Name: amplification of escherichia coli in a continuous-flow-pcr microfluidic chip and its detection with a capillary electrophoresis system
Uploaded: 2023-11-21T12:00:00
Description: Watch this Scientific Journal Video about Amplification of Escherichia coli in a Continuous-Flow-PCR Microfluidic Chip and Its Detection with a Capillary Electrophoresis System at JoVE.com

This work was supported by the Science and Technology Commission of Shanghai Municipality, China (No. 19ZR1477500 and No.18441900400). We gratefully acknowledge financial support from the University of Shanghai for Science and Technology (No.2017KJFZ049).

NOTE: See the Table of Materials for details related to all materials, reagents, and equipment used in this protocol.
1. Fabrication of CF-PCR microfluidic chip
<ol>
	<li>Heat the silicon wafer at 200 &#176;C for 25 min to remove the moisture.</li>
	<li>Dispense 1 mL of SU-8-2075 photoresist per inch of the wafer. Spin it on the silicon wafer using a spin coater at 500 rpm for 5-10 s with an acceleration of 100 rpm/s, and then at 2,000 rpm for 30 s with an a...

<ol>
	<li>Li, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=All-in-one+microfluidic+device+for+on-site+diagnosis+of+pathogens+based+on+an+integrated+continuous+flow+PCR+and+electrophoresis+biochip.">All-in-one microfluidic device for on-site diagnosis of pathogens based on an integrated continuous flow PCR and electrophoresis biochip.</a> Lab on a Chip. 19 (16), 2663-2668 (2019).</li><li>Crews, N., Wittwer, C., Gale, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous-flow+thermal+gradient+PCR.">Continuous-flow thermal gradient PCR.</a> Biomedical Microdevices. 10 (2), 187-195 (2008).</li><li>Li, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+fabrication+of+portable+continuous+flow+PCR+microfluidic+chip+for+DNA+replication.">Design and fabrication of portable continuous flow PCR microfluidic chip for DNA replication.</a> Biomedical Microdevices. 22 (1), 5 (2019).</li><li>Kim, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+and+characterization+of+a+PDMS-glass+hybrid+continuous-flow+PCR+chip.">Fabrication and characterization of a PDMS-glass hybrid continuous-flow PCR chip.</a> Biochemical Engineering Journal. 29 (1-2), 91-97 (2006).</li><li>Shen, K., Chen, X., Guo, M., Cheng, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microchip-based+PCR+device+using+flexible+printed+circuit+technology.">A microchip-based PCR device using flexible printed circuit technology.</a> Sensors and Actuators B: Chemical. 105 (2), 251-258 (2005).</li><li>Harstad, R. K., Johnson, A. C., Weisenberger, M. M., Bowser, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capillary+Electrophoresis.">Capillary Electrophoresis.</a> Analytical Chemistry. 88 (1), 299-319 (2016).</li><li>Redman, E. A., Mellors, J. S., Starkey, J. A., Ramsey, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+intact+antibody+drug+conjugate+variants+using+microfluidic+capillary+electrophoresis-mass+spectrometry.">Characterization of intact antibody drug conjugate variants using microfluidic capillary electrophoresis-mass spectrometry.</a> Analytical Chemistry. 88 (4), 2220-2226 (2016).</li><li>Britz-Mckibbin, P., Kranack, A. R., Paprica, A., Chen, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+assay+for+epinephrine+in+dental+anesthetic+solutions+by+capillary+electrophoresis.">Quantitative assay for epinephrine in dental anesthetic solutions by capillary electrophoresis.</a> Analyst. 123 (7), 1461-1463 (1998).</li><li>Maeda, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+real-time+PCR+using+TaqMan+and+SYBR+Green+for+Actinobacillus+actinomycetemcomitans,+Porphyromonas+gingivalis,+Prevotella+intermedia,+tetQgene+and+total+bacteria.">Quantitative real-time PCR using TaqMan and SYBR Green for Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, tetQgene and total bacteria.</a> FEMS Immunology and Medical Microbiology. 39 (1), 81-86 (2003).</li><li>Hajba, L., Guttman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+column+coatings+for+capillary+electrophoresis+of+proteins.">Recent advances in column coatings for capillary electrophoresis of proteins.</a> TrAC Trends in Analytical Chemistry. 90, 38-44 (2017).</li><li>Kleparnik, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+combination+of+capillary+electrophoresis+with+mass+spectrometry:+methodology+and+theory.">Recent advances in combination of capillary electrophoresis with mass spectrometry: methodology and theory.</a> Electrophoresis. 36 (1), 159-178 (2015).</li></ol>

Both PCR and CE are two popular biotechnologies in the analysis of nucleic acids. This paper describes the amplification of E. coli and the detection of the PCR products using the CF-PCR and CE systems, both built in-house. The target gene of E. coli was successfully amplified within 10 min because of the high heat transfer rates. The DNA fragments smaller than 1,500 bp were separated within 8 min (Figure 5). The great advantage of these two techniques is that it can greatl...

Polymerase chain reaction (PCR) is a molecular biology technique used to amplify specific DNA fragments, thereby amplifying trace amounts of DNA hundreds of millions of times. It has been widely used in clinical diagnosis, medical research, food safety, forensic identification, and other fields. The PCR process mainly consists of three steps: denaturation at 90-95 &#176;C, annealing at 50-60 &#176;C, and extension at 72-77 &#176;C. Thermal cycling is an important part of the PCR process; however, the traditional PCR thermal cycler is not only bulky but also inefficient, requiring approximately 40 min to complete 25 cycles. To overcome these limitations, a continuous-flow PCR (CF-PCR) system was built in-house, based on a microfluidic chip. CF-PCR can greatly save time by driving the PCR solution into microchannels placed on heaters at different temperatures1,2,3,4,5.
As capillary electrophoresis (CE) has many advantages, such as high resolution, high speed, and excellent reproducibility6,7,8,9,10,11, it has become a popular tool in the lab for the analysis of nucleic acids and proteins. However, most labs, especially labs in the developing world, cannot afford this technology because of the high price of the CE instrument. Herein, we have outlined protocols for how to fabricate the CF-PCR microfluidic chip and how to build a versatile CE system in the lab. We also demonstrate the process of amplification of E. coli by this CF-PCR system and the detection of the PCR products by the CE system. By following the procedures described in this protocol, users should be able to fabricate microfluidic chips, prepare PCR solutions, build a CF-PCR system for nucleic acid amplification, and set up a simple CE system, even with limited resources, to separate DNA fragments.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>100 bp DNA ladder</td>
			<td>Takara Bio Inc.</td>
			<td>3422A</td>
			<td></td>
		</tr>
		<tr>
			<td>10x Fast Buffer I</td>
			<td>Takara Bio Inc.</td>
			<td>RR070A</td>
			<td></td>
		</tr>
		<tr>
			<td>10x TBE</td>
			<td>Beijing Solarbio Science &#38; Technology Co., Ltd.</td>
			<td>T1051</td>
			<td></td>
		</tr>
		<tr>
			<td>developer solution</td>
			<td>Alfa Aesar, USA</td>
			<td>L15459</td>
			<td></td>
		</tr>
		<tr>
			<td>dNTP mixture (2.5 &#956;M)</td>
			<td>Takara Bio Inc.</td>
			<td>RR070A</td>
			<td></td>
		</tr>
		<tr>
			<td>EC-F</td>
			<td>Sangon Biotech, Shanghai, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>EC-R</td>
			<td>Sangon Biotech, Shanghai, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HEC,1300K</td>
			<td>Sigma-Aldrich, USA</td>
			<td>9004-62-0</td>
			<td></td>
		</tr>
		<tr>
			<td>isopropanol</td>
			<td>Aladdin, Shanghai, China</td>
			<td>67-63-0</td>
			<td></td>
		</tr>
		<tr>
			<td>microscope</td>
			<td>Olympus, Japan</td>
			<td>BX51</td>
			<td></td>
		</tr>
		<tr>
			<td>photolithography&#160;</td>
			<td>SUSS MicroTec, Germany</td>
			<td>MJB4</td>
			<td></td>
		</tr>
		<tr>
			<td>photomultiplier tube&#160;</td>
			<td>Hamamatsu Photonics, Japan</td>
			<td>R928</td>
			<td></td>
		</tr>
		<tr>
			<td>photoresist</td>
			<td>MicroChem, USA</td>
			<td>SU-8 2075</td>
			<td></td>
		</tr>
		<tr>
			<td>PID temperature controllers&#160;</td>
			<td>Shanghai, China</td>
			<td>XH-W2023</td>
			<td></td>
		</tr>
		<tr>
			<td>plasma cleaner&#160;</td>
			<td>Harrick Plasma</td>
			<td>PDC-32G-2</td>
			<td></td>
		</tr>
		<tr>
			<td>polyvinyl pyrrolidone (PVP)</td>
			<td>Aladdin, Shanghai, China</td>
			<td>P110608</td>
			<td></td>
		</tr>
		<tr>
			<td>pump</td>
			<td>Harvard Apparatus</td>
			<td>PHD2000</td>
			<td></td>
		</tr>
		<tr>
			<td>silicone tubing&#160;</td>
			<td>BIO-RAD,USA</td>
			<td>7318210</td>
			<td></td>
		</tr>
		<tr>
			<td>solid-state relays</td>
			<td>KZLTD, China</td>
			<td>KS1-25LA</td>
			<td></td>
		</tr>
		<tr>
			<td>SpeedSTAR HS DNA Polymerase&#160;</td>
			<td>Takara Bio Inc.</td>
			<td>RR070A</td>
			<td></td>
		</tr>
		<tr>
			<td>steel needle</td>
			<td>zhongxinqiheng,Suzhou,China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SYBR GREEN <img alt="Equation 1" src="/files/ftp_upload/63523/63523eq01.jpg" style="margin: auto;" /></td>
			<td>Solarbio, Beijing, China</td>
			<td>SY1020</td>
			<td></td>
		</tr>
		<tr>
			<td>temperature sensors</td>
			<td>EasyShining Technology, Chengdu, China</td>
			<td>TCM-M207</td>
			<td></td>
		</tr>
		<tr>
			<td>Template (E. coli)</td>
			<td>Takara Bio Inc.</td>
			<td>AK601</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Aladdin, Shanghai, China</td>
			<td>T104863</td>
			<td></td>
		</tr>
		<tr>
			<td>voltage power supply&#160;</td>
			<td>Medina, NY, USA</td>
			<td>TREK MODEL 610E</td>
			<td></td>
		</tr>
	</tbody>
</table>

amplification of escherichia coli in a continuous-flow-pcr microfluidic chip and its detection with a capillary electrophoresis system

Polymerase chain reaction (PCR) is a traditional method employed for the amplification of a target gene that has played an important role in biomolecular diagnostics. However, traditional PCR is very time-consuming because of the low-temperature variation efficiency. This work proposes a continuous-flow-PCR (CF-PCR) system based on a microfluidic chip. The amplification time can be greatly reduced by running the PCR solution into a microchannel placed on heaters set at different temperatures. Moreover, as capillary electrophoresis (CE) is an ideal way to differentiate positive and false-positive PCR products, a CE system was built to achieve efficient separation of the DNA fragments. This paper describes the process of amplification of Escherichia coli (E. coli) by the CF-PCR system built in-house and the detection of the PCR products by CE. The results demonstrate that the target gene of E. coli was successfully amplified within 10 min, indicating that these two systems can be used for the rapid amplification and detection of nucleic acids.

Figure 5&#160;represents the electropherogram of the PCR products and the DNA markers. Trace (Figure 5A) is the CE result of the CF-PCR amplified product, trace (Figure 5B) is the CE result of the product amplified by thermal cycling, and trace (Figure 5C) is the CE result of the 100 bp DNA ladder. We first amplified the target gene of E. coli in the CF-PCR system; the PCR solution took ~10 min...

Watch this Scientific Journal Video about Amplification of Escherichia coli in a Continuous-Flow-PCR Microfluidic Chip and Its Detection with a Capillary Electrophoresis System at JoVE.com

Amplification of Escherichia coli in a Continuous-Flow-PCR Microfluidic Chip and Its Detection with a Capillary Electrophoresis System

This protocol describes how to build a continuous-flow-polymerase chain system based on a microfluidic chip and how to build a capillary electrophoresis system in the lab. It presents a simple method for the analysis of nucleic acids in the lab.

Amplification of Escherichia coli in a Continuous-Flow-PCR Microfluidic Chip and Its Detection with a Capillary Electrophoresis System

Polymerase chain reaction (PCR) is a traditional method employed for the amplification of a target gene that has played an important role in biomolecular ...

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