S-Cool Revision Summary

S-Cool Revision Summary

Genetic engineering aims to remove a desired gene and transfer it to another organism where it can be expressed. This means that the required protein can be synthesised within the new organism.

There are various methods that are used...

  1. If the amino acid sequence of the desired protein is known, the DNA code can be worked out and the DNA made in the lab by stringing together the correct order of nucleotides.

    Note: Many proteins are extremely large, therefore this would be a tedious process.

  2. Isolate the messenger RNA (mRNA) for the desired gene and make a single copy of the complementary DNA using the enzyme reverse transcriptase and another copy is made by adding DNA polymerase so that a doubled stranded length of DNA is made. (The original reverse transcription enzymes were first discovered in retro viruses.)
  3. Isolate the gene from the entire genome. To do this the DNA must first be cut into fragments and the one containing the desired gene must be identified. The enzymes used to cut the DNA are called restriction enzymes or restriction endonucleases.

The fragments need to be put into an organism that will express the protein.

There are several ways of getting DNA fragments into host cells:

  1. Microinjection.
  2. Electroporation - temporary holes are made in the membrane using electrical pulses.
  3. Microprojectiles - tiny beads of tungsten or gold coated with DNA fired by a gun into the cell.
  4. Using viruses as vectors.
  5. DNA in liposomes - fuse with the cell membrane.

Note: These methods only introduce DNA into the cell; they do not incorporate it into the host DNA.

The main requirement of the organism is that large quantities of a protein can be produced as cheaply and easily as possible, it needs to be an organism that:

  1. Grows fast.
  2. Is easily manipulated.
  3. Has a simple chromosome (prokaryotic cells do not have a nuclear envelope).
  4. Contains naturally occurring vectors (see later).

A good option therefore is to use yeasts or bacteria.

How to get the fragment into bacteria

A vector is a carrier DNA molecule into which the desired gene can be inserted.

Most commonly, this vector is a plasmid. This is a small, extra-chromosomal, circular piece of DNA often found in bacteria in addition to their functional DNA.

Agrobacterium tumefaciens (Crown gall disease)

Viruses and bacteria as vectors:

Viruses and some bacteria are known to transfer some of their DNA or RNA into the cells of a host. This genetic material integrates into the host genome, causing the production of disease or deformity such as Galls.

Galls are large tumour-like growths in plants, usually produced as a response by the plant to some invasion by microbe or insects.

The purpose of this PCR is to produce huge numbers of copies of a gene. The enzyme DNA polymerase is added to the sample and when suitably treated, will catalyse many millions of copies of the small sample.

PCR is used as the starting point, or template, for sequencing.

When gene transfer is attempted, there must be a way of detecting which microbe has taken up the new gene arrangements. Those that have been unsuccessful have to be eliminated.

Genetic engineering in agriculture

Viruses and some bacteria are known to transfer some of their DNA or RNA into the cells of a host and this genetic material integrates into the host genome, causing the production of disease or deformity such as Galls.

Galls are large tumour-like growths in plants, usually produced as a response by the plant to some invasion by microbe or insects.

The Ti plasmid of a particular bacterium called Agrobacterium tumefaciens has been used to insert DNA into plant genomes.

Genes coding for the tolerance to herbicides, resistance to insect pests, viral disease and other factors have been introduced to various plant species.

Cells can be taken from the meristem of a plant of the species in question and kept in a sterile nutrient medium so that the cells divide and form a tissue. This can be split several times and each group of cells can go on to form an individual plant (meristem tissue culture or micropropagation).

Gene therapy

Treating a genetic disease by altering an individuals natural genotype by:

  1. Germ cell therapy of sperm, egg or early embryo.
  2. Somatic cell therapy.

Number 1 is technically possible but ethically unacceptable.

There are 3 ways in which gene therapy can work:

  1. Repair of the defective gene.
  2. Replacement of the faulty gene with a normal one.
  3. Addition of a normal gene, leaving the defective one in position.

Much of your DNA is non - coding (i.e. it doesn't code for a protein), but rather it contains regions of highly repetitive sequences of bases (Variable Number Tandem Repeats - VNTRs)

The number of repeats, and hence the size of the VNTRs, varies markedly between individuals. Some will come from one parent; the rest will come from the other.

Only identical twins have the same number of VNTRs. Genetic fingerprinting reveals the differences in the size of the VNTRs in different individuals.