TCOGE: Gene technologies and in vivo cloning

Question 1

What is recombinant DNA?

Answer 1

The combined DNA of 2+ organisms.

Question 2

Organisms that contain recombinant DNA are ______.

Answer 2

Transgenic

Question 3

Why can transgenic organisms translate recombinant DNA?

Answer 3

Genetic code is universal

Question 4

What is recombinant DNA used for?

Answer 4

Gene therapy

Question 5

Name 3 issues associated with recombinant DNA use in gene therapy:

Answer 5

• Expensive
• Side effects
• Ethics

Question 6

How do restriction endonucleases work?

Answer 6

• Identifies recognition sequence (specific set of bases).
• Breaks phosphodiester bonds between adjacent nucleotides.
• This cuts the DNA at the recognition sequence.
• Occurrence of two recognition sequences produces a DNA fragment.

Question 7

What type of molecule is a restriction endonuclease?

Answer 7

Enzyme

Question 8

Explain the process of how reverse transcriptase produces DNA fragments:

Answer 8

• Reverse transcriptase first adds DNA nucleotides to an mRNA molecule- forming complementary DNA (cDNA).
• After a complementary DNA strand is produced, an enzyme destroys the mRNA molecule.
• A second DNA strand is built by DNA polymerase.

Question 9

How does a gene machine produce a DNA fragment?

Answer 9

• At the starting point of interest, the amino acid coded for by 3 bases is identified.
• From this scientists then work backwards to identify the corresponding mRNA sequence.
• Using the mRNA sequence, the complementary DNA sequence can be identified.
• This DNA sequence is checked for safety.
• The gene machine assembles short strands of the DNA, one nucleotide at a time, which are then assembled into one complete DNA fragment.

Question 10

Why is using a gene machine the fastest method to produce a DNA fragment?

Answer 10

Does not rely on reactions carried out by enzymes.

Question 11

What are the 3 methods that can be used to obtain a DNA fragment?

Answer 11

• Restriction endonuclease
• Reverse transcriptase
• Gene machine

Question 12

What is ‘in vivo’ gene cloning?

Answer 12

Gene cloning that occurs inside a living organism

Question 13

What is ‘in vitro’ gene cloning?

Answer 13

Gene cloning that occurs outside a living organism.

Question 14

What do scientists use as a host cell for in vivo cloning?

Answer 14

Bacteria

Question 15

What does a vector do?

Answer 15

Carries the DNA fragment into the host cell.

Question 16

In bacteria, a commonly used vector is a _______.

Answer 16

Plasmid

Question 17

What bonds between the complementary bases of the DNA fragment and plasmid?

Answer 17

Hydrogen

Question 18

What does DNA ligase do?

Answer 18

Attaches DNA nucleotides to the plasmids nucleotides, forming phosphodiester bonds

Question 19

In vivo gene cloning uses DNA fragments that have been produced by the method of ______ ________.

Answer 19

Restriction endonuclease

Question 20

DNA fragments from in vivo cloning have ______ ends.

Answer 20

Sticky

Question 21

What type of molecule is DNA ligase?

Answer 21

Enzyme

Question 22

Where about on a gene does transcription start?

Answer 22

Promoter region

Question 23

Where about on a gene does transcription end?

Answer 23

Terminator region

Question 24

What is the promoter region?

Answer 24

A sequence of bases that acts as a binding site for RNA polymerase and transcription factors.

Question 25

What stage of photosynthesis are promoters and terminators involved in?

Answer 25

Transcription

Question 26

How does a terminator region cause transcription to end?

Answer 26

Acts as a detachment site for RNA polymerase and transcription factors.

Question 27

What is transformation of a plasmid?

Answer 27

The process of adding a plasmid to a host cell

Question 28

Explain how plasmids enter a cell via transformation:

Answer 28

- Calcium ions are added, which bring the plasmid close to the cell membrane. - Heat shock

Question 29

Name 3 reasons why some bacteria in 'in vivo' gene cloning may not contain a recombinant plasmid:

Answer 29

- Some of the plasmids may rejoin without incorporating a DNA fragment. - Some DNA fragments will join to each other, rather than a plasmid. - Some bacteria won't take up a recombinant plasmid during transformation.

Question 30

What are the two marker genes that can be used to identify taken up plasmids by bacteria?

Answer 30

- Antibiotic resistance - Flourescence

Question 31

An antibiotic resistance gene can be added to a plasmid. What will happen to bacteria with this plasmid in an antibiotic solution?

Answer 31

Will survive

Question 32

Two antibiotic resistance marker genes can be added to a plasmid, antibiotic reistance gene 1 and 2. What will happen if a bacteria has taken up the DNA fragment on antibiotic resistance gene 2?

Answer 32

Bacteria will be killed by antibiotic solution, that the other desired gene has replaced the antibiotic resistance gene of.

Question 33

What is replica plating?

Answer 33

- Involves transferring a sample of the bacteria onto a new plate. - Comparing two samples of the same bacteria.

Question 34

Why are two antibiotic solutions used?

Answer 34

- 1st antibiotic will identify if the bacteria has taken up a plasmid at all (as it will have the antibiotic resistance gene) - 2nd antibiotic will identify if the bacteria has taken up the recombinant plasmid (antibiotic resistance gene for the second antibiotic will be disrupted by the recombinant plasmid)

Question 35

What other 2 marker genes can be used instead of the antibiotic reisistance gene?

Answer 35

- Fluorescence - Enzyme

Question 36

Bacteria that have taken up the recombinant plasmid _____ show the effects of the marker gene.

Answer 36

Wont (maker gene is disrupted by the recombinant plasmid)

Question 37

What would a scientist add to a plasmid to identify whether a DNA fragment has been incorporated into the plasmid?

Answer 37

Marker gene

Question 38

What are the benefits of using a fluorescent marker gene over an antibiotic resistance marker gene?

Answer 38

- Fluorescence is an observable property - No risk of antibiotic resistance being passed in to other bacteria - Fluorescence marker genes are more economical - There is no need for replica so it is faster