Lab 5 - GMO Investigator Flashcards
(29 cards)
What does PCR stand for?
polymerase chain reaction
We used PCR to look for what in this lab?
for a DNA sequence common to GM foods found in the grocery store
In short, what did we do?
week 1:
extract genomic DNA from food samples
run PCR reactions to amplify GMO and natural plant sequences from the DNA
week 2:
electrophorese the amplified samples to visualize the DNA
What does PCR allow us to do?
amplify specific sections of DNA and make millions of copies of the target sequence
PCR locates specific DNA sequences using:
primers that are complementary to the DNA template
primers are:
short strands of DNA called oligonucleotides
How many primers are needed to amplify a DNA sequence?
2: one forward and one reverse primer
Components needed to set up a reaction for DNA amplification by PCR (6)
- Template DNA
- Oligonucleotide Primers
- DNA Polymerase
- MgCl2
- dNTPs
- Buffer
TODMdB
What is the purpose of the template DNA?
This is the DNA molecule containing the segment of DNA we want to amplify.
What is the purpose of the Taq DNA polymerase?
This is used to replicate DNA and adds bases onto the 3’ ends of the annealed primers.
stable at high temp.s, isolated from hot spring bacteria
What is the purpose of the forward and reverse primers?
They serve as the replication starting point and amplify the DNA segment.
they bind to complementary regions on opposite strands of the template
What is the purpose of dNTPs?
They are building blocks (monomers) for template DNA replication
deoxynucleoside triphosphate
added by Taq DNA polymerase onto the 3’ ends of the annealed primers
What is the purpose of MgCl2?
Mg2+ is a cofactor for DNA polymerase. Without it, the enzyme does not work.
What is the purpose of buffer?
It maintains the pH of the reaction at a level where the DNA polymerase is most active. (70°C)
What are the reagents present in the Master Mix?
- Buffer
- MgCl2
- dNTPs
- Forward and Reverse Primers
- Taq DNA Polymerase
Numerate one cycle of PCR
- denaturation: high temp, break H-bonds b/w complementary bases
- annealing: low temp, 2 PCR primers anneal to template strands
- extension: intermediate temp, DNA polymerase adds nucleotides onto the ends of annealed primers
Temperature cycling is performed in an instrument called:
thermal cycler
Exponential amplification describes the rate at which:
new products accumulateby the expression 2^n
n = nber of cycles
For this experiment, 2 PCR reactions were set up for each DNA sample. What are their purpose?
- Plant Master Mix: control to determine whether or not we extracted plant DNA from food. (amplifies a section of a chloroplast gene) size: 455bp green
- GMO Master Mix: determines whether or not sample contains GM DNA sequences (identifies sequences common to most of all GM plants) size: 255bp red
What components of the PCR reaction are different between the PMM and GMM?
They use different primers
P: chloroplast genes
GMO: GMO
Inside the cell, the enzyme called helicase is used to separatre the DNA strands to be replicated. In PCR, what technique is used to separate template strands?
Denaturation using heat
Define what a “positive control” is
Experimental control that produces the expected results to ensure that the experiment is working properly. It receives a treatment known to produce a particular response.
What was the positive control in this experiment?
Running a PCR with the PPM to identify a DNA sequence common to all plants. It ensures we have plant DNA.
Define “negative control”
Control group that is known not to produce a response to a treatment. This ensures we don’t get false positives.
to establish a baseline level for comparison and rule out the possibility that any observed effects are simply due to chance or interference from extraneous variables
