BME100 f2018:Group3 T0800 L4

BME 100 Fall 2018	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: Gisselle Beltran Name: Jenny Wong Name: Angie Quinonez Name: Lesly Romero Name: John Nguyen Name: Mason Haynie LAB 4 WRITE-UP Protocol Materials Lab coat and disposable gloves PCR reaction mix, 8 tubes, 50 μL each: Mix contains Taq DNA polymerase, MgCl2, and dNTP’s (http://www.promega.com/resources/protocols/product‐information‐sheets/g/gotaq‐colorless‐master‐mix‐m714‐protocol/) DNA/ primer mix, 8 tubes, 50 μL each: Each mix contains a different template DNA. All tubes have the same forward primer and reverse primer A strip of empty PCR tubes Disposable pipette tips: only use each only once. Never reuse disposable pipettes tips. If you do, the samples will become cross‐contaminated Cup for discarded tips Micropipettor OpenPCR machine: shared by two groups PCR Reaction Sample List Tube Label PCR Reaction Sample Patient ID G3 + Positive Control None G3 - Negative Control None G3 1-1 Patient 1, Replicate 1 83872 G3 1-2 Patient 1, Replicate 2 83872 G3 1-3 Patient 1, Replicate 3 83872 G3 2-1 Patient 2, Replicate 1 97753 G3 2-2 Patient 2, Replicate 2 97753 G3 2-3 Patient 2, Replicate 3 97753 DNA Sample Set-up Procedure Extract DNA from a cell Move the extracted DNA into a special PCR tube (The PCR tube will evenly distribute heat inside the tube) Add Primer 1 to the PCR tube (Primer 1 will attach to the sites on the DNA strands that are at either end of the segment that is being copied) Add Primer 2 to the PCR tube (Primer 2 will attach to the second site) Add nucleotides Adenine (A), Thymine (T), Guanine (G), and Cytosine (C). to the PCR tube Add DNA polymerase to the PCR tube (The DNA polymerase will read the DNA code and then attach to match the nucleotides to create DNA copies) Place the PCT tube into the thermal cycler (The machine will heat and cool the tube at specific times in order to make the reaction work) OpenPCR program The following values will be used to run a heating and cooling program on the thermal cycler. Heated 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 In order for a PCR reaction to take place there are different components that must serve their individual functions. The template DNA is one of the components. It is a strand used by the DNA polymerase to attach complementary bases during DNA replication or RNA transcription. Primers are another component that serve an individual function within a PCR reaction. They are short pieces of DNA that are made in a laboratory. Two primers are designed to match to the segment of DNA you want to copy. Through complementary base pairing, one primer attaches to the top strand at one end of your segment of interest, and the other primer attaches to the bottom strand at the other end. In most cases, two primers that are twenty or so nucleotides long will target just one place in the entire genome. Primers are also necessary because DNA polymerase cannot attach anywhere and start copying away. It can only add onto an existing piece of DNA. Taq polymerase, another component, attaches nucleotides to a DNA template, therefore copying the DNA. Lastly, there is Deoxyribonucleotides (dNTP’s) which are split into two groups, the purines and pyrimidines. The function of the dNTP's is to synthesize an unlimited amount of specific stretch of double stranded DNA. It has to supply to the polymerase enzyme. These components that are part of the PCR reaction undergo certain changes or reactions during thermal cycling. Initially, when it is held at 95°C for two minutes the DNA double helix separates, creating two single-stranded DNA molecules. At the same temperature for thirty seconds denaturation occurs which is when the double-stranded template DNA is heated to separate it into two single strands. These two strands which will act as templates for the production of the new strands of DNA. When the temperature is lowered to enable the DNA primers to attach to the template DNA. When annealing at 57°C for thirty seconds, primers serve as the starting point for DNA synthesis. The polymerase enzyme can only add DNA bases to a double strand of DNA. Only once the primer has bound can the polymerase enzyme attach and start making the new complementary strand of DNA from the loose DNA bases. At 72°C in thermal cycling, it begins to extend and the temperature is raised and the new strand of DNA is made by the Taq polymerase enzyme. The result is a brand new strand of DNA and a double-stranded molecule of DNA.The new fragments of DNA that are made during PCR also serve as templates to which the DNA polymerase enzyme can attach and start making DNA. In its final step at 72°C the polymerases to finish reading whatever strand they are currently on. This optional step can help reduce the number of truncated copies in your final product. The final hold is at 4°C, the temperature allows one to use or analyze the product. DNA is made up of four types of molecules called nucleotides, designated as A (Adenine), T (Thymine), C (Cytosine) and G (Guanine). Base‐pairing, driven by hydrogen bonding, allows base pairs to stick together. The Adenine and Thymine bases anneal to each other (A & T) and the Cytosine and Guanine bases anneal to each other (G & C). The two steps of thermal cycling that base-pairing occurs was deduced. In the first cycle, The thermal cycler cools down to 50°C, about 122°F. At this temperature, single stranded DNA molecules naturally attempt to pair up. There are many primer sequences than DNA strands in the tube. Primers will crowd their way in and lock onto their target before strands can rejoin. Polymerase Chain Reaction SNP Information & Primer Design Background: About the Disease SNP In order to understand what single nucleotide polymorphism (SNP) is, we need to define the terms nucleotide and polymorphism. Nucleotides are the building blocks of nucleic acids such as DNA and RNA. A few examples of a nucleotide would be Adenine, Thymine, Guanine, and Cytosine. Polymorphism is a discontinuous genetic variation resulting in the occurrence of several different forms or types of individuals of a single species. The SNP investigated was rs721710. The species this varation is found in is Homo sapiens and is located on chromosome 12:40315266. There is an uncertain clinical significance of this SNP but it is associated with Parkinson's Disease. Primer Design and Testing In order to find the DNA sequence of the SNP and the surrouding sequence LRRK2 was investigated. Leucine Rich Repeat Kinase 2 (LRRK2) is related to ATP binding, GTP binding, and Actin Binding. It was found that the non-disease allele, a possible form of the gene, contains the codon GTG. However, a change in this allele at the T position to an A is linked to the disease. The disease-associated allele contains the codon GAG and the numerical position of the SNP is 40315266. To design a non-disease forward primer, a sequence of 20 bases where the SNP with numerical position 40315266 ends up in the 3' position. The following is the oligonucleotide sequence of the non-disease forward primer (20 nt): 5' - T T A A G T G A C T T G T A C T T T G T - 3' Because every PCR reaction needs two primers to amplify DNA, a non-disease reverse primer was also designed. The numerical position exactly 200 bases to the right of the disease SNP was used. The numerical position 200 basses to the right of the SNP is 40315466. To design the non-disease reverse primer, a sequence of 20 bases that has where the numerical position 40315466 ends up in the 5' position. The following is the oligonucleotide sequence of the non-disease reverse primer (20 nt): 5' - T G A A G C T C T T C A A G T A G T C T - 3' In order to design a pair of disease SNP-specific primers, the final base of the non-disease forward primer is changed so that it is identical to the disease SNP nucleotide. The following is the oligonucleotide sequence of the disease forward primer (20 nt): 5' - T T A A G T G A C T T G T A C T T T G A - 3' The following is the oligonucleotide sequence of the disease reverse primer (20 nt): 5' - T G A A G C T C T T C A A G T A G T C T - 3' The non-disease primers were validated using an online non-disease human genome sequence. The non-disease primers designed resulted in a 220 bp sequence for the chromosome 12: 40315266. Non-Disease Primer Validation Then the diseased-specific primers that were designed were validated using an online non-disease human genome sequence. The results showed that there were no matches which makes sense because a diseased-specific primer would not show up in a non-disease human genome sequence. Disease Primer Validation