3.5 Genetic modification and biotechnology

Question 1

What is gel electrophoresis used for?

Answer 1

Separating proteins or fragments of DNA according to size.

Question 2

How does gel electrophoresis work?

Answer 2

Gel electrophoresis involves separating charged molecules in an electric field, according to their size and charge. Samples are placed in wells cast in a gel. This gel is immersed in a conducting fluid and an electrical field is applied. Molecules in the sample that are charged will move through the gel. Molecules with opposite charges will move in different directions. Proteins may be negatively or positively charged and are separated according to their charge.
The DNA is broken up into fragments so that it is small enough to move through the mesh of filaments in the gel. All DNA molecules are negative and so move in the same direction but not all at the same rate. Small fragments move faster than long ones so they move further in a given time. Therefore gel electrophoresis can be used to separate fragments of DNA according to size.

Question 3

How do you amplify DNA?

Answer 3

PCR can be used to amplify small amount of DNA. PCR copies DNA sequences. A sequence is selected for copying by using a primer that binds to the start of the desired sequence. The primer binds by complementary base pairing.

Question 4

When do we used PCR?

Answer 4

PCR can be used to amplify small amounts of DNA. Starting with just one molecule of DNA, within an hour or two millions can be made. This makes it possible to study DNA from for example a forensic scene or fossils without the risk of using up a limited sample.

Question 5

What else can we used PCR for?

Answer 5

Testing the presence of genetically modified ingredients in food. For example, we put in a primer that binds to the genetically modified ingredient and if it is present it is amplified by the PCR but if it is not then there is no effect.

Question 6

How can we DNA profile?

Answer 6

DNA profiling involves comparison of DNA.

• A sample of DNA is obtained, either from a known individual or a crime scene or fossil.
• Sequences in the DNA that vary considerably between individuals are selected and copied by PCR.
• The copied DNA is split into fragments using restriction endonucleases.
• The fragments are separated using gel electrophoresis.
• This produces a pattern of bands that is always the same with DNA taken from one individual. This is the individuals DNA profile.
• The profiles of different individuals can be compared to see which bands are the same and which are different.

Question 7

How do we split DNA in DNA profiling?

Answer 7

Using endonucleases

Question 8

What actually does a DNA profile look like?

Answer 8

A pattern of bands in gel, from gel electrophoresis that is always the same which DNA taken from one individual.

Question 9

How does DNA profiling help with forensic investigations?

Answer 9

If you take blood, semen, hair or something else from a crime scene and then DNA profile it. Then DNA profile the suspect, and victim and see which bands match then you can identify if they are the same or not.

Question 10

What can DNA profiling help with?

Answer 10

• Paternity investigations

- Forensic investigations

Question 11

How does DNA profiling help with paternity tests?

Answer 11

• DNA from the child, mother and potential father are taken, they are all profiled.
• The bands are compared and if any band appears in the child that is not present in the mother or potential father then another man must be the father.

Question 12

How do you carry out genetic modification?

Answer 12

Genetic modification is carried out by gene transfer between species. Transferring genes is possible because the genetic code is universal and whatever DNA you put into the nucleus will be made using the organisms ribosomes to produce those proteins.
One example is putting the human genes for making insulin into bacteria. So this can be created and used for treating diabetes.
It can also be used to produce new varieties of crop plant.

Question 13

How do we transfer genes to bacteria?

Answer 13

• We use plasmids, these are a small extra circle of DNA in bacteria. The most abundant plasmids are ones with genes for their own replication in the cytoplasm so that they replicate when the bacterium does. Bacteria used plasmids to exchange genes and so naturally absorb them and incorporate them into their main circular DNA molecule. So this is why they are so useful in genetic engineering.
• Restriction enzymes, also known as endonucleases, are enzymes that cut DNA molecules at specific base sequences. They can be used to cut out desired genes from larger DNA molecules and also to cut open plasmids. Some endonucleases have the useful property of cutting the two strands of DNA at different points. This leaves single-stranded sections called sticky ends. The sticky ends created by any one particular restriction enzyme have complementary base sequences and so can be used to link together pieces of DNA by hydrogen bonding between the bases.
• DNA ligase is an enzyme that joins DNA molecules firmly together by making sugar-phosphate bonds between nucleotides. When the desired gene has been inserted into a plasmid using sticky ends there are still nicks in each sugar-phosphate backbone of DNA but DNA ligase can be used to seal these nicks.

Question 14

What are restriction enzymes?

Answer 14

• Restriction enzymes, also known as endonucleases, are enzymes that cut DNA molecules at specific base sequences. They can be used to cut out desired genes from larger DNA molecules and also to cut open plasmids. Some endonucleases have the useful property of cutting the two strands of DNA at different points. This leaves single-stranded sections called sticky ends. The sticky ends created by any one particular restriction enzyme have complementary base sequences and so can be used to link together pieces of DNA by hydrogen bonding between the bases.

Question 15

What are plasmids?

Answer 15

• We use plasmids, these are a small extra circle of DNA in bacteria. The most abundant plasmids are ones with genes for their own replication in the cytoplasm so that they replicate when the bacterium does. Bacteria used plasmids to exchange genes and so naturally absorb them and incorporate them into their main circular DNA molecule. So this is why they are so useful in genetic engineering.

Question 16

What are endonucleases?

Answer 16

• Restriction enzymes, also known as endonucleases, are enzymes that cut DNA molecules at specific base sequences. They can be used to cut out desired genes from larger DNA molecules and also to cut open plasmids. Some endonucleases have the useful property of cutting the two strands of DNA at different points. This leaves single-stranded sections called sticky ends. The sticky ends created by any one particular restriction enzyme have complementary base sequences and so can be used to link together pieces of DNA by hydrogen bonding between the bases.

Question 17

What does DNA ligase do?

Answer 17

• DNA ligase is an enzyme that joins DNA molecules firmly together by making sugar-phosphate bonds between nucleotides. When the desired gene has been inserted into a plasmid using sticky ends there are still nicks in each sugar-phosphate backbone of DNA but DNA ligase can be used to seal these nicks.

Question 18

What is the process of gene transfer to bacteria?

Answer 18

1) mRNA extracted from human pancreatic cells
2) mRNA is treated with the reverse transcript to make complementary DNA

3) A plasmid is obtained from bacteria
4) The plasmid is cut with a restriction enzyme

5) The plasmid and DNA are fused using DNA ligase.
6) The new plasmid is introduced into host cells.
7) Bacteria multiply in a fermenter and produce insulin
8) Separation and purification of human insulin occur
9) It can be used.

Question 19

What are the benefits of GM crops?

Answer 19

ENVIRONMENTAL:

• Pest resistant crop varieties can be produced, this means that then less insecticide then has to be sprayed on to the crop so fewer bees and other beneficial insects are harmed.
• Pest resistant crops reduce the need for plowing and spraying crops and so less fuel is used for farm machinery.
• The shelf life of fruit and vegetables can be improved, reducing wastage and reducing the area of crops that have to be grown.

HEALTH

• The nutritional value of crops can be improved by increasing the vitamin content.
• Varieties of crops could be produced lacking allergens or toxins that are naturally present in them.
• Crops could be engineered so that they produce edible vaccines so by eating the crop a person could be vaccinated against a disease.

AGRICULTURAL:

• Varieties can be resistant to drought, cold and salinity can be produced by gene transfer, expending the range over which crops can be produced and increasing total yields.
• A gene for herbicide resistance can be transferred to crop plants allowing all other plants to be killed in the growing crop by spraying with herbicide. With less weed the crop yields will be higher.
• Crop varieties can be produced that are resistant to diseases caused by viruses. These viruses currently reduce crop yields and the only method of control at the moment is to kill some of the insects that carry them.

Question 20

What are the risks of GM crops?

Answer 20

HEALTH

• Proteins produced by transcription and translation of transferred genes could be toxic or cause allergic reactions in humans or livestock that eat them.
• Antibiotic resistant genes used as markers during gene transfer could spread to pathogenic bacteria.
• Transferred genes could mutate and cause unexpected problems that were not risk-assessed during development of GM crops.

ENVIRONMENTAL

• Non-target organisms could be affected by toxins that are intended to control pests in GM crop plants.
• Genes transferred to crop plants to make them herbicide resistant could spread to wild plants, turning them into uncontrollable super-weeds.
• Biodiversity could be reduced if a lower proportion of sunlight passes to weed plants, plant eating insects and organisms that feed on them where GM rather than non-GM crops are being grown.

AGRICULTURAL:

• Some seed from a crop is always spilt and germinated to become unwanted volunteer plants that must be controlled, but this could be difficult if it is resistant to herbicide.
• Widespread used of GM crops that contain a toxin that kills insect pests will lead to the resistance to the toxin in the pests that were the initial problem and also to the spread of secondary pests that are resistant to the toxin but were previously scarce.
• Farmers are not permitted by patent law to save and re-sow GM seed from crops they have grown so strains adapted to local conditions cannot be developed.

Question 21

What are clones?

Answer 21

Clones are groups of genetically identical organisms, derived from a single original parent cell.

Question 22

What are the natural methods of cloning?

Answer 22

Many plant species and some animal species have natural methods of cloning. The methods used by plants are very varied and can involve stems, roots, leaves or bulbs. Two examples are given here:

• A single garlic bulb, when planted, uses its food stores to grow leaves. These leaves produce enough food by photosynthesis to grow a group of bulbs. All the bulbs in the group are genetically identical so they are a clone.
• A strawberry plant grows long horizontal stems with plant-lets at the end. These plantlets grow roots into the soil and photosynthesise using their leaves, so can become independent of the parent plant. A healthy strawberry plant can produce ten or more genetically identical new plants in this way during a growing season.
• Animals - hydra clones itself by a process called budding.
• Female aphids can give birth to offspring that have been produced entirely from diploid egg cells that were produced by mitosis rather than meiosis. The offspring are therefore clones of their mother.

Question 23

What are examples of the natural methods of cloning method in plants?

Answer 23

• A single garlic bulb, when planted, uses its food stores to grow leaves. These leaves produce enough food by photosynthesis to grow a group of bulbs. All the bulbs in the group are genetically identical so they are a clone.
• A strawberry plant grows long horizontal stems with plant-lets at the end. These plantlets grow roots into the soil and photosynthesise using their leaves, so can become independent of the parent plant. A healthy strawberry plant can produce ten or more genetically identical new plants in this way during a growing season.

Question 24

What are examples of the natural methods of cloning method in animals?

Answer 24

• Animals - hydra clones itself by a process called budding.
• Female aphids can give birth to offspring that have been produced entirely from diploid egg cells that were produced by mitosis rather than meiosis. The offspring are therefore clones of their mother.

Question 25

How can we clone animal embryos?

Answer 25

At an early stage of development it is theoretically possible for the embryo to divide into two or more parts and each part to develop into a separate individual with all body parts as all the cells are capable of differentiating into anything.
In livestock an egg can be fertilised in vitro and allowed to develop into a multicellular embryo. Individual cells can be separated from the embryo while they are still pluripotent and transplanted into surrogate mothers. Only a limited amount of clones can be obtained this way, because after a certain number of divisions the cells are no longer pluripotent (capable of developing into all types of tissues). There has been little interest in this method of artificial cloning because at the embryo stage it is not possible to assess whether a new individual produced by sexual reproduction has desirable characteristics.

Question 26

Why is there little interest in embryo cloning?

Answer 26

In livestock an egg can be fertilised in vitro and allowed to develop into a multicellular embryo. Individual cells can be separated from the embryo while they are still pluripotent and transplanted into surrogate mothers. Only a limited amount of clones can be obtained this way, because after a certain number of divisions the cells are no longer pluripotent (capable of developing into all types of tissues). There has been little interest in this method of artificial cloning because at the embryo stage it is not possible to assess whether a new individual produced by sexual reproduction has desirable characteristics.

Question 27

What does pluripotent mean?

Answer 27

If a cell is pluripotent it is capable of developing into all types of tissues

Question 28

How can we clone adult animals?

Answer 28

Methods have been developed for cloning adult animals using differentiated cells. Once the embryos have grown into adults it is easy to see their characteristics and access if they want to be cloned however it is much harder to clone them. This is because the cells that make up the body are already differentiated.
You remove the nuclei from body cells and transplant them into egg cells taken from another animal of the same species without the nucleus, then you fuse them together and place them into another sheep.

Question 29

How was the first sheep cloned?

Answer 29

Dolly was a clone and was a large development in animal cloning. The method that was used was called somatic cell nuclear transfer.

1) Adult cells were taken from the udder of an ewe and were grown in a laboratory, using a medium containing a low concentration of nutrients. This made the genes in the cell inactive and so the pattern of differentiation was lost.
2) Unfertilised eggs were taken from the ovaries of another ewe. The nuclei were removed from these eggs The cultured cells were placed inside the protective coating of the egg. A small electric pulse caused the two cells to fuse together.
3) The embryos were then injected when about seven days old into the uteri of other ewes that could act as surrogate mothers.

Question 30

What is a somatic cell?

Answer 30

Used in the name somatic cell nuclear transfer which was the method of cloning dolly. It means a normal body cell with a diploid nucleus.