BME103

BME 103 Fall 2012	Home People Lab Write-Up 1 Lab Write-Up 2 Lab Write-Up 3 Course Logistics For Instructors Photos Wiki Editing Help
Introduction to Biomedical Engineering Learning Objectives: Students should leave this unit equipped with a theoretical understanding of how to detect DNA biomarkers and the relevance of this technology to human healthcare. Emphasis will be on good lab/experimental controls and the collection of statistically valid measurements. Students will also understand how commonly used lab devices function, and explore the recent efforts to simplify experiments and to lower costs. At the end of the section, students will explore creative new biosensor designs based on PCR and fluorescent imaging. GETTING STARTED Create an OpenWetWare account: Fill in the form at OpenWetWare:How to join Edit your group's wiki: Find your group's Wiki template in one of the Lab Write-Up sections. There are further instructions there. Be creative and have fun editing! THE DNA LABS IN REVIEW Set-up: OpenPCR Building Before this unit began, a group of ~10 upper level undergrads and graduate students assembled the OpenPCR machines. This was a great experience for the graduate students, and saved our Freshmen engineers the time and trouble of assembling the delicate pieces from scratch in a very limited amount of time. Thanks to our assembly team and Dr. Pizziconi's Design Studio team for your help! Week 1: Introduction - DNA as a Biomarker, 10/17/12 Students were introduced to basic DNA science and its relationship to diagnostics and health. Sequence-specific DNA hybridization uses primers designed to base-pair with a target disease-associated marker. This leads to exponential amplification of an invisible DNA target. A mismatch (non-disease DNA sequence) does not produce amplification. Team members chose roles as Open PCR machine tester/ engineer, Experimental protocol planner, and Research and development scientist. Snapshot of the worksheet for planning PCR reactions. In concurrent work sessions... Open PCR machine testers partially disassembled, reassembled, and tested the OpenPCR thermal cycler with the guidance of a worksheet and the OpenPCR machine manual. Experimental protocol planners planned a Polymerase Chain Reaction (PCR) protocol for the Open PCR system and programmed the machine for thermal cycling with the guidance of a worksheet. Research and development scientists learned how the Polymerase Chain Reaction works so that they could explain the process to their teammates. This was done with the guidance of the instructor and the OpenPCR Virtual Lab tutorial. Short pieces off DNA called primers can be designed specifically bind to a target DNA sequence. Image: Thurs 1:30pm Group 5 Taq polymerase docks at the hybridization sites. Image: Thurs 1:30pm Group 5 Taq polymerase produces several copies of the DNA which can be visualized with a fluorescent dye. Image: Thurs 1:30pm Group 5 Week 2: DNA Amplification Reactions, 10/24/12 Students used their experience from the previous week to set up and run a PCR experiment. The students were provided with personal protective equipment, 8 tubes of 50 μL PCR reaction mix, 8 tubes of 50 μL diluted template + primers, and disposable transfer pipettes. The instructors provided positive and negative "patient" samples so that some samples would test positive for a DNA marker (produce amplification), and others would test negative (no amplification). Experimental protocol planners set up and ran the PCR reactions. These were set aside to run for ~2 hours. A camera phone is placed in front of the fluorimeter. Photo: Thurs 9:30am Group 9 A sample droplet is placed on a hydrophobic slide. Photo: Thurs. 9:30am Group 17 Digital drawing of the fluorimeter set-up. Image: Wed 9:30am Group 7 Students were introduced to a Single Drop Fluorimeter fluorescence-based DNA detection device that was designed by Dr. Garcia. When a natural or PCR-amplified double-stranded DNA sample is stained with SYBR green and exposed to a blue LED light, the drop fluoresces green. The signal is captured as an image with the user's camera phone. Week 3: Computer-Aided Design with SolidWorks, 10/31/12 The Open PCR Solidworks file shows all of the features of the machine. Image: Thurs 1:30pm Group 2 Students were instructed on navigating the NCBI dsSNP database to find disease-associated SNP's and disease prediction information. Students were also introduced to Wiki web page editing. Concurrent work sessions: Open PCR machine engineers used SolidWorks to explore the computer-aided design file of the Open PCR system (provided by Josh Perfetto, Open PCR). Experimental protocol planners added the protocols for the Polymerase Chain Reaction and Fluorimeter Measurements to their group’s Wiki page. Research and development scientists reported information that makes it clear to a non-specialist why a cancer mutation gives a positive PCR signal, and why a non-cancer sequence gives no signal. Week 4: Measuring DNA Using Fluorescence, 11/7/12 Samples were mixed with SYBR green dye and analyzed on Single Drop Fluorimeters. Students used 2 μg/mL purified calf thymus DNA to calibrate the DNA measurement process. Using this value, they were able to convert the PCR results from brightness into DNA concentration. Summaries of their results are available on the Lab Report 1 page. Negative control water droplet. Photo credit: Wed 9:30am Group 2 Double stranded DNA positive control. Photo credit: Wed 9:30am Group 2 Information about the human single nucleotide polymorphism (SNP) rs17879961 was used to demonstrate how sequence-specific DNA hybridization could be used to detect a disease-linked DNA marker (allele). The SNP is a missense mutation on chromosome 22 that replaces a Thymine with a Cytosine. The mutation affects gene CHK2, and is linked to colorectal cancer. Lab Report 1: Each team created a Wiki page write-up of their learning experiences. Week 5: Designing a New System, 11/14/12 The class discussed some of the strengths and areas for possible improvement of the DNA amplification and detection system. Each team then conceptualized a new DNA detection system based on OpenPCR and the Single Drop Fluorimeter. Concurrent work sessions: Open PCR machine engineers used SolidWorks to identify, illustrate, and describe a portion of the OpenPCR system their team proposes to improve/ redesign. Experimental protocol planners created protocols for their group's re-designed system. Research and development scientists gathered and report information (from the NCBI dbSNP database) that would enable real-world application of their DNA biomarker detection system. Proposal to make the lid easier to use. Image: Thurs. 9:30am Group 10 Redesigned transparent casing to visualize & confirm proper assembly Image: Wed. 9:30am Group 1 Lab Report 2: Each team created a Wiki page write-up of their machine designs and protocols. Week 6 & 7: Advertisement Videos, 11/28/12 & 12/6/12 The instructors presented PCR and DNA detection systems that are currently on the market. The systems included super-compact personal PCR machines to very large systems that were capable of both amplifying and measuring the levels of DNA in real time. Lab Report 3: Each team created an advertisement video for their new system. These videos were showcased in class. How Well Did OpenPCR + Single Drop Imaging Perform? The instructors compared traditional gel electrophoresis with the results from the new single drop Fluorimeter approach. Each Sample either has template DNA or is blank. Only DNA Samples should produce amplification (visible band as a Gel result, or high Fluorimeter value). The OpenPCR system successfully amplified products of the expected size, and the Single Drop Fluorimeter measurements agreed with gel electrophoresis data. Overall, the system is a success. Furthermore, the Fluorimeter is much quicker and scales up for large class settings much more easily than gel electrophoresis. Samples from Wednesday groups 1 and 2 resolved on a 1% agarose etBr-stained gel.     1 2 3 4 5 6 7 8 Group 1 Sample DNA blank DNA DNA DNA blank blank blank Gel band none band band band none none none Fluorimeter (μg/mL) 1.77 0.24 1.39 1.54 2.39 0.40 0.47 0.62 Group 2 Sample blank DNA blank blank blank DNA DNA DNA Gel none band none none none band band band Fluorimeter (μg/mL) n/d n/d 0.00 1.10 1.2 4.00 0.70 2.2