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: only use each only once. Be sure not to use pipette tips twice because
samples will be cross-contaminated
Waste cup for diposal tips
Cup of ice
OpenPCR machine: shared by two groups
PCR Reaction Sample List
PCR Reaction Sample
Patient 1, replicate 1
Patient 1 replicate 2
Patient 1, replicate 3
Patient 2, replicate 1
Patient 2, replicate 2
Patient 2, replicate 3
DNA Sample Set-up Procedure
Gather all of the materials listed above
Label all 8 50μL tubes(G8 P, G8 N, G8 1-1, G8 1-2, G8 1-3, G8 2-1, G8 2-2, G8 2-3)
After gathering samples place all DNA samples, Primers, etc. on the cup of ice
Place no more than 50μL of each DNA/Primer mix into the corresponding tubes using a micropipette set at the correct volume
Using a micropipette add 50μL of PCR mix (primers, nucleotides, and DNA polymerase) to the PCR tube
Insert the PCR tube in to the thermal cycler
Repeat Steps above with each DNA sample
Place all samples in thermal cycler and start the machine
HEATED LID: 100°C
INITIAL STEP: 95°C for 2 minutes
NUMBER OF CYCLES: 25
Denature at 95°C for 30 seconds
Anneal at 57°C for 30 seconds
Extend at 72°C for 30 seconds
FINAL STEP: 72°C for 2 minutes
FINAL HOLD: 4°C
Research and Development
PCR - The Underlying Technology
What is the function of each component of a PCR reaction?
The components of a PCR reaction include the DNA template, primers, Taq Polymerase, and deoxyribonucleotides.
The function of the DNA template (represented below) is the basic genetic code meant to be copied and replicated. picture source: http://learn.genetics.utah.edu/content/labs/pcr/
The primers (shown below) are small fragments of DNA which bind to specific sites on the DNA template which facilitate the attachment of the Taq polymerase and start DNA replication at specific sites. picture source: http://learn.genetics.utah.edu/content/labs/pcr/
The Taq Polymerase(shown below)is a heat-activated polymerase, which attaches to the primers and builds and replicates a second DNA strand from the template DNA. picture source: http://learn.genetics.utah.edu/content/labs/pcr/
The deoxribonucleotides (shown below) are components of DNA (adenine, guanine, cytosine, thymine) which are needed to replicate the DNA. They are used as building blocks of DNA, and are added to the replicated strand by the Taq Polymerase. picture source: www.shmoop.com
What happens to the components (listed above) during each step of thermal cycling?
INITIAL STEP: 95°C for 3 minutes: This step activates hot-start primers and leads into the denaturation step.
Denature at 95°C for 30 seconds: This breaks the hydrogen bonds and separates the two DNA strands.
Anneal at 57°C for 30 seconds: The primers bind to their complementary sequence in the DNA template.
Extend at 72°C for 30 seconds: DNA polymerase extends the primers by sequencing complementary nucleotides.
FINAL STEP: 72°C for 3 minutes: This is done to ensure that any remaining single strands are extended.
FINAL HOLD: 4°C: The DNA is kept at this temperature for analysis and storage.
Which base anneals to each base below?
Adenine always binds with Thymine.
Guanine always bind with Cytosine.
During which two steps of thermal cycling does base-pairing occur? Explain your answers.
Base-pairing occurs during annealing and extension. During the annealing step, the primers must use base-pairing to bind to their sequence. In extension, base-pairing occurs as DNA polymerase completes the DNA strand.
SNP Information & Primer Design
Background: About the Disease SNP
SNP is commonly known as "single nucleotide polymorphism." This particular SNP is referred to as clear cell renal cell carcinoma or kidney cancer.To distinguish this SNP on the cellular level, the malignant epithelial cells have clear cytoplasm and a distinguished pattern of growth. On the genetic level, SNP is characterized by a specific 5' group of DNA located at the chromosome position 16:89919736 and associated with the MC1R gene. The MC1R (melanocortin 1 receptor (alpha melanocyte stimulating hormone receptor) gene is responsible for coding the receptor protein for melanocyte-stimulating hormone. What causes the gene to malfunction and eventually lead to SNP is when the allele at rs1805008 position 1858 is changed from CGG to TGG.
Primer Design and Testing
With the primer test, it was discovered that everyone has the non-diseased primers. However, what gives someone a specific disease are the unique changes in their DNA sequence. Someone may have the same disease as another, but it may have been caused by a different genetic mutation.
The non-disaesed primers used were:
The diseased primers used were:
There were no matching primers because it is a diseased primer and would not be included in the normal human genome.