DNA technology

Question 1

What are the 5 steps of making a protein using DNA technology?

Answer 1

1. Isolation
2. Insertion
3. Transformation
4. Identification
5. Growth/ Cloning

Question 2

Reverse Transcriptase

Answer 2

1. A cell that readily produces the protein is selected
2. These cells have large quantities of the relevant mRNA, which is extracted
3. Reverse transcriptase is then used to make DNA from RNA. This cDNA doesn’t include introns.
4. DNA polymerase is used to build up the complementary nucleotides on the cDNA template

Question 3

Restriction Endonucleases

Answer 3

Cut a DNA double strand at a recognition sequence.

Question 4

Blunt ends

Answer 4

When the DNA is cut between two opposite base pairs.

Question 5

Sticky ends

Answer 5

When the DNA is cut in a staggered fashion so that the bases of the sticky ends are a palindrome

Question 6

What is the importance of sticky ends?

Answer 6

If the same restriction endonuclease is used to cut DNA, then all the fragments produced will have ends that are complimentary to one another. Once the complimentary bases of 2 sticky ends have paired up, DNA ligase is used to join the phosphate-sugar framework. This allows the DNA of one organism to be combined with that of any other organism.

Question 7

Plasmid

Answer 7

Circular lengths of DNA, found in bacteria, which are separate from the main bacterial DNA.

Question 8

Why are Plasmids used as vectors?

Answer 8

Almost always contain genes for antibiotic resistance, restriction endonucleases are used at one of the antibiotic- resistance genes to break the plasmid loop.

Question 9

Transformation

Answer 9

Involves the plasmids and bacteria cells being mixed together in a medium containing calcium ions. The calcium ions and changes in temp. make the bacteria permeable, allowing the plasmids to pass through the cell membrane into the cytoplasm.

Question 10

Why won’t all the bacterial cells have the DNA fragment?

Answer 10

1. Only a few bacterial cells take up the plasmid when the 2 are mixed together
2. Some plasmids will have closed up again without incorporating the DNA fragment

Question 11

What method is used to find out which bacterial cells have taken up the plasmid?

Answer 11

1. All the bacterial cells are grown on a medium that contains ampicillin
2. Bacterial cells that have taken up the plasmids will have acquired the gene for ampicillin resistance
3. These bacterial cells are able to break down the ampicillin and therefore survive
4. The bacterial cells that have not taken up the plasmids will not be resistant to ampicillin and therefore die

Question 12

Outline Replica Plating

Answer 12

1. The bacterial cells that survived the treatment with ampicillin are known to have taken up the plasmid
2. These cells are cultured, spreading thinly on nutrient agar plates
3. Each separate cell on plate will grown into a genetically identical colony
4. A tiny sample of each colony is transferred onto a replica plate in exactly the same position as the colonies on original plate
5. Replica plate contains tetracycline
6. The colonies killed by the antibiotic will be the ones that have taken up the required gene
7. The colonies in the exact same position on original plate are the ones that possess the required gene

Question 13

Fluorescent markers

Answer 13

Green fluorescent protein (GFP) derives from a jellyfish. The gene to be cloned is transplanted into the centre of the GFP gene. Any bacterial cell that has taken up the plasmid with the gene is unable to produce GFP.

Question 14

Advantages of Fluorescent markers over replica plating?

Answer 14

Bacterial cells with the desired gene aren’t killed so no need for replica plating. This makes the process more rapid.

Question 15

Enzyme markers

Answer 15

Lactase will turn a particular colourless substrate blue. The required gene is transplanted into the gene that makes lactase. If a plasmid with the required gene is present in bacterial cell, the colonies grown from it will not produce lactase. Therefore, when these bacterial cells are grown on the colourless substrate they won’t change its colour.

Question 16

Outline polymerase chain reaction

Answer 16

1. The DNA fragments, primers and DNA polymerase are placed in a vessel in the thermocycler. The temp. is increased to 95C, causing the 2 strands of the DNA fragments to separate.
2. The mixture is cooled to 55C causing the primers to anneal to their complimentary bases at the end of the DNA fragment. The primers provide the starting sequences for DNA polymerase to begin DNA copying because DNA polymerase can only attach nucleotides to the end of an existing chain. Primers also prevent the 2 separate strands from simply rejoining.
3. The temp. is increased to 72C. This is the optimum temp. for the DNA polymerase to add complementary nucleotides along each of the separated DNA strands.

Question 17

What are primers?

Answer 17

Short sequences of nucleotides that have a set of bases complementary to those at one end of each of the two DNA fragments.

Question 18

What are the advantages of in vitro gene cloning?

Answer 18

1. Extremely rapid production of genes. Particularly valuable where there is only a minute amount of DNA available e.g. crime scene -> forensics
2. It doesn’t require living cells or complex culturing techniques.

Question 19

What are the advantages of in vivo gene cloning?

Answer 19

1. It is useful where we wish to introduce a gene into another organism. Plasmid can be used to deliver the gene into another organism.
2. It involves almost no risk of contamination. A gene has to be cut by the same restriction endonuclease. Contaminant DNA won’t be taken up by the plasmid.
3. Very accurate. DNA copied has few errors.
4. It cuts out specific genes. Culturing of transformed bacteria produces many copies of a specific gene.
5. It produces transformed bacteria that can be used to produce large quantities of gene products. -> proteins for commercial or medical use.

Question 20

How do genetic modifications benefit humans?

Answer 20

1. Increasing the yield from animals or plant crops
2. Improving the nutrient content of foods
3. Introducing resistance to disease and pests
4. Making crop plants tolerant to herbicides
5. Developing tolerance to environmental conditions
6. Making vaccines
7. Producing medicines for treating diseases

Question 21

What are 3 main groups of substances produced using genetically modified bacteria?

Answer 21

1. Antibiotics -> bacteria that increase quantitity of antibiotics produced
2. Hormones -> Insulin produced is identical to human insulin and avoids killing animals and need to modify insulin before injection
3. Enzymes - food industry eg. amylases to break down starch during beer prod. / proteases to tenderise meat

Question 22

Examples of genetically modified plants

Answer 22

1. Herbicide-resistant crops
2. Disease-resistant crops
3. Pest-resistant crops -> gene tat allows plant to make a toxin (harmless to animals)
4. GM Tomatoes -> prevents the mRNA coding for softening to be expressed. So softening enzyme not produced -> allows tomatoes to develop flavour without problems associated with harvesting, transporting and storing fruit

Question 23

Example of genetically modified animals

Answer 23

Transfer of genes from an animal that has natural resistance to a disease to a disease in a different animals.

Question 24

Outline extraction of anti-thrombin from goats milk

Answer 24

1. Mature eggs are removed from female goats + fertilised
2. The normal gene for anti-thrombin prod. from a human is added to the fertilised eggs with gene that codes for proteins in goats’ milk
3. Genetically transformed eggs are implanted into female goats
4. Resulting goats with anti-thrombin gene are crossbred, to give a herd with rich anti-thrombin milk prod.
5. Anti-thrombin is extracted from milk, purified and given to humans unable to manufacture their own

Question 25

What are the benefits of recombinant DNA technology?

Answer 25

1. Microorganisms can be modified to produce a range of substances
2. Microorganisms can be used to control pollution
3. GMP’s can be transformed to produce a specific substance in a particular organ of the plant. These organs can then be harvested and the desired substance extracted.
4. GMC’s can be engineered to have econ. and environ. advantages. e.g. able to survive drought
5. GMC’s can help prevent certain diseases. e.g. Rice Vit A
6. GMA’s can produce expensive drugs, antibiotics, hormones/ enzymes cheaply

Question 26

What are the risks of recombinant DNA technology?

Answer 26

1. Impossible to predict ecological consequences of releasing genetically engineered organisms into environ,
2. A recombinant gene may pass from the organism it was placed in, to a completely different one
3. Any manipulation of the DNA of a cell will have consequences for the metabolic pathways within that cell.
4. GMB often have antibiotic resistance marker genes added. Horizontal transmission -> conjugation
5. All genes mutate

Question 27

What is the first stage of PCR?

Answer 27

Exponential amplification: At every cycle, the amount of product is doubled (assuming 100% reaction efficiency). The reaction is very sensitive: only minute quantities of DNA must be present.

Question 28

What is the second stage of PCR?

Answer 28

Levelling off stage: The reaction slows as the DNA polymerase loses activity and as consumption of reagents such as dNTPs and primers causes them to become limiting.

Question 29

What is the third stage of PCR?

Answer 29

No more product accumulates due to exhaustion of reagents and enzyme.