Role(s): Responsible for Research and Development
Role(s): Father of Four BME Students
Role(s): Supreme Leader
Role(s): Responsible for DNA Sample Set-up Procedure
LAB 4 WRITE-UP
- Lab coat and disposable gloves
- PCR reaction mix, 8 tubes, 50μl each.
- The mixes contain taq DNA polymerase, MgCl₂, and dNTP's.
- DNA / primer mix, 8 tubes, 50μl each.
- Each 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
- OpenPCR Machine
PCR Reaction Sample List
| Tube Label || PCR Reaction Sample || Patient ID
| G9 + || Positive control || 12307
| G9 - || Negative control || 19722
| G9 1-1 || Patient 1, replicate 1 || 12307
| G9 1-2 || Patient 1, replicate 2 || 12307
| G9 1-3 || Patient 1, replicate 3 || 12307
| G9 2-1 || Patient 2, replicate 1 || 19722
| G9 2-2 || Patient 2, replicate 2 || 19722
| G9 2-3 || Patient 2, replicate 3 || 19722
DNA Sample Set-up Procedure
- Gather all appropriate lab materials for the experiment and adhere to the lab safety protocol by wearing the correct lab safety clothing.
- Take 4 of the 50ul test tubes and place the DNA of patient 1 into each container using a micropipettor with disposable tips. Be sure to label each of the tubes according to the data table above. Repeat these procedures using a new micropipette tip to add the DNA of patient 2 into 4 new tubes.
- Dispose of the micropipette tips in a cup for the discarded tips.
- Add the reaction mix to each of the 50ul tubes that are not the control group.
- Place all of the 50ul test tubes into an OpenPCR Machine.
- Heat the contents at 95 degrees Celsius for 3 minutes. The mixture must be allowed to denature for 30 additional seconds after the initial heating.
- After the DNA has been broken down into two specific strands by waiting for the allotted time, add the primer mix and cool the mixtures at 57 degrees Celsius for 30 seconds.
- To extent the DNA replication, heat the mixtures again at 72 degrees Celsius for 30 seconds. The final heating step is to continue heating at this temperature for 3 more minutes.
- The last step is to hold the DNA mixtures that contain the primers at 4 degrees Celsius.
- Repeat steps 6-9 for the test tubes labeled "replicate 2 and replicate 3".
- Repeat steps 6-9 another time using the test tubes labeled "replicate 3".
- Heated Lid: 100 degrees Celsius
- Initial Step: 95 degrees Celsius for two minutes
- Number of Cycles:
- Denature at 95 degrees Celsius for 30 seconds
- Anneal at 57 degrees Celsius for 30 seconds
- Extend at 72 degrees Celsius for 30 seconds
- Final Step: 72 degrees Celsius for two minutes
- Final Hold: 4 degrees Celsius
Research and Development
PCR - The Underlying Technology
Function of Components
The Template DNA in a PCR reaction is the target section of DNA that is copied. Primers are short segments of DNA that are designed to match the target section of DNA. In the PCR reaction, two different primers are designed to attach to the top strand and the bottom strand of the target section of DNA. The Taq Polymerase is what attaches to the end of the primer and adds Deoxyribonucleotides (dNTP's) to create the DNA base pairs. The dNTP's are what is used to create the DNA base pairs on both the top and bottom strand of the target sequence to create two double-stranded DNA molecules.
The initial step in thermal cycling is the step of heating the Template DNA and all materials from room temperature to 95 degrees Celsius. In the denaturing step of thermal cycling, the Template of DNA is separated into two single-stranded DNA molecules. In the annealing step of thermal cycling, the temperature is dropped to 57 degrees Celsius for 30 seconds and the primers attach to the two single-stranded DNA molecules. In the extending step of thermal cycling, the temperature is raised to 72 degrees Celsius for 30 seconds and the Taq Polymerase attaches to the end of the primers and begins attaching dNTP's to the single-stranded DNA molecules to create two double-stranded DNA molecules, and the Taq Polymerase eventually falls off. The final step of keeping the temperature at 72 degrees for 2 minutes is to ensure the extending step of thermal cycling is completed. The final hold at 4 degrees Celsius is to ensure that the double-stranded DNA molecules do not break down.
Base pairing in DNA is driven by hydrogen bonding and allows base pairs to stick together. Out of the four base pairs in DNA, the ones that bind are:
- Adenosine -> Thymine (AT) or (TA)
- Guanine -> Cytosine (CG) or (GC)
Base pairing occurs during the thermocycling steps of annealing and extending. This is when the temperature is dropped to 57 degrees Celsius which allows the primers to attach to the two single-stranded DNA molecules. The Taq DNA Polymerase adds base pairs to the strands when the temperature rises to 72 degrees Celsius.
Digital Illustration of PCR
SNP Information and Primer Design
Background: About the Disease SNP
The disease SNP, or single nucloetide polymorphism, causes one allele to mutate and replace new mutated nucleotides. A nucleotide is defined as one of the structural components, or building blocks, of DNA and RNA. A nucleotide consists of a base (one of four chemicals: adenine, thymine, guanine, and cytosine) plus a molecule of sugar and one of phosphoric acid. Polymorphism refers to variant of a gene that may lead to the abnormal expression or to the production of an abnormal form of the gene; this may cause or be associated with disease.
The variation rs1805008 is found in Homo sapiens on chromosome 16 and is associated with the MC1R gene. MC1R stands for Melanocortin 1 Receptor, and is also known as the alpha melanocyte stimulating hormone receptor. The MC1R gene is responsible for producing the hormone that normalizes pigmentation such as those in human skin, hair and eye color. The clinical significance of this SNP is pathogenic. The diseases linked to this SNP are most commonly renal cell carcinoma and melanoma.
MC1R Gene Ontology
An allele is one of multiple forms of a gene that arise from a mutation at the same chromosome. The allele associated with this SNP contains the sequence TGG, where a non-diseased allele would contain the sequence CGG. The numerical position of the SNP where the mutation occurs is 89919736.
Primer Design and Testing
The results on the primer test showed that the designed primers would indeed attach the RNA, each primer at a slightly different temperature as they should be.
Two DNA strand has been designed to test the mutated (Disease) gene compared to the normal gene,
- Non-disease forward primer (20nt): 5'-CAGCATCGTGACCCTGCCGC
- Non-disease reverse primer (20nt): 5'-CTTGTGGAGCCGGGCGATGC.
The numerical position exactly 200 bases to the right of the disease SNP is: 899199936
- Disease forward primer (20nt): 5'-CAGCATCGTGACCCTGCCGT
- Disease reverse primer (20nt): 5'-GTCGTAGCACTGGGATGGCA.
It must be mentioned that the difference between the normal primer and the diseased primer occurs in the last nucleotide where the thymine (T) replaces the cytosine (C) in the normal primer.
The UCSC In-Silco PCR website was used to test the DNA disease strand, the result was (NO MATCHES) that indicate that this DNA strand has no match in the normal human genome confirming that it is a mutation in the human genome. The Feb 2009 data base was specifically used to get no matching result that could lead to the fact that this mutation was discovered recently.