PCR reaction mix, 8 tubes, 50 uL each: Mix contains Taq DNA polymerase, MgCl2, and dNTP's
DNA/ Primer mix, 8 tubes 50 uL each: Each mix contains a different template DNA. All tubes have the same forward primer and reverse primer
A strip of empty PCR tubes
Dsposable pipette tips
Cup for discarded tips
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
Label sides of empty PCR tubes with specific tube labels and put tubes in rack
Transfer 50 uL of PCR mix into positive control tube and discard disposable tip
With a new tip, transfer 50 uL of DNA/ primer mix into the positive control tube, making the total volume in this tube 100 uL
Repeat two previous steps for the negative control, patient 1 replicates 1,2, 3, and patient 2 replicates 1, 2, 3, using the proper DNA/primer mix for the specific tubes. Each tube should have 50 uL of DNA/primer mix and 50 uL of PCR mix, totaling in 100 uL.
Close the lids securely on the PCR reaction tubes and place the tubes into the heating slots in the assigned PCR machine.
Research and Development
PCR - The Underlying Technology
The temperature is raised to 95 degrees in order to break the double helix of the DNA.
The temperature is cooled to allow the primers to bind to the DNA.
The temperature is raised to allow the Taq DNA Polymerase to begin transcription and replicate the target DNA.
What is the function of each component of a PCR reaction?
Template DNA: This is the initial DNA copy that you are trying to replicate. It contains the target sequence. Primers: Attach to sites on the DNA strands that are at either end of the segment you copy. Primers are complimentary to the target sequence. Taq Polymerase: Enzyme that can read DNA code and attach matching nucleotides to create copies of the target DNA. Deoxyribonucleotides: dNTPs are single units of bases A, T, C and G, which are the building blocks for new DNA strands.
What happens to the components during each step of thermal cycling?
Initial Step (95°C for 3 minutes): Activates polymerases Denature step (95°C for 30 seconds): DNA double helix separates, creating two single-stranded DNA molecules Anneal (57°C for 30 seconds): DNA molecules try to pair up. Polymerase binds to primer/template DNA complex Extend (72°C for 30 seconds): Activates DNA polymerase which will copy template DNA Final step (72°C for 3 minutes): Polymerases finish reading whatever strand they are currently working on, helping to reduce the number of prematurely developed copies Final hold (4°C): Keeping the sample at 4°C until ready to be analyzed, as to preserve the sample
Which base anneals to each base?
Adenine (A): T Thymine (T): A Cytosine (C): G Guanine (G): C
During which two steps of thermal cycling does base-pairing occur?
Base pairing occurs during the annealing and extension steps. During annealing, the sample is cooled and target DNA binds with short DNA primers that serve as starting positions for replication. During extension, taq polymerase matches the base primers with their new pairs, forming new complimentary nucleotide strands.
SNP Information & Primer Design
Background: About the Disease SNP
The disease, SNP (short nucleotide polymorphism), is found in humans and the variation is located on Chromosome 7. A nucleotide is the subunit of DNA and polymorphism is the genetic variation within a population. This means that SNP is a disease involving the variation of the building blocks of DNA. The disease is pathogenic meaning it is caused by a microorganism such as bacteria or a virus. Cystic Fibrosis has been linked to SNP.
Primer Design and Testing
After locating the allele change that causes CFTR (cystic fibrosis transmembrane conductance regulator), the first primer was made by including the nineteen bases before the allele change and that allele. The reverse primer was made by locating an allele 200 bases away from the CFTR allele and using the 20 bases from the reverse strand of DNA. When the CFTR allele is changed from A to C, cystic fibrosis occurs because the gene regulates transport pathways. To test the primer, the UCSC In-Silico PCR website was used to show that the non-diseased primer codes for the proper 220 base pair sequence (pictured below). With the diseased primer, the primer does not code for a sequence.