integration with isothermal nucleic acid amplification technologies and visual readout.
Additional Information
© Royal Society of Chemistry 2010. Received 22nd March 2010, Accepted 9th June 2010. First published on the web 1st July 2010. This work was supported by ONR grant No. N00014-08-1-0936, the Camille Dreyfus Teacher-Scholar Awards program, the Chicago Center for Systems Biology (funded by the National Institute of General Medical Sciences at the NIH), and by the NIH Director's Pioneer Award program, part of the NIH Roadmap for Medical Research (1 DP1 OD003584). Part of this work was performed at the Materials Research Science and Engineering Centers microfluidics facility (funded by the National Science Foundation). We thank Elena M. Lucchetta for additional experimental validation and Elena K. Davydova for helpful suggestions in experimental design. We thank Heidi Park for contributions to writing and editing this manuscript.Attached Files
Published - Ismagilov_LOC_2010_digital_PCR_FS_JEK_10_2666_2672.pdf
Accepted Version - nihms216770.pdf
Supplemental Material - Ismagilov_LOC_2010_digital_PCR_FS_JEK_10_2666_2672_supp_info.pdf
Cover Image - Ismagilov_LOC_2010_digital_PCR_FS_JEK_inside_front_cover.pdf
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Additional details
- PMCID
- PMC2948063
- Eprint ID
- 40865
- Resolver ID
- CaltechAUTHORS:20130821-160730931
- N00014-08-1-0936
- Office of Naval Research (ONR)
- Camille and Henry Dreyfus Foundation
- National Institute of General Medical Sciences
- 1DP1 OD003584
- NIH
- NSF
- Created
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2013-08-26Created from EPrint's datestamp field
- Updated
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2021-11-10Created from EPrint's last_modified field