DNA/Primer mix- contains different template DNAs; all tubes have same forward primer and reverse primer (8 tubes- 50 μL each)
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
G5 P
Positive control
none
G5 N
Negative control
none
G5 1-1
Patient 1, replicate 1
87717
G5 1-2
Patient 1, replicate 2
87717
G5 1-3
Patient 1, replicate 3
87717
G5 2-1
Patient 2, replicate 1
56788
G5 2-2
Patient 2, replicate 2
56788
G5 2-3
Patient 2, replicate 3
56788
DNA Sample Set-up Procedure
1. Obtain a human sample and place into a special PCR tube
2. To the other PCR tube add primer 1
3. Add Primer 2 to the second site
4. To the PCR tube add the nucleotides
5. To the PCR tube add DNA polymerase
6. Since the PCR tube has all the components and add the tube to the DNA Thermal Cycler
OpenPCR program
HEATED LID: 100°C
1. INITIAL STEP: 95°C for 2 minutes
2. NUMBER OF CYCLES: 25
Denature at 95°C for 30 seconds: the double-stranded DNA to split apart
Anneal at 57°C for 30 seconds: primers to attach to DNA
Extend at 72°C for 30 seconds: taq polymerase attaches to primer and adds deoxyribonucleotides to complementary strand
3. FINAL STEP: 72°C for 2
4. FINAL HOLD: 4°C
Research and Development
The Underlying Technology
PCR - Important Components
Polymerase Chain Reaction is a process by which a specific segment of DNA can be copied a billion times. The specific segment of DNA that is being copied serves as the template DNA. Primers bind to the DNA segment that is being replicated. One primer will attach to the top strand of DNA and a second primer will attach to the bottom strand of DNA. Primers show DNA polymerase where to start copying the strand. Taq polymerase is the DNA polymerase used in PCR. The Taq polymerase will attach to the primer of the DNA strand and create a complementary strand. The DNA strands are made of four deoxyribonucleotides called Adenine, Thymidine, Cytosine, and Guanine.(http://learn.genetics.utah.edu/content/labs/pcr/)
PCR - Thermal Cycling
The template DNA and the other materials necessary for PCR are heated up to 95 Celsius for two minutes. When the strand reaches a hot enough temperature, the template DNA splits into two strands as the hydrogen bonds holding the DNA strand together is broken down a result of the high temperature. This process is called denaturing and takes thirty seconds. After the denaturing, the reaction is cooled down 57 Celsius for thirty seconds. This process of annealing allows a primer to attach to each DNA strand. The two strands created from denaturing are complementary to each other so the two primers that attach to the DNA strands run in opposite directions to each other. The temperature of the reaction is then heated to 72 Celsius in thirty seconds which allows the Taq polymerase to attach to the primers on the DNA strands. It adds deoxyribonucleotides to build a complementary strand to the DNA strand with the primer.This results in a new double-stranded DNA. This process is called extending and the temperature will be held for two minutes after reaching 72 Celsius. After the reaction is finished, the DNA will be kept at a temperature of four Celsius.
(https://www.yourgenome.org/facts/what-is-pcr-polymerase-chain-reaction)
PCR - Base-Pairing
As the Taq polymerase adds deoxyribonucleotides to the complementary strand, Thymidine can only base pair with Adenine and Cytosine can only base pair with Guanine. Adenine can only base pair with Thymidine and Guanine can only base pair with Cytosine. During the annealing and extending step of PCR, base-pairing occurs. During annealing, the primer base pairs to the DNA strand that it wants to copy. During extending, base pairing occurs as the Taq polymerase creates the new complementary strand. (https://www.yourgenome.org/facts/what-is-pcr-polymerase-chain-reaction)
SNP Information & Primer Design
Background: About the Disease SNP
Using a database that provides information about single nucleotide polymorphisms, we searched a reference SNP id number to learn more about a specific single nucleotide polymorphism. A nucleotide is a structural component of DNA and RNA (https://www.ncbi.nlm.nih.gov/Class/MLACourse/Original8Hour/Genetics/nucleotide.html). A polymorphism is the presence of genetic variations within a certain population (http://www.sciencedirect.com/topics/neuroscience/polymorphism). The reference SNP id number, rs769452, occurs in Homo sapiens and is located on the chromosome 19:44907853. This SNP affects the gene Apolipoprotein E. The clinical significance of this gene is that it appears in 30-50% of patients with late-onset Alzheimer (https://www.ncbi.nlm.nih.gov/pubmed/22530123). It also has a role in strokes (https://www.ncbi.nlm.nih.gov/pubmed/17672902). The gene Apolipoprotein E is also written as APOE. The main function of this variation is lipid binding, protein binding, and phospholipid binding. An allele is an alternate view of a certain mutation on a chromosome. The disease containing codon is CTG because it is transcripted into CCG. The numerical position of the SNP is 44907853.
Primer Design and Testing
The results of our Non-disease primers matched with the 220 bp sequence from chromosome 19 (from question 4), which validated that we had the correct primers before we tested our disease-specific primers. We then tested our disease-specific primers with the changed base and the results displayed that there were no matches. This is because this DNA base pair does not naturally exist in DNA; a C and C would never naturally pair together.
BME 100 Fall 2017
