PCR & GMO

Question 1

What’s the primary use of polymerase chain reaction?

Answer 1

To amplify a specific sequence or sequences of DNA

Question 2

What are the six main “ingredients” to running PCR?

Answer 2

(1) Thermally stable DNA polymerase
(2) pH buffer
(3) Mg2+ cofactor for DNA polymerase
(4) Template DNA
(5) Forward and reverse primers
(6) Deoxynucleoside triphosphates (dNTPs)

Question 3

Why are primers important for specificity in PCR?

Answer 3

They provide the free 3’ OH (hydroxyl) group needed for DNA polymerase to attach incoming dNTPs

Question 4

Primers should be in large _______________________ of the template to be amplified and should __________________ the sequence to be amplified

Answer 4

Excess

Flank

Question 5

Why are forward and reverse primers required for PCR?

Answer 5

To flank the sequence to be amplified

Question 6

Amplification in PCR is _______________ (linear or exponential)

Answer 6

Exponential

Question 7

There are six overall steps to PCR. What are they?

Answer 7

(1) Initial denaturation of DNA
(2) Continued denaturation of DNA
(3) Annealing of primer sequences
(4) Extension of DNA
(5) Incubation to finish remaining fragments
(6) Soak

Question 8

Initial denaturation occurs at _____ (temperature)

Answer 8

95 degrees Celsius

Question 9

Continued denaturation occurs at _____ (temperature)

Answer 9

95 degrees Celsius

Question 10

Annealing occurs at ______ (temperature)

Answer 10

Dependent upon the primer

Question 11

Extension occurs at _______ (temperature)

Answer 11

68 to 72 degrees Celsius

Question 12

Soaking occurs at ______ (temperature)

Answer 12

4 degrees Celsius

Question 13

What’s the quick sequence of PCR events?

Answer 13

Denaturation - denaturation - annealing - extension - incubation - soaking

Question 14

What are the temperatures associated with each PCR step?

Answer 14

95 - 95 - varied - (68 - 72) - 4

Question 15

How many times are steps 2 (continued denaturation) through 4 (extension) completed in PCR?

Answer 15

30X

Question 16

What is a GMO?

Answer 16

A GMO is a genetically modified organism that has been modified through the insertion of foreign genetic material

Question 17

What are five cons of GMOs?

Answer 17

(1) Horizontal gene transfer of unfavorable genes
(2) Changed relationship between organism and environment
(3) Changed offspring viability
(4) Allergy development
(5) Reduced diversity

Question 18

What is horizontal gene transfer?

Answer 18

Acquisition of foreign genes via transformation, transduction, and conjugation by organisms in a variety of environmental situations

Question 19

Why is horizontal gene transfer a con of GMOs?

Answer 19

Because unfavorable genes can spread to different species

Question 20

What are seen pros of GMOs?

Answer 20

(1) Elimination of manual weeding
(2) Enhanced nutritional value of food
(3) Reduced and/or elimination of pesticide use
(4) Vaccine delivery
(5) Longer shelf life
(6) Microbial resistance

Question 21

Approximately 85% of GMO plants use a specific promoter or terminator. What are they?

Answer 21

Promoter: CaMV 35S
Terminator: Agrobacterium tumefaciens nopaline synthase terminator (NOS terminator)

Question 22

What’s the size of the CaMV 35S promoter?

Answer 22

203 base pairs

Question 23

What’s the size of the NOS terminator?

Answer 23

225 base pairs

Question 24

GMO plant primers contain forward and reverse primers for __________________ promoter and __________________ terminator

Answer 24

CaMV 35S promoter

Agrobacterium tumefaciens nopaline synthase terminator (NOS terminator)

Question 25

In the GMO lab, what was used as the positive control?

Answer 25

Photosystem II chloroplast gene

Question 26

What’s the size of the photosystem II gene?

Answer 26

455 base pairs

Question 27

What does it mean if a sample doesn’t show isolation of photosystem II?

Answer 27

It indicates problems in DNA extraction, reaction set up, or both

Question 28

In the GMO lab, what was used as the negative control?

Answer 28

Certified GMO-free grains (oats)

Question 29

Should photosystem II primers work on a non-GMO plant sample?

Answer 29

Yes, because all plants have photosystem II genes