GENETIC ENGEERING

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GENETIC ENGEERING

GENETIC ENGEERING

Genetic engineering, also known as recombinant DNA technology, means altering the genes in a living organism to produce a Genetically Modified Organism (GMO) with a new genotype. Various kinds of genetic modification are possible: inserting a foreign gene from one species into another, forming a transgenic organism; altering an existing gene so that its product is changed; or changing gene expression so that it is translated more often or not at all.

TECHNIGUES OF GENETIC ENGEERING

Genetic engineering is a very young discipline, and is only possible due to the development of techniques from the 1960s onwards. These techniques have been made possible from our greater understanding of DNA and how it functions following the discovery of its structure by Watson and Crick in 1953. Although the final goal of genetic engineering is usually the expression of a gene in a host, in fact most of the techniques and time in genetic engineering are spent isolating a gene and then cloning it. This table lists the techniques that we'll look at in detail.

RESTRICTION ENZYMES

These are enzymes that cut DNA at specific sites. They are properly called restriction endonucleases because they cut the bonds in the middle of the polynucleotide chain. Most restriction enzymes make a staggered cut in the two strands, forming sticky ends.

The cut ends are "sticky" because they have short stretches of single-stranded DNA. These sticky ends will stick (or anneal) to another piece of DNA by complementary base pairing, but only if they have both been cut with the same restriction enzyme. Restriction enzymes are highly specific, and will only cut DNA at specific base sequences, 4-8 base pairs long.Restriction enzymes are produced naturally by bacteria as a defence against viruses (they "restrict" viral growth), but they are enormously useful in genetic engineering for cutting DNA at precise places ("molecular scissors"). Short lengths of DNA cut out by restriction enzymes are called restriction fragments. There are thousands of different restriction enzymes known, with over a hundred different recognition sequences. Restriction enzymes are named after the bacteria species they came from, so EcoR1 is from E. coli strain R.

DNA LIGASE

This enzyme repairs broken DNA by joining two nucleotides in a DNA strand. It is commonly used in genetic engineering to do the reverse of a restriction enzyme, i.e. to join together complementary restriction fragments.

The sticky ends allow two complementary restriction fragments to anneal, but only by weak hydrogen bonds, which can quite easily be broken, say by gentle heating. The backbone is still incomplete. 

DNA ligase completes the DNA backbone by forming covalent bonds. Restriction enzymes and DNA ligase can therefore be used together to join lengths of DNA from different sources

VECTORS

In biology a vector is something that carries things between species. E.g. the mosquito is a vector that carries the malaria parasite into humans. In genetic engineering a vector is a length of DNA that carries the gene we want into a host cell. A vector is needed because a length of DNA containing a gene on its own won’t actually do anything inside a host cell. Since it is not part of the cell’s normal genome it won’t be replicated when the cell divides, it won’t be expressed, and in fact it will probably be broken down pretty quickly. A vector gets round these problems by having these properties:

  • It is big enough to hold the gene we want 
  • It is circular (or more accurately a closed loop), so that it is less likely to be broken down 
  • It contains control sequences, such as a transcription promoter, so that the gene will be replicated or expressed. 
  • It contain marker genes, so that cells containing the vector can be identified.

PLASMIDS (THE MOST COMMON VECTORS)

Plasmids are by far the most common kind of vector, so we shall look at how they are used in some detail. Plasmids are short circular bits of DNA found naturally in bacterial cells. A typical plasmid contains 3-5 genes and there are around 10 copies of a plasmid in a bacterial cell. Plasmids are copied when the cell divides, so the plasmid genes are passed on to all daughter cells. They are also used naturally for exchange of genes between bacterial cells (the nearest they get to sex), so bacterial cells will take up a plasmid. Because they are so small, they are easy to handle in a test tube, and foreign genes can quite easily be incorporated into them using restriction enzymes and DNA ligase.

One of the most common plasmids used is the R-plasmid (or pBR322). This plasmid contains a replication origin, several recognition sequences for different restriction enzymes (with names like EcoRI), and two marker genes, which confer resistance to different antibiotics (ampicillin and tetracycline).

The diagram below shows how DNA fragments can be incorporated into a plasmid using restriction and ligase enzymes. The restriction enzyme used here (PstI) cuts the plasmid in the middle of one of the marker genes (we’ll see why this is useful later). The foreign DNA anneals with the plasmid and is joined covalently by DNA ligase to form a hybrid vector (in other words a mixture or hybrid of bacterial and foreign DNA). Several other products are also formed: some plasmids will simply re-anneal with themselves to re-form the original plasmid, and some DNA fragments will join together to form chains or circles. Theses different products cannot easily be separated, but it doesn’t matter, as the marker genes can be used later to identify the correct hybrid.

GENE TRANSFER

Vectors containing the genes we want must be incorporated into living cells so that they can be replicated or expressed. The cells receiving the vector are called host cells, and once they have successfully incorporated the vector they are said to be transformed. Vectors are large molecules which do not readily cross cell membranes, so the membranes must be made permeable in some way. There are different ways of doing this depending on the type of host cell. The most important one have the symbol the others are less commonly used

  • Heat Shock. Cells are incubated with the vector in a solution containing calcium ions at 0°C. The temperature is then suddenly raised to about 40°C. This heat shock causes some of the cells to take up the vector, though no one knows why. This works well for bacterial and animal cells. 
  • Electroporation. Cells are subjected to a high-voltage pulse, which temporarily disrupts the membrane and allows the vector to enter the cell. This is the most efficient method of delivering genes to bacterial cells. 
  • Viruses. The vector is first incorporated into a virus, which is then used to infect cells, carrying the foreign gene along with its own genetic material. Since viruses rely on getting their DNA into host cells for their survival they have evolved many successful methods, and so are an obvious choice for gene delivery. The virus must first be genetically engineered to make it safe, so that it can’t reproduce itself or make toxins.

Three viruses are commonly used:

  • Bacteriophages also called phages) are viruses that infect bacteria. They are an effective way of delivering large genes into bacteria cells in culture.
  • Adenoviruses are human viruses that causes respiratory diseases including the common cold. Their genetic material is double-stranded DNA, and they are ideal for delivering genes to living patients in . Their DNA is not incorporated into the host’s chromosomes, so it is not replicated, but their genes are expressed. The adenovirus is genetically altered so that its coat proteins are not synthesised, so new virus particles cannot be assembled and the host cell is not killed..  
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  • Retroviruses are a group of human viruses that include HIV. They are enclosed in a lipid membrane and their genetic material is double-stranded RNA. On infection this RNA is copied to DNA and the DNA is incorporated into the host’s chromosome. This means that the foreign genes are replicated into every daughter cell. After a certain time, the dormant DNA is switched on, and the genes are expressed in the host cells.

  • Plant Tumours. This method has been used successfully to transform plant cells, which ...

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