MG (Genetic engineering)

Question 1

what’s DNA ligase

Answer 1

enzyme that catalyses the joining of sugar and phosphate groups (phosphodiester) within DNA via condensation reaction

Question 2

DNA polymerase vs DNA ligase

Answer 2

DNA polymerase adds nucleotides to the template strand (5’ to 3’), and ligase seals the gaps between the nucleotides on lagging strand (3’ to 5’) by joining Okazaki fragments

Question 3

what are plasmids

Answer 3

small loops of DNA in prokaryotic cells

Question 4

what is Recombinant DNA

Answer 4

a composite DNA molecule created in vitro by joining foreign DNA with a vector molecule (plasmid)

Question 5

What’s genetic engineering

Answer 5

when genes are isolated from one organism and inserted into another organism, using vectors.

Question 6

What are the main steps in genetic engineering

Answer 6

1. The required gene is obtained
2. A copy of the gene is placed inside a vector
3. The vector carries the gene into a recipient cell
4. The recipient expresses the novel gene

Question 7

Step 1 - How is the required gene obtained in genetic engineering

Answer 7

• mRNA obtained from cells where the required gene is being expressed. Reverse transcriptase catalyses formation of a single strand of complementary DNA (cDNA) using the mRNA as a template. The addition of primers and DNA polymerase can make this cDNA into a double-stranded length of DNA, whose base sequence codes for the original protein.
• If scientists know the nucleotide sequence of the gene, then the gene can be synthesised using an automated polynucleotide synthesiser.
• If scientists know the sequence of the gene, they can design PCR primers to amplify the gene from the genomic DNA.
• A DNA probe can be used to locate a gene within the genome and the gene can then be cut out using restriction enzymes.

Question 8

what’s a vector

Answer 8

in gene technology anything that can carry/insert DNA into a host organism; examples of such vectors include plasmids, viruses and certain bacteria.

Question 9

Step 2 - How is the gene placed into a vector

Answer 9

• Plasmids mixed with restriction enzymes that will cut the plasmid at specific recognition sites.
• The cut plasmid has exposed unpaired nucleotide bases, called sticky ends
• If free nucleotide bases, complementary to the sticky ends of the plasmid, are added to the ends of the gene to be inserted, then the gene and cut plasmid should anneal. DNA ligase enzyme catalyses the annealing.
• A gene may be sealed into an attenuated (weakened) virus that could carry it into a host cell.

Question 10

What are the 5 methods used to insert vector into the recipient cell

Answer 10

• Heat shock treatment
• Electroporation
• Electrofusion
• Transfection
• T1 (recombinant) plasmids

Question 11

What’s electrofusion

Answer 11

electrical fields help to introduce DNA into cells.

Question 12

What’s electroporation

Answer 12

a pulse of electricity makes the recipient cell membrane more porous.

Question 13

what’s heat shock treatment ?

Answer 13

• bac subjected to alternating periods of cold and hot in the presence of calcium chloride,
• their walls and membranes will become more porous and allow in the recombinant vector.
• Ca2+ reduces repulsion between -vely charged parts of DNA and phospholipids

Question 14

what’s transfection

Answer 14

DNA can be packaged into a bacteriophage, which can then transfect the host cell.

Question 15

How are T1 (recombinant) plasmids used to get vector into recipient cell

Answer 15

the plasmids are inserted into the bacterium Agrobacterium tumefaciens, which infects some plants and naturally inserts its genome into the host cell genomes.

Question 16

How is reverse transcriptase used

Answer 16

• Find a cell that produces the protein you require (e.g β cells of the Islets of Langerhans in the pancreas produce insulin).
• Extract the mRNA from the cells.
• Use reverse transcriptase to make cDNA from the RNA.

Question 17

what’s the direct method of introducing gene into recipient

Answer 17

• If plants are not susceptible to A. tumefaciens, then direct methods can be used.
• Small pieces of gold or tungsten are coated with the DNA and shot into the plant cells. This is called a “gene gun”.

Question 18

What’s genetic engineering also known as?

Answer 18

• recombinant DNA technology
• genetic modification

Question 19

function of reverse transcriptase found in retroviruses (HIV)

Answer 19

• HIV contains RNA that they can inject into the hosts genome
• reverse transcriptase catalyses formation of cDNA using RNA as template
• useful in genetic engineering

Question 20

restriction enzymes create ……… and ……….. ends

Answer 20

• sticky (staggered cuts)
• blunt (cuts not staggered)

Question 21

How is insulin obtained from GM bacteria

Answer 21

1. adding reverse transcriptase enzyme makes a single strand of cDNA and treatment with DNA polymerase makes a double strand - the gene
2. addition of free unpaired nucleotides at the ends of DNA produces sticky ends
3. with the help of ligase enzyme, the insulin gene can be inserted into plasmids extracted from E.coli bac ( now recombinant DNA)
4. Bac mixed with recombinant plasmids and subjected to heat shock in presence of CaCl2, so they’ll take up the plasmids

Question 22

what are the two antibiotic resistance genes in bacterial plasmids

Answer 22

• resistance to tetra
• resistance to amp

Question 23

How do you find the plasmids that have taken up the desired gene?

Answer 23

• desired gene is inserted into the plasmid midway through the gene for tetra resistance.
• Some plasmids take up the gene some do not
• all colonies grow in amp plate (as all plasmids have gene for amp resistance)
• Bacteria only grow on the tetracycline plate if they have NOT got the insulin gene.

Question 24

How is the risk of transgenic bacteria escaping into the wild controlled?

Answer 24

• genes knocked out so they can’t synthesise particular nutrients so will only survive in the lab where they are supplied those nutrients in growth medium

Question 25

Benefits of GM microorganisms (E.coli)

Answer 25

• insulin production for treatment of diabetes
• make human growth hormone to treat children with pituitary dwarfism

Question 26

Hazards of GM microorganisms (E.coli)

Answer 26

• can escape into wild + transfer marker genes for antibiotic resistance to other bac
• HOWEVER, bac modified so can’t grow outside of lab

Question 27

Benefits of GM Bt tobacco and Bt maize

Answer 27

• genetically modified to produce toxin to kill pests do don’t need to spray it, other organisms not contaminated

Question 28

Hazards of GM Bt tobacco and Bt maize

Answer 28

• Bt is toxic to monarch butterflies
• HOWEVER, they don’t take nectar from maize or tobacco plants in the wild

Question 29

Benefits of GM soya beans

Answer 29

genetically modified to be resistant to herbicide

Question 30

Hazards of GM soya beans

Answer 30

• superweeds may arise
• HOWEVER, this hasn’t happened

Question 31

Benefits of GM golden rice

Answer 31

rice modified to contain beta carotene (precursor to vitamin A ), preventing blindness

Question 32

Hazards of GM golden rice

Answer 32

• concerns about farmers having to buy seeds every year
• HOWEVER, companies offered free licenses

Question 33

Benefits of GM plantain

Answer 33

• enhanced to contain more zinc to combat zinc deficiency
• zinc is an enzyme cofactor

Question 34

Hazards of GM plantain

Answer 34

• inserted genes will be expressed in us
• HOWEVER, all the food we eat contains genes + we digest them

Question 35

Benefits of GM crop plants resistant to pests

Answer 35

• no need to use pesticides

Question 36

Hazards of GM crop plants resistant to pests

Answer 36

• people die due to exposure when applying them
• concerns that farmers wouldn’t want GM seeds and wouldn’t have the choice to buy non-GM seeds.
• HOWEVER, non-GM seeds are stills sold

Question 37

Benefits of GM pathogens

Answer 37

• used as vectors in gene therapy
• modified to have no virulence so can be used in vaccines without making patient ill

Question 38

Hazards of GM pathogens

Answer 38

• in gene therapy, allele may be inserted into the genome in a way that increase risk of cancer or interfere with gene regulation

Question 39

Benefits of GM mice

Answer 39

• used to develop therapies for breast and prostate cancer
• certain genes knocked out in them so their function can be determined

Question 40

Hazards of GM mice

Answer 40

• some object to the use of animals for testing
• HOWEVER, there are strict regulations that govern their welfare + we have benefited from usage of animals

Question 41

what’s ‘pharming’

Answer 41

1. Creating animal models.
   - Adding/removing genes so that the animal develops certain diseases to act as models in the development of new treatments
   - Eg. ‘Knock out mice’ have genes deleted so that they develop cancer. Treatments can then be trailed.
2. Creating human proteins.
   - Human gene can be inserted into a fertilised egg (cow/pig/sheep).
   - Promotor sequence could mean that the gene is only expressed in the mammary glands – milk will then contain the required human protein.

Question 42

What are palindromic sequences?

Answer 42

sections of DNA that consist of ANTIPARALLEL BASE PAIRS (base pairs that read in the opposite direction).

Question 43

Explain cutting out DNA fragments using restriction enzymes

Answer 43

• Restriction enzymes recognise specific palindromic sequences (known as recognition sequences) and cut the DNA at these places. Different restriction enzymes cut at different specific palindromic sequences, because the shape of the recognition sequence is complementary to an enzyme’s active site.
• If the recognition sites are present at either side of the DNA fragment you want, you can use restriction enzymes to separate it from the rest of the DNA.

Question 44

what’s the role of Restriction endonucleases

Answer 44

cut DNA at certain recognition points via hydrolysis