DNA Technology

Question 1

What is recombinant DNA?

Answer 1

The combined DNA of two different organisms

Question 2

What is the process of using reverse transcriptase?

Answer 2

Reverse transcriptase catalyses DNA production from RNA

1) A host cell with desired protein is selected
2) Reverse transcriptase produces DNA from the mRNA in the host cell
3) Complementary DNA is produced (cDNA)
4) DNA polymerase forms a double helix

Question 3

What do restriction endonucleases do?

Answer 3

The cut a double stand of segment of DNA at a specific recognition sequence

Question 4

What is a recognition sequence?

Answer 4

A 6 base sequence that is read the same both forwards and backwards i.e. a palindrome

Question 5

What is a vector?

Answer 5

A means of transporting DNA to the host cell

Question 6

Which enzyme is used to join the sugar phosphate backbone?

Answer 6

DNA Ligase (DNA polymerase bind the nucleotides together)

Question 7

What is the process of transformation?

Answer 7

It involves the plasmids and bacterial cells being mixed together in a medium containing Ca2+ ions

Question 8

How do you check which bacterial cells have taken up the vector?

Answer 8

1) Grow all bacterial cells in a medium containing ampicillin
2) Any cells which survive will have taken up the plasmid containing a gene for ampicillin resistance
3) Only bacteria that have taken up the plasmid will survive

Question 9

How do you insure which bacterial cells have both ampicillin and tetracycline resistance?

Answer 9

1) The original surviving cells are cultured on an agar plate
2) A tiny sample of each colony is placed on a different plate in the exact same position
3) Plate 2 will contain tetracycline
4) The colonies not killed on plate 2 will be those which contained the modified gene

Question 10

What are 2 other ways of identifying the desired species?

Answer 10

1) Fluorescent markers- using GFP

2) Enzyme markers- using lactase which turns are desired substrate blue

Question 11

What is DNA polymerase and why is it used in PCR?

Answer 11

An enzyme that joins nucleotides, used as it doesn’t denature at high temperatures

Question 12

What is a primer?

Answer 12

Short sequence of bases, complementary to those as the end pf a DNA strand

Question 13

What is the process of PCR?

Answer 13

1) The DNA sample, DNA polymerase and primers are placed in the machine and heated to around 95 degrees which separates the DNA stand.
2) Cool the mixture to around 55 degrees which allows the primers to join with their complementary bases.
3) Raise temperature to optimum of DNA polymerase around 70 degrees which will join up nucleotides between the primer and the end of the DNA molecule.
4) Cycle is repeated and each cycle yield double the amount of DNA.

Question 14

What are some advantages and disadvantages of in vivo (vectors)?

Answer 14

Advantages 
1) Very accurate 
2) Produces useful GM products (vaccines)
3) Little risk of contamination
Disadvantages
1) Could harm the host
2) Host may not take up the vector
3) Detection may fail

Question 15

What are some uses of DNA technology?

Answer 15

1) GM of crops i.e. increase in yield
2) Medicinal i.e. antibiotics, enzymes, hormones
3) GM animals

Question 16

What is cystic fibrosis and how is it caused?

Answer 16

A disorder where epithelial membranes become defective and secrete thick heavy mucus. It is caused by a deletion mutation.

Question 17

What are the treatments for CTFR using gene therapy?

Answer 17

1) Gene replacement
2) Gene supplementation- adding copies of the healthy gene alongside the defective one but the healthy gene is dominant.
3) Germline cell therapy- replacing the defective gene whilst in the fertilised egg so all daughter cells will have the healthy gene.
4) Somatic cell therapy- targets only the affected tissue and so cannot be passed to offspring. This treatment is not permeant.

Question 18

How do you deliver CTFR genes?

Answer 18

Use adenovirus as the host, which takes up the normal CF gene as a vector.

Question 19

How do the CF genes pass the phospholipid bilayer?

Answer 19

The genes are wrapped in lipid molecules forming a liposome

Question 20

What is DNA hybridisation?

Answer 20

The use of a DNA probe attached to a radioactive label with a complementary sequence to the gene you wish to identify.

Question 21

What is gel electrophoresis?

Answer 21

The technique used to separate DNA fragments in order of length.

Question 22

What is restriction mapping?

Answer 22

Electrophoresis only works for short fragments of DNA, therefore DNA must be cut my restriction enzymes

Question 23

What are the 5 stages of genetic fingerprinting?

Answer 23

1) Extraction and amplification of DNA
2) Digestion- uses restriction enzymes which will isolate core sequences
3) Separation- uses of electrophoresis to separate fragments
4) Hybridisation- complementary DNA probes to the core sequences are added
5) Development- X-ray films