BME103 s2013:T900 Group2 L3

BME 103 Spring 2013	Home People Lab Write-Up 1 Lab Write-Up 2 Lab Write-Up 3 Course Logistics For Instructors Photos Wiki Editing Help
OUR TEAM Name: Joe Sansone Role(R&D Scientist) Name: Shang Ruan Open PCR Machine Engineer Name: William Scott Role(R&D Scientist) Name: Student Role(s) Name: Student Role(s) LAB 3 WRITE-UP Original System: PCR Results PCR Test Results Sample Name Ave. INTDEN* Calculated μg/mL Conclusion (pos/neg) Positive Control 518016 0.199 N/A Negative Control 375033 0.032 N/A Tube Label:2 Patient ID: 91562 rep 1 384551 0.043 neg Tube Label:3 Patient ID: 91562 rep 2 312171 -0.042 neg Tube Label:4 Patient ID: 91562 rep 3 371384 -0.031 neg Tube Label:5 Patient ID: 25235 rep 1 467127 0.139 pos Tube Label:6 Patient ID: 25235 rep 2 406664 0.064 neg Tube Label:7 Patient ID: 25235 rep 3 484088 0.154 pos * Ave. INTDEN (Patient 91562)= 356035.3 *Ave. INTDEN (Patient 25235)= 452626.3 Bayesian Statistics These following conditional statistics are based upon all of the DNA detection system results that were obtained in the PCR lab for 20 hypothetical patients who were diagnosed as either having cancer or not having cancer. Bayes Theorem equation: P(A|B) = P(B|A) * P(A) / P(B) Calculation 1: The probability that the sample actually has the cancer DNA sequence, given a positive diagnostic signal. A = [frequency of cancer-positive conclusions] = [9/20] = [0.45] B = [frequency of positive PCR reactions] = [26/60] = [0.43] P (B|A) = [frequency of positive PCR given cancer-positive conclusion] = [24/27] = [0.89] P(A|B) = [0.931 0r 93.1%] Calculation 3: The probability that the patient will develop cancer, given a cancer DNA sequence. A = [frequency of "yes" cancer diagnosis] = [6/9] = [0.67] B = [frequency of pos test conclusions] = [9/20] = [0.45] P (B|A) = [frequency of pos given "yes"] = [0.67] P(A|B) = [0.47 or 47%] New System: Design Strategy We concluded that a good system Must Have: [Must have #1 - Be easy to determine the result. Normally we have bunches of tubes waiting to run the test. Efficiency is important for the test. If it takes sophisticated procedure to determine the result,the test engineer must be highly qualified which is not that common. ] [Must have #2 - Fast imaging results. Efficiency is essential when the test we are running is based on tons of samples.] [Must have #3& #4 - Simple OpenPCR software to operate and easy sample loading OpenPCR. Basically the same reason as #1. ] [Must have #5 - Small sample volume. Operating the whole test is easier with small samples. We don't have to waste time on collecting big amount of sample and then disposing it. ] We concluded that we would Want a good system to have: [Want #1 - why? short, ~4 or 5 sentences] [Want #2 - why? short, ~4 or 5 sentences] We concluded that a good system Must Not Have: [Must Not Have #1 - why? short, ~4 or 5 sentences] [Must Not Have #2 - why? short, ~4 or 5 sentences] We concluded that a good system Should Avoid: [Should Avoid #1 - why? short, ~4 or 5 sentences] [Should Avoid #2 - why? short, ~4 or 5 sentences] [[Image:]]==New System: Machine/ Device Engineering== SYSTEM DESIGN KEY FEATURES We chose to include these new features Feature 1 - explanation of how this addresses any of the specifications in the "New System: Design Strategy" section Feature 2 - explanation of how this addresses any of the specifications in the "New System: Design Strategy" section Etc. [OR] We chose keep the devices the same as the original system Feature 1 - explanation of how a pre-existing feature addresses any of the specifications in the "New System: Design Strategy" section Feature 2 - explanation of how a pre-existing feature addresses any of the specifications in the "New System: Design Strategy" section Etc. INSTRUCTIONS New System: Protocols DESIGN We chose to include these new approaches/ features We thought we would provide the PCR with an additional flourimeter apperatus (designed like a mini-box that a smart phone can be incorperated into) as well as the necessary chemicals. The user would have to provide some DNA sample (saliva or blood maybe) and we would provide an app for smart phone that the user buys. The app would in theory do all the necessary calculations in one easy step (pic->result), basically skipping the image j analysis. MATERIALS Supplied In Kit Supplied by User dNTPs Sample DNA MgCl2 Forward and Reverse Primers Reaction buffers Fluorescent, cancer-specific probe Taq DNA Polymerase SYBR Green dye PROTOCOLS PCR Protocol The first step was to program the PCR machine and create the Thermal Cycler Program. The set-up used for the program is as follows Stage one: 1 cycle, 95 degrees Celsius for 3 minutes Stage two: 35 cycles, 95 degrees Celsius for 30 seconds, 57 degrees Celsius for 30 seconds, 72 degrees Celsius for 30 seconds Stage three: 72 degrees Celsius for 3 minutes Final Hold: 4 degrees Celsius The second step is to add reagents to the tubes. Start by gathering the necessary materials to set-up the DNA samples (pipette, PCR reaction mix, 8 transfer pipettes) Set the pipette to 50 microliters Place the transfer pipette onto the pipette to prevent cross contamination (never re-use). DNA Measurement and Analysis Protocol Step 1 Step 2 Etc. New System: Research and Development BACKGROUND Polymerase Chain Reaction (PCR) is a scientific method that utilizes DNA Polymerase to create a complimentary base strand from a template strand of DNA. Triphosphate nucleotides align with open DNA strands and DNA polymerase works to link the complementary nucleotide bases together growing strands through both condensation and hydroysis reactions. Through these mechanisms it is possible to target specific positions on the template DNA sequence that a scientist intends to amplify(PCR 1). When the PCR process is completed the targeted DNA sequence containing the single-nucleotide polymorphism (SNP) will have manufactured over a billion copies (amplicons). A SNP essentially is a type of gentic variation among organisms which represents a difference in a single nucleotide. For example, a SNP may replace a nucleotide cytosine (C) with a nucleotide thymine (T) in a certain part of an organisms DNA. These SNPs can be utilized as biological markers which in turn can help locate genes that have associative properties that contribute to the formation of harmful diseases. The targeted SNP for this research was rs17879961. This SNP is found in Humans (Homo sapiens) and represents a variation class SNV, which stands for single nucleotide variation. Furthermore, This SNP is a variant of the CHEK2 gene (Checkpoint kinase 2) which if present in a person's genome may increase their risk of developing breast cancer. This SNV signifies a single base change from a Thymine (T) to a Cytosine (C) located on chromosome 22 and its clinical significance is classified as a pathogenic allele. For example, this mutation would alter the normal alelle ATT and the middle position resulting the cancer associated allele ACT. DESIGN Primers for PCR Cancer allele forward primer: -> TTGAGAATG[TCA]CGTATGTAT Cancer allele reverse primer: -> AACTCTTAC[AGT]GCATACATA Disease alleles will yield PCR products because the target amplicon is only associated with the cancer DNA sequences. Thus primer annealing will following base pairing rulese when it binds with the template strand. For example, triphosphate nucleotides align with open DNA strands and DNA polymerase works to link the complementary nucleotide bases together growing strands through both condensation and hydroysis reactions. The presence of a primer is required so that polymerase can proceed with directing the new nucleotides in place. Through these mechanisms it is possible to target specific positions on the template DNA sequence that a scientist intends to amplify. When the PCR process is completed the targeted DNA sequence containing the single-nucleotide polymorphism (SNP) will have manufactured over a billion copies (amplicons). Sources: http://www.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi?rs=17879961 http://hmg.oxfordjournals.org/content/16/15/1794.full.pdf http://www.ncbi.nlm.nih.gov/projects/genome/probe/doc/TechPCR.shtml Our primers address the following design needs Design specification 1 - explanation of how an aspect of the primers addresses any of the specifications in the "New System: Design Strategy" section Design specification 2 - explanation of how an aspect of the primers addresses any of the specifications in the "New System: Design Strategy" section Etc. New System: Software [THIS SECTION IS OPTIONAL. If your team has creative ideas for new software, and new software is a key component included in your new protocols, R&D, or machine design, you may describe it here. You will not receive bonus points, but a solid effort may raise your overall page layout points. If you decide not to propose new software, please delete this entire section, including the ==New System: Software== header.]