BME100 f2017:Group15 W1030 L4

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OUR TEAM

Name: Cole Alvarez
Name: Kylie Mercer
Name: Nathan Moorman
Name: Kasandra Sanchez
Name: Lauren Seliger)

LAB 4 WRITE-UP

Lab A: Protocol

Materials

  • Lab coat and disposable gloves
  • PCR reaction mix, 8 tubes, 50 μL each: Mix contains Taq DNA polymerase, MgCl2, and dNTP’s
  • 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
  • Cup for discarded tips
  • Micropipettor
  • OpenPCR machine

PCR Reaction Sample List

Tube Label PCR Reaction Sample Patient ID
G15 + Positive control none
G15 - Negative control none
G15 1-1 Patient 1, replicate 1 76014
G15 1-2 Patient 1, replicate 2 76014
G15 1-3 Patient 1, replicate 3 76014
G15 2-1 Patient 2, replicate 1 66716
G15 2-2 Patient 2, replicate 2 66716
G15 2-3 Patient 2, replicate 3 66716


DNA Sample Set-up Procedure

  1. HEATED LID: 100°C
  2. INITIAL STEP: 95°C for 2 minutes
  3. 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
  4. FINAL STEP: 72°C for 2 minutes
  5. FINAL HOLD: 4°C



Lab A: Research and Development

PCR - The Underlying Technology

Components in a PCR Reaction

Four components of a Polymerase Chain Reaction include Template DNA, Primers, Taq Polymerase, and Deoxyribonucleotides. Template DNA is sample DNA that contains the target sequence. During the beginning of the reaction, high temperatures are applied to original double stranded DNA molecules and separates the strands. Primers are short pieces of single stranded DNA that work in unison with the target sequence. Taq Polymerase is an isolated, heat-loving enzyme that copies DNA. It is heat resistant and can handle the high temperatures of the polymerase chain reaction. Deoxyribonucleotides are single units of the bases A, T, G, and C which work as "building blocks" for the new DNA strands.


What happens to the components (listed above) during each step of thermal cycling? There are 6 stages in thermal cycling. The initial step occurs when the temperature is 95°C for two minutes. During the stage, the Template DNA and all the other components heats up which causes the hydrogen bonds to break the DNA into separate strands. The Denaturing stage then occurs at 95°C for 30 seconds. The result of this stage is two single strands of DNA. The annealing stage occurs next where the temperature is at 57°C for 30 seconds. The cooling in this stage enables the primers to attach to a specific location on the single-stranded template DNA through the use of Hydrogen Bonding. The next stage is to extend. At this stage, the temperature is at 72°C for 30 seconds. The heat is increased to enable the new DNA to be made by Taq polymerase. This special polymerase adds DNA bases. The result of this stage is a brand new strand of DNA and a double-stranded molecule. In the next step, the temperature is kept at 72°C for two minutes. In this step, the taq polymerase begins to add deoxyribonucleotides right up until it reaches the end of the strand. The final step is where the temperature is held at 4°C. At this stage, the entire polymerase reaction comes to a stop.

Source: “Steps Involved in Polymerase Chain Reaction.” Study.com, Study.com, study.com/academy/lesson/pcr-synthesizing-dna-using-polymerase-chain-reaction.html.

Base anneals

Adenine (A) Thymine (T)
Thymine (T) Adenine (A)
Cytosine (C) Guanine (G)
Guanine (G) Cytosine (C)

During which 2 steps of thermal cycling does base pairing occur? The action of base pairing occurs between the steps of annealing and extending. The stage of annealing is when the primers attach to the DNA template. These DNA primers take the role of the starting position for replication. After this, the temperature is raised slightly and the stage of extension begins. In this stage, the taq polymerase appears and matches the base primers with their pairs. This results in the formation of new nucleotide strands for the desired target sequences of DNA.

Source: “Steps Involved in Polymerase Chain Reaction.” Study.com, Study.com, study.com/academy/lesson/pcr-synthesizing-dna-using-polymerase-chain-reaction.html.

Background: About the Disease SNP A SNP is a Single Nucleotide Polymorphism. A SNP is an alteration in a nucleotide that takes places at a certain point in the genome. The different variations of a SNP is what makes a persons hair and eye color the way that it is. A SNP can also affect how a person reacts to certain medications and drugs. The SNP is also responsible for identifying genetic effects that generate susceptibility to autoimmune diseases. The disease SNP is a pathogenic disease in which the codon AGT is replaced with CGT. The disease associated with the SNP is Alzheimers and Subarachnoid Hemorrhages.

Primer Design and Testing The disease associated SNP is occupying the top strand of the DNA sequence. This means that the strand is going 5' with a 20 base long sequence. The non disease forward primer was 5'-AGCGGCCAGCGCTGGGAACT. The second primer done in this lab was the non-disease reverse primer. This is located exactly 200 bases to the right of where the original disease SNP was situated. 200 bases to the right brings about the numerical position of 44908053. 5'-CAGGCCCCCCAAGACTTAGC was the non disease reverse primer. After the primers were found for the non-diseased, the forward and reverse primers for the disease were found. The process was the same, except that the last base of the non-disease forward primer was replaced with the disease SNP. The disease forward primer came out to 5'-AGCGGCCAGCGCTGGGAACC. The disease reverse primer came out to 5'-CAGGCCCCCCAAGACTTAGT.

Non Disease Forward Primer.PNG Disease Specific Primer.PNG

Lab B: Disease SNP - Specific Primer Design

Part 1: Use the NCBL Database to find a disease associated sequence.

What is a Nucleotide A nucleotide is the building block and basic structural unit for DNA. Nucleotides are composed of a phosphate group, the bases adenine, cytosine, guanine, and thymine, and a pentose sugar.
What is a Polymorphism? A polymorphism is a discontinuous genetic variation resulted in the occurrence of several different forms or types of individuals in a single species.
What species is this variation found in? (Latin name) The variation is found under the Latin name: Homo Sapiens.
What chromosome is the variation located on? The variation is located on the chromosome 19.
What is listed as the Clinical significance of this SNP? Clinical Significance is listed as "with pathogenic allele."
Click the PubMed link to view summaries of research associated with the SNP. What condition is linked to this SNP? The conditions linked to this SNP is Alzheimer's and Subarachnoid Hemorrhage.

Source: “National Center for Biotechnology Information.” National Center for Biotechnology Information, U.S. National Library of Medicine, www.ncbi.nlm.nih.gov/.


Part 2: Find the DNA sequence of the SNP and the surrounding sequence.

What does APOE​ stand for? APOE stands for apolipoprotein E.
What is the function of APOE? The function of APOE includes cholesterol binding, lipoprotein particle binding, and antioxidant activity.
What is an allele? An allele is a variant form of a given gene.
The disease-associated​ ​allele contains what codon? The disease-associated allele contains the CCG codon.
The numerical​ ​position​ ​of​ ​the​ ​SNP​ is: 44907853


Part 3: Design primers for PCR

Non-disease​ ​forward​ ​primer​ ​(20​ ​nt): 5'-AGCGGCCAGCGCTGGGAACT
The numerical position​ ​exactly​ ​200​ ​bases to​ ​the​ ​right​ ​of​ ​the​ ​disease​ ​SNP​ is: 44908053
Non-disease​ ​reverse​ ​primer​ ​(20​ ​nt)​: 5'-CAGGCCCCCCAAGACTTAGC
Disease​ ​forward​ ​primer​ ​(20​ ​nt): 5'-AGCGGCCAGCGCTGGGAACC
Disease​ ​reverse​ ​primer​ ​(20​ ​nt): 5'-CAGGCCCCCCAAGACTTAGT