Body Temperature Lab Learning Objectives: Students will learn to design a thermal shirt to monitor body temperature and to ascertain and develop to a market a unique solution with benefits over state-of-the-art technologies. Students will identify those technologies and highlight their improvements in the design. Accuracy of the thermo shirt will be assessed by calculating accuracy and reproducibility of the new thermal shirt and comparing these values to a gold standard, such as an oral or tympanic thermometer.
DNA Lab 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.
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, 10/23/13
Snapshot of an OpenPCR plugged into a computer. Photo from AM Group 7 Snapshots of a PCR video. Photo from AM Group 8
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 a DNA target. A mismatch (non-disease DNA sequence) does not produce amplification.
Quiz: students answered 5-10 questions at the beginning of the class and received assistance from the TA's to assess and reinforce comprehension of the pre-lab material.
LAB ACTIVITIES:
Open PCR Machine Testing - Students partially disassembled and identified parts in a thermal cycling machine with the guidance of a worksheet and the OpenPCR machine manual. Students tested the machine for proper function.
Research and Development - Students learned how the Polymerase Chain Reaction can be used to detect cancer-associated mutations with the guidance of a step-by-step worksheet.
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, a 200 μL micropipettor, and disposable pipette tips. The instructors provided positive and negative "patient" samples so that some samples would test positive (produce amplification) for a specific single nucleotide polymorphism (SNP), and others would test negative (no amplification).
Finding a Cancer-Associated Mutation Online - Follow the steps shown in the slides in CancerSNP.pptx (from Blackboard) to explore the NCBI SNP database.
Quiz: students answered 5-10 questions at the beginning of the class and received assistance from the TA's to assess and reinforce comprehension of the pre-lab material. Students also completed a colored dye pipetting exercise to practice good technique.
LAB ACTIVITIES:
Pipetting Practice - Students practiced good micropipetting technique, using food coloring.
PCR Reaction - Students ran PCR reactions on real DNA samples.
Cancer-specific Primer Design - Students learned how PCR can be applied to detect a cancer-associated DNA sequence (SNP) with a hands-on exercise (guided by a worksheet) in designing a SNP-specific primer pair.
Week 3: Computer-Aided Design with TinkerCAD, 11/06/13
Activities:
Open PCR machine engineers used Tinker CAD to design PCR strip tubes.
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.
The students were provided with this default design.
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.
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.
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.
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.
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.
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
Fluorimeter (μg/mL)
n/d
n/d
0.00
1.10
1.2
4.00
0.70
2.2
CONCLUSIONS
Amplification of a single band. No non-specific background
No false positives in the absence of template DNA
The Fluorimeter is a quick and simple-to-use alternative to gel electrophoresis.
OpenPCR + the Single Drop Fluorimeter system makes PCR labs scalable (~200 students in this class).
BME 100 Fall 2013
