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DNA separation and ...
DNA separation and fluorescent detection in an optofluidic chip with sub-base-pair resolution
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- Pollnau, Markus (författare)
- KTH,Material- och nanofysik,Univ Twente, Netherlands
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Hammer, Manfred (författare)
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Dongre, Chaitanya (författare)
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Hoekstra, Hugo J. W. M. (författare)
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(creator_code:org_t)
- SPIE - International Society for Optical Engineering, 2015
- 2015
- Engelska.
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Ingår i: Microfluidics, BioMEMS, and Medical Microsystems XIII. - : SPIE - International Society for Optical Engineering. - 9781628414103
- Relaterad länk:
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https://urn.kb.se/re...
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https://doi.org/10.1...
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Abstract
Ämnesord
Stäng
- DNA sequencing in a lab-on-a-chip aims at providing cheap, high-speed analysis of low reagent volumes to, e.g., identify genomic deletions or insertions associated with genetic illnesses. Detecting single base-pair insertions/deletions from DNA fragments in the diagnostically relevant range of 150-1000 base-pairs requires a sizing accuracy of S < 10(-3). Here we demonstrate S = 4x10(-4). A microfluidic chip was post-processed by femtosecond-laser writing of an optical waveguide. 12 blue-labeled and 23 red-labeled DNA fragments were separated in size by capillary electrophoresis, each set excited by either of two lasers power-modulated at different frequencies, their fluorescence detected by a photomultiplier, and blue/red signals distinguished by Fourier analysis. Different calibration strategies were tested: a) use either set of DNA molecules as reference to calibrate the set-up and identify the base-pair sizes of the other set in the same flow experiment, thereby eliminating variations in temperature, wall-coating and sieving-gel conditions, and actuation voltages; b) use the same molecular set as reference and sample with the same fluorescence label, flown in consecutive experiments; c) perform cross-experiments based on different molecular sets with different labels, flown in consecutive experiments. From the results we conclude: Applying quadratic instead of linear fit functions improves the calibration accuracy. Blue-labeled molecules are separated with higher accuracy. The influence of dye label is higher than fluctuations between two experiments. Choosing a single, suitable dye label combined with reference calibration and sample investigation in consecutive experiments results in S = 4x10(-4), enabling detection of single base-pair insertion/deletion in a lab-on-a-chip.
Ämnesord
- TEKNIK OCH TEKNOLOGIER -- Annan teknik (hsv//swe)
- ENGINEERING AND TECHNOLOGY -- Other Engineering and Technologies (hsv//eng)
Nyckelord
- DNA separation
- optofluidic chip
- capillary electrophoresis
- fluorescent detection
- single base-pair insertion/deletion
- sub-base-pair sizing accuracy
Publikations- och innehållstyp
- ref (ämneskategori)
- kon (ämneskategori)
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