Lab 5 - GMO Investigator

Question 1

What does PCR stand for?

Answer 1

polymerase chain reaction

Question 2

We used PCR to look for what in this lab?

Answer 2

for a DNA sequence common to GM foods found in the grocery store

Question 3

In short, what did we do?

Answer 3

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

Question 4

What does PCR allow us to do?

Answer 4

amplify specific sections of DNA and make millions of copies of the target sequence

Question 5

PCR locates specific DNA sequences using:

Answer 5

primers that are complementary to the DNA template

Question 6

primers are:

Answer 6

short strands of DNA called oligonucleotides

Question 7

How many primers are needed to amplify a DNA sequence?

Answer 7

2: one forward and one reverse primer

Question 8

Components needed to set up a reaction for DNA amplification by PCR (6)

Answer 8

1. Template DNA
2. Oligonucleotide Primers
3. DNA Polymerase
4. MgCl2
5. dNTPs
6. Buffer

TODMdB

Question 9

What is the purpose of the template DNA?

Answer 9

This is the DNA molecule containing the segment of DNA we want to amplify.

Question 10

What is the purpose of the Taq DNA polymerase?

Answer 10

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

Question 11

What is the purpose of the forward and reverse primers?

Answer 11

They serve as the replication starting point and amplify the DNA segment.

they bind to complementary regions on opposite strands of the template

Question 12

What is the purpose of dNTPs?

Answer 12

They are building blocks (monomers) for template DNA replication

deoxynucleoside triphosphate

added by Taq DNA polymerase onto the 3’ ends of the annealed primers

Question 13

What is the purpose of MgCl2?

Answer 13

Mg2+ is a cofactor for DNA polymerase. Without it, the enzyme does not work.

Question 14

What is the purpose of buffer?

Answer 14

It maintains the pH of the reaction at a level where the DNA polymerase is most active. (70°C)

Question 15

What are the reagents present in the Master Mix?

Answer 15

1. Buffer
2. MgCl2
3. dNTPs
4. Forward and Reverse Primers
5. Taq DNA Polymerase

Question 16

Numerate one cycle of PCR

Answer 16

1. denaturation: high temp, break H-bonds b/w complementary bases
2. annealing: low temp, 2 PCR primers anneal to template strands
3. extension: intermediate temp, DNA polymerase adds nucleotides onto the ends of annealed primers

Question 17

Temperature cycling is performed in an instrument called:

Answer 17

thermal cycler

Question 18

Exponential amplification describes the rate at which:

Answer 18

new products accumulateby the expression 2^n

n = nber of cycles

Question 19

For this experiment, 2 PCR reactions were set up for each DNA sample. What are their purpose?

Answer 19

1. 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
2. GMO Master Mix: determines whether or not sample contains GM DNA sequences (identifies sequences common to most of all GM plants) size: 255bp red

Question 20

What components of the PCR reaction are different between the PMM and GMM?

Answer 20

They use different primers
P: chloroplast genes
GMO: GMO

Question 21

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?

Answer 21

Denaturation using heat

Question 22

Define what a “positive control” is

Answer 22

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.

Question 23

What was the positive control in this experiment?

Answer 23

Running a PCR with the PPM to identify a DNA sequence common to all plants. It ensures we have plant DNA.

Question 24

Define “negative control”

Answer 24

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

Question 25

The positive GMO was another example of positive control. What is the purpose of the positive GMO control DNA sample?

Answer 25

It allows us to verify that the test actually checks for GMO, if it does indeed show GMO. If it doesn’t show GMO, then results are likely invalid.

Question 26

Why was the non-GMO food-control prepared prior to the test food sample?

Answer 26

To avoid cross-contamination in the mortar: better to have non-GMO food in the GMO food than vice versa because we don’t want a false positive in the non-GMO control.

Question 27

Explain why DNA fragments separate according to size in an electrophoresis gel.

Answer 27

DNA fragments are force, in an electric field, to travel through a porous gel material where shorter fragments of DNA will move at a faster rate than longer ones.

Question 28

Why do we need a molecular weight ruler alongside the samples?

Answer 28

The molecular weight ruler allows for an estimate of the size of each PCR sample bands. If a sample has a bond that travels the same distance as the GMO-associated sequence on the MWR, then that sample contains a GM sequence.

Question 29

Why was EZ-Vision TM added to each sample?

Answer 29

• tracks gel progress
• buffer
• weigh down sample
• safer tracking dye