Biotechnology

Question 1

What can genetically engineered organisms can be used for?

Answer 1

1. Protein production
2. DNA production
3. Research

Question 2

What does knowing DNA sequence helps identify?

Answer 2

genetic alterations

Question 3

What is an alteration that may alter phenotype?

Answer 3

Antibiotic resistance in bacteria

Question 4

What are two examples of alterations that may result in disease?

Answer 4

1. Sickle cell anemia (1 base-pair change in gene)

2. Cystic fibrosis (3 base-pair deletion)

Question 5

How does DNA sequence analysis assist in studying evolutionary relatedness?

Answer 5

1. Organism with similar sequence are closely related

2. Organism with vastly different sequence are probably only distantly related

Question 6

A Dideoxy Chain Termination DNA Sequencing in vitro DNA Synthesis reaction
requires what 4 elements?

Answer 6

1. Single stranded DNA template
2. A Primer that anneals to template
3. DNA polymerase
4. Each of the nucleotide bases

Question 7

Why does Dideoxy Chain Termination DNA Sequencing in vitro DNA Synthesis require a primer?

Answer 7

DNA polymerase needs a primer because it can only add to the 3’ OH

Question 8

How is one nucleotide base in Dideoxy Chain Termination DNA Sequencing labeled for detection?

Answer 8

The tag can be either radioactive, or more recently fluorescent

Question 9

What are the 4 deoxynucleotides that are used in DNA synthesis?

Answer 9

1. dATP
2. dGTP
3. dCTP
4. dTTP

Question 10

How do we stop a Dideoxy Chain Termination DNA Sequencing reaction before it copies the entire strand?

Answer 10

Dideoxynucleotides (ddNTP) are added

Question 11

What are dideoxynucleotides (ddNTP)? What do they do?

Answer 11

They are identical to nucleotides, but lack 3’ OH. When a dideoxynucleotide is incorporated, the DNA molecule can not be extended further, resulting in chain termination.

Question 12

What are the steps of a 4 in vitro DNA Synthesis reaction?

Answer 12

1. A small amount of different ddNTP (ddATP, ddGTP, ddCTP, ddTTP) is added to each of 4 tubes, along with template DNA and primer.
2. The tubes are incubated at an appropriate temperature so that primers can anneal to template DNA.
3. When ddNTP is incorporated (infrequently), synthesis stops
4. Chains can be read, via electrophoresis, based upon the location of each tagged ddNTP.

Question 13

Electrophoresis utilizes seive-like action to separate DNA chains by _______. It is driven by the force of ________.

Answer 13

1. Length between ddNTP stops

2. Electricity

Question 14

In 1983, Kary Mullis “invented” what?

Answer 14

Polymerase Chain Reaction (PCR)

Question 15

When would one use PCR?

Answer 15

If you need a large amount of DNA, ex. to sequence it

Question 16

How fast can PCR make millions of copies of a given region of DNA?

Answer 16

Within a matter of hours

Question 17

What are the 3 basic steps of PCR?

Answer 17

1. Double stranded DNA is “opened” to single stranded DNA by heating to 95ºC
2. Specific short single stranded primers are allowed to anneal by lowering temperature to 50ºC
3. DNA synthesis occurs at 72ºC

Question 18

Who choses DNA primers for PCR, and why?

Answer 18

Primers are chosen by you to amplify the region you are interested in.

Question 19

How does DNA synthesis occur in PCR?

Answer 19

By adding nucleotides to the 3’ OH of the primers

Question 20

Where does the DNA polymerase used in PCR come from, and why?

Answer 20

The polymerase comes from the thermophile, Thermus aquaticus. A thermophile is used so that more polymerase does not have to be used during PCR. The bacteria can withstand high temperatures without their proteins breaking down. Therefore, the entire process can be automated.

Question 21

Which thermophile polymerase is associated with a higer error rate, and which is associated with a lower error rate?

Answer 21

TAC= higher error rate; PFU= lower error rate

Question 22

When is it favorable to incur an error during the PCR process? Why?

Answer 22

It is better for an error to occur later in the process, so that it effects less copies of DNA.