LAB 4 WRITE-UP
- Lab Coat
- Disposable Gloves
- PCR reaction mix, 8 tubes, 50 μL each
- DNA/ primer mix, 8 tubes, 50 μL each
- PCR tubes
- Disposable pipette tips
- Cup for discarded tips
- Open PCR machine
PCR Reaction Sample List
| Tube Label || PCR Reaction Sample || Patient ID
| G14 + || Positive control || none
| G14 - || Negative control || none
| G14 1-1 || Patient 1, replicate 1 || 76291
| G14 1-2 || Patient 1, replicate 2 || 76291
| G14 1-3 || Patient 1, replicate 3 || 76291
| G14 2-1 || Patient 2, replicate 1 || 64291
| G14 2-2 || Patient 2, replicate 2 || 64291
| G14 2-3 || Patient 2, replicate 3 || 64291
DNA Sample Set-up Procedure
- Using the micropipettor, place a sample of the extracted DNA into the PCR tube.
- Add the first primer to the same PCR tube so that it comes into contact with the DNA sample. This is also done with the micropipettor.
- Add the second primer in the same fashion.
- Add nucleotides to the mixture in the PCR tube.
- Add the DNA polymerase to the PCR tube.
- Place the PCR tube into the Thermocycler and run the proper program using the parameters described below.
Heated Lid: 100°C
Initial Step: 95°C for 2 minutes. The DNA strands are prepared for the PCR process
Number of Cycles: 25. Each cycle allows for more replications to occur.
Denature at 95°C for 30 seconds. This level of heat causes the DNA strand to separate from its antiparallel double helix into two individual strands.
Anneal at 57°C for 30 seconds. This temperature cools the DNA, making the strands want to come back together. However, during PCR, at this point the primers attach to the strands instead.
Extend at 72°C for 30 seconds. DNA polymerase is able to attach to the DNA strands at this temperature.
Final Step: 72°C for 2 minutes. The DNA polymerase now attached to the strands adds complimentary nucleotides to the strand.
Final Hold: 4°C. Lowered temperatures cause the individual strands to come back together to form a full double helical DNA strand. This would occur at slightly higher temperatures but stability is ensured at even lower temperatures.
Research and Development
PCR - The Underlying Technology
Function of each component in PCR reaction
For a Polymerase Chain Reaction to take place a number of components need to be present. A Template DNA is used as a stencil for the DNA copies to be produced. Primers, short pieces of DNA that are made in a laboratory and can have any sequence of nucleotides, are used as a foundation for the attachment of Taq Polymerase. Taq Polymerase are a naturally occurring complex of proteins whose function is to copy a cell DNA before making new copies. These new copies of DNA are built using Deoxyribonucleotides (dNTP’s). And the DNA lives happily ever after, the end.
Stages of Thermal Cycling
The Initial Step of thermal cycling is to heat the DNA at 95oC for 2 minutes. This gets the DNA hot, and prepares it to denature in the next step. The second step is Denature, where an additional 30 seconds at 95oC where the DNA template actually denatures, and the helix separates into two single stranded DNA. Next is the Anneal step, where the DNA is cooled to 57oC for 30 seconds so that the DNA will attempt to reattach, but the primers will attach instead since they are more abundant. The Extend step heats the DNA to 72oC for 30 seconds since this temperature is ideal to allow Taq Polymerase to bind to the primers. In the Final Step the DNA is kept at 72oC for 2 minutes to allow the polymerase to attach complementary nucleotides to each DNA strand. Final Hold cools the DNA to 4oC to ensure stability after the cycling is done.
In DNA Adenine and Thymine are base pairs, as well as Guanine and Cytosine.
Base Pairing in Thermal Cycling
Base pairing occurs during the Anneal and Final Step stages of thermal cycling. During Anneal the primers attach to the DNA template through base pairing. In Final Step the DNA polymerase uses base pairing to attach nucleotides to each DNA strand.
SNP Information & Primer Design
Background: About the Disease SNP
Disease SNP, or Single-nucleotide polymorphism, is when a single nucleotide change occurs in an amino acid. The change or mutation is known as an allele. An allele is a variation by mutation in genetic expression found in the same place on the chromosome as the original gene. These variations can have mass effects on a population; They code for how a species responds to stimuli such as pathogens, drugs, and vaccines. For example, The amino acid CGG may be a perfectly functional amino acid but if it has been mutated and found as TGG in the genome it could then be disease associated and the specific disease could ensue in that individual.
Primer Design and Testing
Nucleotide- The main component structures of DNA, with base chemicals adenine, guanine, thymine, and cytosine
Polymorphism- mutations that occur among the nucleotides in DNA
- What species is this variation found in? (Latin name) - Homo sapiens
- What Chromosome is the variation located on? - 16
- What is listed as the Clinical significance of this SNP? - Pathogenic
- What gene(s) is this SNP associated with? - MC1R
- What disease is linked? - Parkinson disease (marginally associated, in spanish population)
MC1R stands for: melanocortin 1 receptor (alpha melanocyte stimulating hormone receptor)
- Function of MC1R: G-Protein coupled peptide receptor activity, hormone binding, melanocortin receptor activity
Allele -a variation by mutation in genetic expression found in the same place on the chromosome as the original gene.
Disease-associated allele: CGG→ TGG
Numerical position of the SNP - 89919736
Non-disease forward primer (20 nt): 5’ C A G C A T C G T G A C C C T G C C G C
Numerical position 200 base spaces to the right: 89919936
Non-disease reverse primer (20 nt): 5’ C T T G T G G A G C C G G G C G A T G C
Disease forward Primer (20 nt): 5’ C A G C A T C G T G A C C C T G C C G G
Disease reverse primer (20 nt): 5’ C T T G T G G A G C C G G G C G A T G G