Topic 21 - Recombinant DNA Technology

Question 1

What is recombinant DNA?

Answer 1

Introduction of a foreign gene into the DNA of another organism. The resulting organism is known as a genetically modified organism.

Question 2

Why does DNA fragment from one organism, inserted into another, still produce the same protein?

Answer 2

The genetic code is universal, it’s the same in all organisms. Transcription and translation is also universal.

Question 3

Method 1: Isolation of DNA fragments - Using reverse transcriptase to produce DNA from mRNA.

Answer 3

1) The mRNA has been transcribed from gene of interest.
2) Reverse transcriptase used to synthesise a single strand of complementary DNA (cDNA) from mRNA molecule.
3) DNA polymerase then forms the other strand of DNA from free nucleotides = double strand.

Question 4

What is the advantage of using mRNA rather than DNA?

Answer 4

• Quantity: cells that make this protein will have lots of RNA.
• Searching: Don’t have to find gene on DNA in chromosome.
• The genetic code: Introns have already been removed.

Question 5

Method 2: Isolation of DNA fragments using restriction endonucleases.

Answer 5

Restriction endonucleases found in bacteria that cut DNA at specific base sequences, so can be used to cut out a desired gene from the rest of genome.

Sticky ends:
- Cut ends of DNA, one strand longer.
- Have a strand of single stranded DNA, can attach to complementary DNA bases.
- Will join with another sticky end but only if cut with same RE.

Restriction Endonucleases:
- Highly specific active sites that catalyse hydrolysis of sugar-phosphate backbone of both strands of DNA molecule.

Question 6

Why might some recognition sequences not be useful when isolating a particular gene?

Answer 6

If gene has recognition sequence in the middle, it’s useless as don’t want to cut the middle of the gene.

Question 7

Method 3: Isolation of DNA fragments - fragments of DNA produced by creating gene in a ‘gene machine’ in a lab.

Answer 7

1) Desired nucleotide sequence derived from desired protein.
2) Nucleotide sequence fed into computer, which is checked. Small, single strands formed which assemble to form complete gene.
3) Oligonucleotides are created and joined to make gene.
4) Gene is replicated many times using PCR, makes gene into double-stranded DNA.
5) Using sticky ends, gene may be inserted into bacterial plasmid, acts as vector.

Question 8

What is a plasmid?

Answer 8

Circular piece of DNA, separate from main bacterial DNA, contains only a few genes.

Question 9

How is DNA prepared for insertion?

Answer 9

Before being inserted, extra lengths of DNA must be added to DNA fragment. These include:
- A promotor: length of DNA added before DNA fragment, to which transcriptional factors and RNA polymerase can bind to initiate transcription.
- A terminator: length of DNA added after DNA fragment, which causes RNA polymerase to be released and stop transcription.

Question 10

Why are genes inserted into plasmids?

Answer 10

Plasmid acts as a ‘carrier’, or vector which can then be introduced back into a bacterial cell. Complimentary sticky ends makes this possible.

Question 11

How is recombinant plasmids introduced into host cells?

Answer 11

1) Plasmids mixed with bacterial cells in ice-cold medium containing calcium ions.
2) Heat shock applied for 2 minutes. Bacterial cell membranes increase in permeability allowing plasmids to pass through into cell.
3) Only a few bacterial cells will take up plasmids, so gene markers can identify which bacterial cells have been successful in taking up plasmids.

Question 12

What is the problem with introducing recombinant plasmids into host cells?

Answer 12

• Not all plasmids will take up the gene.
• Plasmids may join together.
• Recombinant DNA may join together.

Question 13

Identification using gene markers: Antibiotic-resistance markers

Question 14

Identification using gene markers: Fluorescent markers

Answer 14

1) Insert gene for a fluorescent protein into plasmid that also has the gene for ampicillin resistance.
2) Insert gene of interest into centre of a gene for a fluorescent protein.
3) Transfer plasmid into bacterial cell and grow in agar containing ampicillin.
4) Any bacterial cells that took up the plasmids will not be killed and will not be able to glow.

Question 15

Identification using gene markers: Enzyme markers

Answer 15

1) Particular substrate that is usually colourless but turns blue when lactase acts upon it.
2) Insert chosen gene into gene that makes lactase, thereby inactivating the lactase gene. This plasmid contains gene for ampicillin resistance.
3) Transfer plasmid into bacterial cells.
4) Grow in agar containing ampicillin and the colourless substrate.
5) Any colonies that have taken up transformed plasmid will not be able to change its colour to blue.
6) Any colourless spots will indicate which cells have been transformed.

Question 16

What is ‘in vivo’?

Answer 16

Performed in a living organism.

Question 17

What is ‘in vitro’?

Answer 17

Performed in a test tube.

Question 18

What is the polymerase chain reaction?

Answer 18

A method of copying or amplifying DNA fragments in the lab. It’s an automated process so is rapid and efficient.

Question 19

What is needed for the polymerase chain reaction?

Answer 19

1) DNA fragments to be copied.
2) DNA polymerase to join together thousands of DNA nucleotides. Found in bacteria found in hot springs.
3) Primers to allow attachment of DNA polymerase and prevent two strands of DNA from joining.
4) Free DNA nucleotides.
5) Thermocycler to regulate and vary temperature over a period of time.

Question 20

What are the stages of the polymerase chain reaction?

Answer 20

1) Strand separation - DNA heated at 95 degrees c for 5 minutes.
2) Mix with primers.
3) Annealing of primers - Mixture cooled to 55 degrees c.
4) Mix with free nucleotides and DNA polymerase.
5) DNA synthesis - Mixture heated to 70 degrees c (optimum for DNA polymerase)
6) Finish with 2 identical DNA molecules.
7) Repeats - With every cycle, the amount of DNA increases by 2x.

Question 21

What are the advantages of using recombinant DNA technology as opposed to selective breeding to improve the productivity of crops and animals?

Answer 21

• Can move one gene at a time so more precise.
• Can make new genetic combinations not previously possible.

Question 22

What are the advantages of recombinant DNA uses to humans?

Answer 22

• Increasing yield and nutritional value.
• Pest and disease resistant crops/animals.
• Crops resistant to herbicides.
• Crops resistant to extreme weather.
• Cultivating microorganisms for medicines.
• Production of active ingredients of medicines and vaccines.

Question 23

What are some examples of genetically modified microorganisms?

Answer 23

• Bacteria and fungi naturally produce antibiotics and can be GMd to produce it in larger quantities.
• Enzymes required for food and beverage production. Brewing industry requires amylase to break down starch to produce glucose as energy source for yeast. Lipase in cheese production.

Question 24

What are some examples of genetically modified plants?

Answer 24

• Tomatoes can be given complementary gene to block translation of softening gene when they ripen to transport.
• Rice crops developed to withstand infection of RBSDV virus and other viruses.
• Some plants given gene to produce toxins that are insecticidal - block respiratory pathways, paralysis.

Question 25

What are some examples of genetically modified animals?

Answer 25

• Genes provide resistance to certain disease can be transferred from one animal to another.
• Genes for rare and expensive proteins can be inserted into animals like goats - protein produced in milk e.g. spider silk in goat milk.
• Growth hormone genes can be added to fish, sheep etc. For salmon, they can grow 30x as big at 10x the rate.

Question 26

What are some issues associated with DNA technology in agriculture?

Answer 26

• ‘Superweeds’ - weeds resistant to herbicides if GM crops interbreed with wild plants.
• Farmers may only plant one type of transformed crop, making them all vulnerable to disease.
• Organic farmers can have their crops contaminated by wind-blown seeds from GM crops and can’t sell their crops as organic, losing income.

Question 27

What are some issues associated with DNA technology in industry?

Answer 27

• Anti-globalisation activists oppose globalisation.
• Few, large biotechnology companies control some forms of GE. Companies get more powerful = smaller companies out of business.
• Some consumer markets like the EU won’t import GM food/products which can cause economic loss to producers.
• Without proper labelling, some people think they won’t have a choice about whether to consume food made using GE crops or not.

Question 28

What are some issues associated with DNA technology in medicine?

Answer 28

• Companies who own genetic engineering technologies may limit use of technologies that could be saving lives.
• Some people worry this technology could be used to make “designer babies” - babies that have characteristics chosen by parents. This is illegal at the moment.

Question 29

What causes cystic fibrosis?

Answer 29

• CFTR - channel protein used to transport chloride ions across epithelial cells.
• Water moves down water gradient by osmosis.
• CF: Recessive allele that codes for CFTR channel protein is mutated.
• Means that one amino acid is omitted from coded protein.
• Therefore, less water moves out of cell by osmosis so mucus becomes thicker and stickier.

Question 30

What is gene therapy?

Answer 30

‘Replacement’ of defective gene by introduction of healthy gene.

Question 31

What is the difference between transfection and transfected?

Answer 31

Transfection - Process of putting a corrected gene into a chromosome.

Transfected - The cell that has received the new gene.

Question 32

Gene therapy: What is somatic-cell therapy?

Answer 32

Targets the affected tissues. Not passed on to future generations. Long-term goal is to target undifferentiated stem cells so treatment is effective for lifespan of individual.

Question 33

Gene therapy: What is germ-line/cell therapy?

Answer 33

Replacing/supplementing the defective gene in the fertilised egg. All offspring will develop normally. Passed on to future generations.

Question 34

How could germ-line therapy be misused?

Answer 34

• Unscrupulous scientist could use the techniques to produce babies with inherited features that they themselves consider to be desirable.
• Producing babies with so called desirable features is called eugenics and was carried out by Nazis.

Question 35

What is a DNA probe and how does it work?

Answer 35

Short, single stranded sections of DNA. Base sequence that is complementary to part of target gene. Easily identifiable as they are labelled in some way e.g. radioactivity or fluorescence. Can’t attach to double-stranded DNA so DNA strands need separating.

Question 36

What is DNA Hybridisation?

Answer 36

• Can only bind with single-stranded DNA.
• Heated until strands separate.
• Add DNA probe and allow to cool. Probe will bind to complementary bases if mutant allele present.
• DNA washed to remove unattached probe.
• Hybridised DNA labelled and detected.

Question 37

The process of genetic fingerprinting

Answer 37

1) Extraction: Cells from sample broken down, DNA extracted.
2) Digestion: DNA cut into small fragments using restriction endonucleases, producing restriction fragments.
3) Separation: Fragments separated using gel electrophoresis. DNA fragments injected into wells and electric current applied along gel, DNA attracted to positive end of gel.
4) Hybridisation: DNA on gel transferred to nylon membrane, immersed in alkali to separate DNA strands. DNA probes attach to VNTRs sequences.
5) Development: Nylon sheet placed under X-ray film, radioactive probes on DNA fragments expose film, producing visible pattern of light and dark bands. Pattern of fragment distribution then analysed, they are unique to every individual expect identical twins.

Question 38

What are the five ways in which genetic fingerprinting can be used?

Answer 38

1) Determining genetic relationships e.g. figuring out paternity.
2) Assessing genetic variation in a population. Very similar genetic fingerprints = low genetic diversity.
3) Forensic analysis: DNA often left at scene of crime and GF can establish whether the person was likely to have been there at time of crime, but doesn’t say if they committed it.
4) Medical diagnosis - Compare someone’s GF to someone who already has the disease.
5) Animal and plant breeding - Prevent undesirable inbreeding during breeding programmes on farms or in zoos. Can also identify plants and animals that have a particular allele or desired gene.