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Droplet centrifugation, droplet DNA extraction, and rapid droplet thermocycling for simpler and faster PCR assay using wire-guided manipulations.

You DJ, Yoon JY - J Biol Eng (2012)

Bottom Line: Following extraction, the 1500 bp sequence of Peptidase D from E. coli was amplified using rapid droplet thermocycling, which took 10 min for 30 cycles.The results of these sequentially executed processes were analyzed using gel electrophoresis.Thus, this work demonstrates the adaptability of the system to replace many common laboratory tasks on a single platform (through re-programmability), in rapid succession (using droplets), and with a high level of accuracy and automation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Agricultural and Biosystems Engineering, The University of Arizona, Tucson, AZ 85721-0038, USA. jyyoon@email.arizona.edu.

ABSTRACT
A computer numerical control (CNC) apparatus was used to perform droplet centrifugation, droplet DNA extraction, and rapid droplet thermocycling on a single superhydrophobic surface and a multi-chambered PCB heater. Droplets were manipulated using "wire-guided" method (a pipette tip was used in this study). This methodology can be easily adapted to existing commercial robotic pipetting system, while demonstrated added capabilities such as vibrational mixing, high-speed centrifuging of droplets, simple DNA extraction utilizing the hydrophobicity difference between the tip and the superhydrophobic surface, and rapid thermocycling with a moving droplet, all with wire-guided droplet manipulations on a superhydrophobic surface and a multi-chambered PCB heater (i.e., not on a 96-well plate). Serial dilutions were demonstrated for diluting sample matrix. Centrifuging was demonstrated by rotating a 10 μL droplet at 2300 round per minute, concentrating E. coli by more than 3-fold within 3 min. DNA extraction was demonstrated from E. coli sample utilizing the disposable pipette tip to cleverly attract the extracted DNA from the droplet residing on a superhydrophobic surface, which took less than 10 min. Following extraction, the 1500 bp sequence of Peptidase D from E. coli was amplified using rapid droplet thermocycling, which took 10 min for 30 cycles. The total assay time was 23 min, including droplet centrifugation, droplet DNA extraction and rapid droplet thermocycling. Evaporation from of 10 μL droplets was not significant during these procedures, since the longest time exposure to air and the vibrations was less than 5 min (during DNA extraction). The results of these sequentially executed processes were analyzed using gel electrophoresis. Thus, this work demonstrates the adaptability of the system to replace many common laboratory tasks on a single platform (through re-programmability), in rapid succession (using droplets), and with a high level of accuracy and automation.

No MeSH data available.


Related in: MedlinePlus

Wire-guided droplet manipulator apparatus. Modular design includes PCR chamber for rapid-PCR thermocycling and a superhydrophobic surface for serial dilution, centrifugation, and DNA extraction.
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Related In: Results  -  Collection

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Figure 1: Wire-guided droplet manipulator apparatus. Modular design includes PCR chamber for rapid-PCR thermocycling and a superhydrophobic surface for serial dilution, centrifugation, and DNA extraction.

Mentions: To demonstrate precise sample handling in a reconfigurable manner, serial dilutions were performed to simulate a diluted bacterial sample. This diluted sample was then re-concentrated using wire-guided droplet centrifugation, which only the target is concentrated while culture media (and potentially other sample matrices) is diluted, thus effectively eliminating much of culture media or sample matrices. The genetic material was extracted using the same droplet manipulation assay surface with only a different pre-programmed algorithm, demonstrating the ease of reconfigurability of the system. Afterwards, the DNA was amplified using rapid droplet thermocycling with wire-guided droplet manipulation. Figure 1 shows the entire apparatus to perform all the aforementioned protocols. The modular base allows for easy repositioning and addition of further components to the system. While only a single confluent assay was demonstrated, the ability to run multiple assays simultaneously, utilizing the necessary delays between steps in the protocols, could easily be implemented.


Droplet centrifugation, droplet DNA extraction, and rapid droplet thermocycling for simpler and faster PCR assay using wire-guided manipulations.

You DJ, Yoon JY - J Biol Eng (2012)

Wire-guided droplet manipulator apparatus. Modular design includes PCR chamber for rapid-PCR thermocycling and a superhydrophobic surface for serial dilution, centrifugation, and DNA extraction.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3526397&req=5

Figure 1: Wire-guided droplet manipulator apparatus. Modular design includes PCR chamber for rapid-PCR thermocycling and a superhydrophobic surface for serial dilution, centrifugation, and DNA extraction.
Mentions: To demonstrate precise sample handling in a reconfigurable manner, serial dilutions were performed to simulate a diluted bacterial sample. This diluted sample was then re-concentrated using wire-guided droplet centrifugation, which only the target is concentrated while culture media (and potentially other sample matrices) is diluted, thus effectively eliminating much of culture media or sample matrices. The genetic material was extracted using the same droplet manipulation assay surface with only a different pre-programmed algorithm, demonstrating the ease of reconfigurability of the system. Afterwards, the DNA was amplified using rapid droplet thermocycling with wire-guided droplet manipulation. Figure 1 shows the entire apparatus to perform all the aforementioned protocols. The modular base allows for easy repositioning and addition of further components to the system. While only a single confluent assay was demonstrated, the ability to run multiple assays simultaneously, utilizing the necessary delays between steps in the protocols, could easily be implemented.

Bottom Line: Following extraction, the 1500 bp sequence of Peptidase D from E. coli was amplified using rapid droplet thermocycling, which took 10 min for 30 cycles.The results of these sequentially executed processes were analyzed using gel electrophoresis.Thus, this work demonstrates the adaptability of the system to replace many common laboratory tasks on a single platform (through re-programmability), in rapid succession (using droplets), and with a high level of accuracy and automation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Agricultural and Biosystems Engineering, The University of Arizona, Tucson, AZ 85721-0038, USA. jyyoon@email.arizona.edu.

ABSTRACT
A computer numerical control (CNC) apparatus was used to perform droplet centrifugation, droplet DNA extraction, and rapid droplet thermocycling on a single superhydrophobic surface and a multi-chambered PCB heater. Droplets were manipulated using "wire-guided" method (a pipette tip was used in this study). This methodology can be easily adapted to existing commercial robotic pipetting system, while demonstrated added capabilities such as vibrational mixing, high-speed centrifuging of droplets, simple DNA extraction utilizing the hydrophobicity difference between the tip and the superhydrophobic surface, and rapid thermocycling with a moving droplet, all with wire-guided droplet manipulations on a superhydrophobic surface and a multi-chambered PCB heater (i.e., not on a 96-well plate). Serial dilutions were demonstrated for diluting sample matrix. Centrifuging was demonstrated by rotating a 10 μL droplet at 2300 round per minute, concentrating E. coli by more than 3-fold within 3 min. DNA extraction was demonstrated from E. coli sample utilizing the disposable pipette tip to cleverly attract the extracted DNA from the droplet residing on a superhydrophobic surface, which took less than 10 min. Following extraction, the 1500 bp sequence of Peptidase D from E. coli was amplified using rapid droplet thermocycling, which took 10 min for 30 cycles. The total assay time was 23 min, including droplet centrifugation, droplet DNA extraction and rapid droplet thermocycling. Evaporation from of 10 μL droplets was not significant during these procedures, since the longest time exposure to air and the vibrations was less than 5 min (during DNA extraction). The results of these sequentially executed processes were analyzed using gel electrophoresis. Thus, this work demonstrates the adaptability of the system to replace many common laboratory tasks on a single platform (through re-programmability), in rapid succession (using droplets), and with a high level of accuracy and automation.

No MeSH data available.


Related in: MedlinePlus