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3.4 – Genetic Engineering and Biotechnology

3.4 – Genetic Engineering and Biotechnology

3.4.1 – Outline the use of polymerase chain reaction (PCR) to copy and amplify minute quantities of DNA

This process is also called DNA amplification, and is used to produce enough DNA for procedures such as:

  • DNA sequencing
  • DNA profiling
  • Diagnose disease
  • Identify bacteria

It produces more DNA when there is only a small sample available, such as from a crime scene or a long-extinct organism. The cycle of replication happens at an exponential rate, and makes billions of copies in a few hours

The Process

Sample obtained (target DNA). DNA denatured by heating at 95°C for 5mins then cooled to 60°C. Primers are bonded (annealed) to each strand. Primers are short strands of DNA which provide a starting sequence for DNA extension. Free nucleotides and DNA polymerase are added. Polymerase binds to primers and synthesises complementary strands of DNA with the free nucleotides, resulting in two copies of the DNA. The process is repeated about 25 times

The Thermal Cycler

Loading Tray

Samples in tiny PCR tubes are put in the loading tray and the lid is closed

Temperature Control

Machine has heating and refrigeration mechanisms to rapidly change the temperature

Dispensing Pipette

Pipettes with disposable tips are used to dispense DNA samples into the PCR tubes

DNA Quantitation

Amount of DNA sample is determined by placing a known volume in the quantitation machine. Often a minimum amount of DNA is required

Controls

Control panel allows a number of different PCR programmes to be stored in the machine’s memory. Usually only one is started to run a PCR

 

3.4.2 – State that, in gel electrophoresis, fragments of DNA move in an electric field and are separated according to their size

Gel electrophoresis is a method that separates large molecules, including nucleic acids or proteins based on size, electric charge, and other physical properties. DNA has a slight negative charge due to the phosphates on the backbone, repelled by the negative electrode. Shorter DNA molecules travel further, whilst larger molecules are subject to more friction against the gel. Once stained, the separated molecules in each lane can be seen as a series of bands

 

The Process

  • Tray is prepared to hold the gel matrix
  • Gel comb is placed in the tray to create wells in the gel
  • Agarose gel powder is mixed with a buffer solution. This is heated until dissolved, then poured into the tray to cool. The buffer is a liquid used to carry the DNA in a stable form
  • Gel tray is placed in the electrophoresis chamber, which is then filled with buffer to cover the gel. This allows the electric current from the electrodes at either end to flow through it
  • DNA samples are mixed with a loading dye to make the DNA visible. This contains glycerol or sucrose to make the DNA heavy so that it sinks to the bottom of the well
  • A safety cover is placed over the gel, electrodes are attached to power supply and turned on
  • Once the dye marker has moved through the gel, the current is turned off and the gel is removed from the tray
  • DNA molecules are made visible by staining the gel with methylene blue or ethidium bromide, which binds to DNA and fluoresces in UV light
  • The gel matrix acts as a sieve for the negatively charged DNA molecules as they move towards the positive terminal
  • Large molecules have difficulty getting through the holes
  • Small molecules move easily

3.4.3 – State that gel electrophoresis of DNA in used in DNA profiling

Chromosomes have simple, repetitive sequences of non-coding DNA that are found scattered throughout the genome. Short sequences are called microsatellites or short tandem repeats, Sequence lengths vary considerably between people in the numbers of the repeating unit

Gel electrophoresis is used in DNA fingerprinting, which is a form of DNA profiling, used to differentiate one individual from another

The technique is used in forensic crime investigations, parentage issues, animal breeding pedigrees and disease detection.

The Process

  • DNA is extracted
  • Microsatellites are amplified using PCR
  • Specific primers for microsatellites are used
  • Fragments separated in a gel electrophoresis

3.4.4 – Describe the application of DNA profiling to determine paternity and also influence in forensic investigations

Paternity Investigation

DNA samples are taken from mother, child and potential fathers. All the DNA fragments from the child must match with either the mother or father. The band on the child’s fragments are either found on the mother or the father (Male 1)

Forensic Investigation

DNA is taken from the victim, crime scene and the suspects. The bands are compared to associate the suspects but to eliminate the victims DNA. In the example, suspect 1 matches the specimen

3.4.5 – Analyse DNA profiles to draw conclusions about paternity or forensic investigations

See above statement

 

3.4.6 – Outline three outcomes of the sequencing of the complete human genome

The Human Genome Project was an international research effort to identify and map all the human genes. We have benefitted greatly from the completion of this project, including:

  • A knowledge of the number of human genes
  • The location of specific genes. There are approximately 30,000 genes in the human genome, which have all been identified.
  • All the information has been stored in a database for future reference and research.
  • Allowed for the discovery of proteins and their specific functions.
  • The technologies that were developed for this research also have uses in other areas.
  • The evolutionary relationships between organisms can be identified using genetics.
  • However, there are ethical, legal and social issues that have arisen from the project.

3.4.7 – State that, when genes are transferred between species, the amino acid sequence of polypeptides translated from them is unchanged because the genetic code is universal

The genetic code is universal, with all known organisms using the same nucleic acids to code for proteins. In principle, if we transfer a gene from one species to another, it should be transcribed and translated into the same protein.

 

3.4.8 – Outline a basic technique used for gene transfer involving plasmids, a host cell (bacterium, yeast or other cell), restriction enzymes (endonucleases) and DNA ligase

In prokaryotic organisms, most of the DNA is in one circular chromosome. They also have plasmids, which are smaller circles of DNA floating freely in the cytoplasm. Plasmids can be removed and cleaved by restriction enzymes at target sequences.

DNA fragments from another organism can also be cleaved by the same restriction enzyme. Pieces can be added to the open plasmid and spliced together by ligase. Recombinant plasmids can be inserted into new host cells and then cloned.

 

Restriction Enzymes 

The plasmid is cut using restriction enzymes, leaving sticky ends, or exposed nucleotide bases.

The DNA containing the desired gene will have been isolated using gel electrophoresis. This DNA strand will also be cut at s specific recognition site using the same restriction enzymes. This produces a fragment with complementary sticky ends to those on the plasmid.

 

Ligation

The plasmids and the DNA fragments are mixed together in the presence of the enzyme ligase. The hydrogen bonds form using complementary base pairing and the sugar-phosphate backbone is joined through annealing.

 

 

3.4.9 – State two examples of the current uses of genetically modified crops or animals

Added Retinol in Rice

Retinol, or Vitamin A1, is essential for the development of an effective immune system, normal vision and growth. Deficiency leads to stunted growth, weakened immune system, loss of night vision and possible blindness. In third world countries, deficiency is coupled with malnutrition and disease. Children are also more likely to die from disease when they are vitamin A deficient.

Beta-carotene is used by the body to make retinol. Normal rice does not contain retinol or beta-carotene, but it does contain a molecule that is normally used to make beta-carotene. However, the gene and enzymes to manufacture retinol are missing.

GM rice contains the gene for the manufacture of beta-carotene. The gene was sourced from Erwinia bacterium or the common daffodil. Transgenic rice is usually yellow due to the presence of beta-carotene, which is crossed with local strains of rice. As a result, communities are able to have more nutrients in their diet.

Herbicide Resistance in Crop Plants

Weeds use soil nutrients that crops need to grow and the competition reduces productivity and efficiency of farming. Herbicides are used to kill weeds. They must be used before planting, as they also kill crops.

Some crops have been made resistant to major herbicides because they can produce an enzyme that breaks down glyphosate, found in a major herbicide. Herbicides can be used after planting to kill weeds.

3.4.10 – Discuss the potential benefits and possible harmful effects of one example of genetic modification

 

 

3.4.11 – Define clone

A group of genetically identical organisms or a group of cells derived from a single parent cell
3.4.12 – Outline a technique for cloning using differentiated animal cells

Dolly the Sheep was cloned using the following process:

Donor somatic cells were taken from the udder of the original sheep that was to be cloned and
cultured in a low-nutrient medium to stop division of the cell, making it dormant. An unfertilised
ovum was taken from a ewe and stimulated to superovulate by hormone FSH, producing a large
number of ova. The nucleus, containing DNA, was removed from the ovum using micromanipulation techniques. The ovum was left with no nucleus, but still had a cytoplasm and cellular machinery to produce an embryo.

The dormant donor cell and egg cell were fused using a gentle electrical pulse. The ovum retained its ability to replicate chromosomes and divide by mitosis. Cell division was triggered and when it reached the 16-cell stage, it was implanted into a surrogate mother sheep. Dolly, the cloned sheep, was genetically identical to the donor sheep. However, they experienced a different set of environmental conditions.

 

3.4.13 – Discuss the ethical issues of therapeutic cloning in humans