DNA/ primer mix, 8 tubes, 50 μL each (each contains a different template DNA but have the same reverse and forward primer)
A strip of empty PCR tubes
Disposable pipette tips (use once in order to avoid cross contamination)
Cup for discarded tips
Micropipettor
OpenPCR machine (per two groups)
PCR Reaction Sample List
Tube Label
PCR Reaction Sample
Patient ID
G9 +
Positive control
none
G9 -
Negative control
none
G9 1-1
Patient 1, replicate 1
14176
G9 1-2
Patient 1, replicate 2
14176
G9 1-3
Patient 1, replicate 3
14176
G9 2-1
Patient 2, replicate 1
91779
G9 2-2
Patient 2, replicate 2
91779
G9 2-3
Patient 2, replicate 3
91779
DNA Sample Set-up Procedure
Using the 8 test tubes, label accordingly:
G9 +
G9 -
G9 1-1
G9 1-2
G9 1-3
G9 2-1
G9 2-2
G9 2-3
In order to perform a Polymerase Chain Reaction (PCR), DNA must be collected from cells from a source such as hair follicles.
Next, DNA should be extracted from the source to a designated PCR tube
Afterwards, the first primer should be extracted and released into the same designated PCR tube
The second primer is then extracted and released into the same designated PCR tube
Nucleotides are then added to the PCR tube in order to provide the needed building blocks when the DNA replicates
The final thing to be added is DNA polymerase that commences the matches the nucleotides in order to make the DNA copies
After everything has been released into the test tube, place the PCR tube into the thermal cycler and begin the cycle
OpenPCR program
An integral part of the Polymerase Chain Reaction is the thermal cycling portion. The cycle begins with heating the lid at approximately 100°C. Then the container is heated where the tubes are placed at 95°C for 2 minutes. The PCR tubes are then placed into the thermal cycler and for 30 seconds are heated at this temperature. This allows the DNA double helix strand to denature (due to the hydrogen bonds breaking) and separate into single strands. Afterwards, the cycler is cooled to 57°C for 30 seconds to allow the primers to bind to the single stranded DNA which is also called primer annealing. Once the primers have attached to the strands, the cycler is heated to 72°C for 30 seconds to allow the DNA polymerase to bind to the primers to activate and begin performing complimentary base pairing. This temperature remains for 2 minutes in order to allow the primer to finish performing complimentary base pairing on the target DNA until it reaches the end of the strand. The final step is to hold and cool the cycler holding the PCR tubes at 4°C. 25 repetitions of the cycle are performed in order to amplify the affected portion of DNA. A brief overview of the process is listed below.
Heat the lid at 100°C
The initial step is to heat to 95°C for 2 minutes
Thermal cycler should perform 25 times
Denature the DNA at 95°C for 30 seconds
Anneal at 57°C for 30 seconds to allow the primers to bind to the target DNA
Extend at 72°C for 30 seconds to activate DNA polymerase to allow
Finally 72°C for 2 minutes the primer to perform complimentary base pairing to the target DNA
The last step is a final hold at 4°C
Research and Development
PCR - The Underlying Technology
Function of each component in a PCR reaction
Template DNA
The template DNA is the DNA that is transcribed. It is the basis for the transcribed messenger RNA
Primer
are short pieces of DNA that are made in a laboratory. Since they're custom built, primers can have any sequence of nucleotides you'd like
Taq Polymerase
The most important enzyme in the PCR reaction, which speeds up the reaction and attaches molecules together
Deoxyribonucleotides (dNTP’s)
A deoxyribonucleotides is a monomer or unit of DNA made of a deoxyribose sugar, a phosphate, and a nitrogenous base
Effects during thermal cycling
INITIAL STEP: 95°C for 3 minutes:
The DNA is heated up and begins to denature.
Denature at 95°C for 30 seconds:
template DNA double helix separates, creating two single-stranded DNA molecules
Anneal at 57°C for 30 seconds:
primers lock onto their targets before strands naturally rejoin
Extend at 72°C for 30 seconds:
The DNA polymerase is activated and locates a primer attached to a single DNA strand. It then begins to add complementary nucleotides onto the strand.
FINAL STEP: 72°C for 3 minutes:
The polymerase continues until the end of the stand and then falls off.
FINAL HOLD: 4°C:
Now you have the fragment you wanted. You can keep cycling and continue to duplicate your template.
Purine and Pyridine Bases
Adenine(A):
T
Thymine(T):
A
Cytosine(C):
G
Guanine(G):
C
Base Pairing Occurrence
During the anneal phase, the temperature is lowered to 57°C for 30 seconds where the primers attach to the target DNA strand. During the extend step is where base pairing begins when the temperature reaches 72°C for 30 seconds, allowing the DNA polymerase to activate from the primer and add complementary bases to the strand. The next step as well, which is holding this temperature for approximately 3 minutes allows the primer to continue adding base pairs until it reaches the end of the strand and falls off the target strand.
As seen by the image, the primers are short pieces of DNA located in certain parts of the template DNA and proceeds to find the sequence on the DNA that it was made for. Then upon reaching a specific heat, the DNA combination of the primers is replicated by the Taq Polymeraze enzyme.
SNP Information & Primer Design
Background: About the Disease SNP
The acronym SNP is an acronym for Single Nucleotide Polymorphism, which occurs when a certain nucleotide (A,T,C, and G) swaps for a different base. In some cases, it can lead to drastic effects and inflict disease upon those affected. In this case, the variation rs1805008 located on chromosome 16:89919736 and is a pathogenic gene found in human beings. The resulting nucleotide base change from cytosine to thymine in this particular variation and the gene MC1R begins to cause problems in melanocortin receptor activity, which can lead to pathogenic diseases such as Clear Cell Carcinoma among other diseases. It goes to show that a single nucleotide has an immense significance on gene expression and can cause lethal diseases.
What is a nucleotide?
Building blocks of DNA that consists of a nucleoside with a phosphate group. The four basic nucleotides are: Adenine (A), Thymine (T), Cytosine (C), and Guanine (G) (For RNA, Thymine is replaced with Uracil (U).)
What is a polymorphism?
When two or more phenotypes exist within the same species.
Primer Design and Testing
What species is this variation found in? (Latin name)
The Latin name for the species this variation is found in is Homo sapiens, also known as humans.
What chromosome is the variation located on?
Variation rs1805008 is located on chromosome 16:89919736.
What is listed as the Clinical significance of this SNP?
What makes Variation rs1805008 a clinical significance is the fact that it is pathogenic, meaning that it is able to cause disease to those who have the variation.
Which gene(s) is this SNP associated with?
Variation rs1805008 is associated with the gene known as MC1R.
What disease is linked to this SNP?
Some of the said diseases that variation rs1805008 can cause are Clear Cell Carcinoma, Renal Cell Carcinoma, and Parkinson’s Disease.
Some of the functions of MC1R include, G-protein coupled peptide receptor activity, Hormone binding, and Melanocortin receptor activity.
What is an allele?
An allele is a form of the same gene from each parent that appear in the same loci of the chromosome that control the same characteristic
The disease-associated allele contains what sequence?
The disease-associated allele contains the sequence CGG -> TGG
The numerical position of the SNP is:
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 3’
The numerical position exactly 200 bases to the right of the disease SNP is:
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 3’
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 T 3’
Disease reverse primer (20 nt)
5’-C G T A G C G G G C C G A G G T G T T C 3’
Upon testing the Non-diseased primers, we received the following 220bp sequence
The temperature calculations are done assuming 50 mM salt and 50 nM annealing oligo concentration. The code to calculate the melting temperature comes from Primer 3.
When testing the diseased primers, the following result was given:
However, upon testing the Diseased primers multiple times, they received no matches at all, this could be a display of only the beginning of the harmful effects from a diseased primer, not only that but it is also worth noting when making the Diseased forward primer, only one of the nucleotides were changed, but upon pairing this up with a Diseased reverse primer, the many different nucleotides were changed. Which shows another display of a diseased SNP's harmful effects.
BME 100 Fall 2015
