PCR reaction mix, 8 tubes, 50 µL each: Mix contains Taq DNA polymerase, MgCl2, and dNTP’s
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. Never reuse disposable pipette tips. If you do, the samples will become cross-contaminated
Cup for discarded tips
Micropipettor
OpenPCR machine: shared by two groups
PCR Reaction Sample List
Tube Label
PCR Reaction Sample
Patient ID
G# +
Positive control
none
G# -
Negative control
none
G# 1-1
Patient 1, replicate 1
27929
G# 1-2
Patient 1, replicate 2
27929
G# 1-3
Patient 1, replicate 3
27929
G# 2-1
Patient 2, replicate 1
34835
G# 2-2
Patient 2, replicate 2
34835
G# 2-3
Patient 2, replicate 3
34835
DNA Sample Set-up Procedure
Step 1 Obtain DNA extracted from cells into a “PCR” tube
Step 2 Add primer 1 into “PCR” test tube
Step 3 Add primer 2 into “PCR” test tube
Step 4 Add nucleotides to “PCR” test tube
Step 5 Add DNA polymerase to “PCR” test tube
Step 6 Put “PCR” tube in thermocycler
OpenPCR program
Inside the thermal cycler, at 95 degrees Celsius, he DNA double helix splits apart. Next, the thermal cycler drops to 50 degrees Celsius. At this point single stranded DNA molecules attempt to pair up, but are unsuccessful because there are more primer sequences than strands of DNA which attach to their target area. The thermal cycler then goes to 72 degrees Celsius and a DNA polymerase attaches to a single DNA strand and adds nucleotides until the DNA strad cuts off. These steps are repeated in future cycles until there are a multitude of copies of the desired sequence.
Research and Development
PCR - The Underlying Technology
DNA is made up of four types of molecules called nucleotides. These are adenine (A), thymine (T), cytosine (C), and guanine (G) and they anneal: A to T, T to A, C to G, and G to C. The template DNA of a PCR reaction is the DNA being replicated. At 95 degrees Celsius it begins to untangle, preparing for denaturization. A primer attaches to the denatured template DNA at the top and bottom of each segment to allow polymerase to copy. This anneal process happens at 72 degrees Celsius. Taq polymerase attaches to the primers and adds nucleotides therefore copying the DNA before it splits into two. Base pairing occurs during anneal and extend steps. The deoxyribonucleotides are what make up DNA and are what allow sequences to be copied enough times to create a billion target sequences. The sequences then begin to degrade slower because they are protected by most bacteria.
SNP Information & Primer Design
Background: About the Disease SNP
A nucleotide is one of the structural components, or building blocks, of DNA and RNA. It consists of a base (adenine, thymine, guanine, and cytosine) plus a molecule of sugar and one of phosphoric acid. A single nucleotide polymorphism is a single nucleotide variation in a genetic sequence that occurs at appreciable frequency in the population. This variation is found in Homo sapiens, in chromosome 4. Its clinical significance is listed as pathogenic and the condition linked to this SNP is cardiac arrhythmia. ANK2 stands for ankyrin 2, which is important in activities such as proliferation, cell motility, activation, contact, and the maintenance of specialized membrane domains. An allele is one alternative form of a gene that is created by mutation and are found on the chromosome at the same place. The disease-associated allele contains the codon ATC. The numerical position of the SNP is 113367751.
Primer Design and Testing
When the designed non-disease forward primer and reverse primer were tested, they resulted in the 220bp sequence from chromosome 4. When the designed disease forward primer and reverse primer were tested, there were no matches.
BME 100 Fall 2016
