BME100 f2017:Group9 W1030 L4

BME 100 Fall 2017	Home People Lab Write-Up 1 | Lab Write-Up 2 | Lab Write-Up 3 Lab Write-Up 4 | Lab Write-Up 5 | Lab Write-Up 6 Course Logistics For Instructors Photos Wiki Editing Help
OUR TEAM Name: Samantha Balconi Name: Cassity Jones Name: Carlos Ruiz Name: Rachel Oberlander Name: Nicholas Lee LAB 4 WRITE-UP Protocol Materials Lab coat Disposable gloves PCR reaction mix, 8 tubes, 50μL each: mix contains Taq DNA polymerase, MgCl2, and dNTP's DNA/prer mix, 8 tubes, 50μL each: each mix contains a different template DNA Strip of empty PCR tubes Disposable pipette tips Cup for discarded tips Micropipettor OpenPCR machine PCR Reaction Sample List Tube Label PCR Reaction Sample Patient ID G9 + Positive control none G9 - Negative control none G9 1-1 Patient 1, replicate 1 54046 G9 1-2 Patient 1, replicate 2 54046 G9 1-3 Patient 1, replicate 3 54046 G9 2-1 Patient 2, replicate 1 94177 G9 2-2 Patient 2, replicate 2 94177 G9 2-3 Patient 2, replicate 3 94177 DNA Sample Set-up Procedure Get DNA sample from patients Put DNA sample into designated PCR tube Add Primers 1 and 2 into the PCR tube Add nucleotides to PCR tube Add DNA polymerase to PCR tube Place PCR tube into DNA Thermal Cycler Remove and analyze DNA Sample Conduct two more trials with same patient DNA Repeat steps 1-7 for the second patient OpenPCR program Heated Lid: 100 CHEATED LID: 100°C INITIAL STEP: 95°C for 2 minutes NUMBER OF CYCLES: 25 Denature at 95°C for 30 seconds, Anneal at 57°C for 30 seconds, and Extend at 72°C for 30 seconds FINAL STEP: 72°C for 2 minutes FINAL HOLD: 4°C Research and Development PCR - The Underlying Technology Components Functions Template DNA DNA sequence which is to be copied in PCR reaction Primers Used to target specific portion of DNA wanted to be copied Taq Polymerase Builds new strand of DNA that was marked by primers Deoxyribonucleotides (dNTP's) Single units for the bases A, T, C, and G Steps Event INITIAL STEP: 95°C for 2 minutes: Heat up the sample Denature​ at 95°C for 30 seconds: Template DNA split into single strands Anneal​ at 57°C for 30 seconds: Primers bind to complimentary matches on DNA sequence Extend​ at 72°C for 30 seconds: Taq Polymerase bind to primers and add nucleotides, extending second strands FINAL STEP: 72°C for 2 minutes: Keep the extend process to continue FINAL HOLD: 4°C: Stop the process and stir Nucleotide Base Pair Adenine (A) T Thymine (T) A Cystosine (C) G Guanine (G) C The base-pairing occurs during the anneal and extend steps of the cycle. The primer attaches to a complementary base during the anneal. The extension step pairs all the bases of the DNA strand starting from the primer. SNP Information & Primer Design Background: About the Disease SNP SNP stands for single nucleotide polymorphism. It accounts for the large portion of genetic variation between individuals by differing the nucleotides in certain sections of the DNA. Most of the time, SNPs are used as biological markers and do not have a damaging effect on the individual. However, when the SNPs lie close to or within a gene they are more likely to play a direct role in disease. The specific SNP, rs769452, is found in Homo sapiens on the chromosome 19:44907853. The clinical significance of this SNP is that it is pathogenic and is linked to Alzheimer's Disease. APOE stands for Apolipoprotein E and is the gene where the SNP rs769452 is located. APOE is responsible for carrying cholesterol through the bloodstream. Three unique terms include amyloid-beta binding, antioxidant activity, and cholesterol binding. An allele is the specific location of a gene on a chromosome. The non-disease allele has the codon CTG while the disease allele has the codon CAG. The numerical position of the SNP is 44907853. Primer Design and Testing The results of the primer test showed that the sequence of DNA that would be replicated would be what lies in the 5' to 3' direction after the forward primer and would end before the start of the reverse primer.