Recombinant DNA Technology

What is Biotechnology?

• Biotechnology uses cellular processes to make products that are of use to humans. Eg. Yeasts to make bread and alcohol, bacteria to make cheese and yoghurt

• Modern biotechnology has dramatically expanded to range of techniques and products that can be used to improve human welfare.

  • Treatments and prevention of disease

  • Food production

  • Production of clean energy

  • Efficiency of manufacturing processes

• Scientists use gene technology to alter genes, remove genes, add extra copies of a genes or even add in genes from another person.

• The definition of biotechnology has recently expanded to include genetic testing, gene manipulation, cell replacement therapies and tissue engineering.

The Human Genome

• The process of mapping the location of genes in chromosomes  - The Human Genome Project 1990

• A genome is defined as the complete set of genetic information of an organism.

• For humans this is the complete set of nucleotides that make up approximately 21 000 genes in our chromosomes

  • An individual will have a Chromosomal Genome and a Mitochondrial Genome

• Gene replacement is a major area of investigation into the treatment and cure of disease.

• New technology is being developed to monitor the expression of the genes involved in particular diseases.

• Monitoring the genes responsible for turning on the different stages of cancer development.

• The information has also been used to develop better genetic tests to screen individuals to determine the risk of developing certain conditions in the future.

What is Recombinant DNA Technology?

• Involves the introduction of DNA into cells that is foreign to the organism or that has been modified in some way.

• Genes from one organism can be placed into the chromosomes of another organism.

  • This process has huge potential to replace faulty genes with healthy ones (theoretically)

• Could be beneficial for individuals suffering with cystic fibrosis, rheumatoid arthritis and certain cancers with identified genes that are causative

• The same technology is another way of identifying mutation and detecting whether a person is affected by, or is a carrier for, hereditary diseases.

• Recombinant techniques can also be used to produce: (really happens!)

  • Proteins e.g. factor VIII

  • Hormones e.g. insulin , HGH

  • Vaccines e.g. Hepatitis B, influenza

• The Process

  • Cutting DNA:

    • One of the essential requirements of genetic engineering is the ability to cut segments of DNA at known sequences.

    • Cutting is done using restriction endonucleases  = "molecular scissors”

  • DNA is universal therefore a restriction enzyme can be used to cut out a section of DNA from one organism and the same restriction enzyme can be used to open up DNA in another organism.

  • Restriction endonucleases (endo= within, nuclease = an enzyme that cleaves nucleic acids) are also commonly called restriction enzymes. These are special enzymes found in bacteria that protect them from foreign DNA (viral) by restricting the duplication of bacteriophages.

  • Restriction enzymes cut DNA in a specific place. This is known as a recognition site  - contains a specific sequence of bases – usually 4-8 base pairs in length that are palindromic (same sequence when read both forward & backward)

    • They are like “molecular scissors” which cut DNA molecules into smaller pieces, called restriction fragments. This process is controlled.

  • Types of Cuts

    • Restriction enzymes can produce different types of cuts in the DNA.

    1. Straight Cut

       • A clean break is made across the two strands of DNA to produce a blunt end where both strands terminate in a base pair.

    2. Staggered Cut

    • A staggered cut produces fragments with sticky ends, where there is a stretch of unpaired nucleotides. These can ’recombine’ with sections of DNA with complimentary endings.

  • As the same restriction enzyme is used in this process, it means the ‘sticky ends’ (if they are present) are complimentary and so the cut out section can be inserted into another organisms DNA.

  • DNA ligase is used to stick the pieces of DNA together, in process called ligation. Like ‘molecular glue’.

• Terminology

  Term	Definition
  Blunt Ends	The ends produced by a straight cut of a sequence of nucleotide bases
  DNA Ligase	An enzyme capable of combining two small components of single-stranded DNA into one single structure
  Phage	Or bacteriophage; a virus that infects bacteria
  Plasmid	A small circular strand of DNA distinct from the main bacterial genome; it is composed of only a few genes and is able to replicate independently within a cell
  Restriction enzyme	An enzyme that cuts a strand of DNA at a specific sequence of nucleotides called the “recognition site”
  Staggered Cut	Produced when a restriction enzyme creates fragments of DNA with unpaired nucleotides that overhang at the break in the strands ; called “sticky ends”
  Straight Cut	Produced when a restriction enzyme makes a clean break across the two strands of DNA so that ends terminate in a base pair; called “blunt ends”
  Sticky Ends	The overhanging ends produced by a staggered cut of a sequence of nucleotide bases; can also be called “cohesive ends”
  Vector	A bacterial plasmid, viral phage or other such agent used to transfer genetic material from one cell to another

• Transgenic Organisms

  Transgenic Organisms are those whose genome has been altered by the transfer of a gene or genes from another organism. This gene is then expressed by the new host.

  The introduced genes become part of the transgenic organism’s DNA and can be passed from one generation to the next.

  A genetically modified organism (GMO) is one whose genome has been modified or altered to give it characteristics it does not normally have.

• Using a Vector

  • Vector: DNA molecule that is used to carry DNA into a cell

    • First step in producing organism with recombinant DNA is to isolate the gene of interest.

  • Gene is then inserted into vector & cloned. This is achieved by:

  1. Identifying desired gene

  2. Using restriction enzyme to cut the DNA on either side of the gene

  3. Using the same restriction enzyme to cut DNA of the vector

  4. Adding desired gene to vector

  5. Using DNA ligase to join 2 sections of DNA

• Bacteria and Plasmids

  • Bacteria have small circular piece of DNA called plasmids. The plasmids are distinct from the main bacterial genome (cytoplasmic DNA) and can replicate independently of chromosomal DNA.

  • Plasmids can be exchanged between bacterial cells.

  • The plasmid is reintegrated into a bacterial cell which is then cloned

  • Once large quantities of the transgenic cell have been produced, they can be introduced into the selected host cells such as bacterial, yeast or mammalian cells.

    • Exchange of plasmids will result in many cells containing the new gene.

  • These host cells will produce a foreign protein using instructions in the gene in the recombinant DNA.

Examples of Recombinant DNA Technology

• Important in diagnosis & treatment of diseases & genetic disorders

• Enabled manufacture of large quantities of pure protein for many medical products including:

  • Insulin

  • Growth hormone

  • Factor VIII

  • FSH

• In the past, substances were extracted from people or animals, they were often impure & of variable strength

• Example: transmission of Creutzfeldt-Jakob disease (a variant of mad-cow disease) by contaminated Growth hormone

• Insulin

  • Type 1 diabetes: elevated blood sugar levels due to impaired insulin production by pancreas. Insulin regulates use & storage of glucose

    • 1921: Patients treated with insulin from pancreas of pigs & cattle

    • 1982: Insulin by genetically engineered bacteria was approved

  • Human gene that has code for insulin production was introduced into bacterial cells

  • These bacteria became insulin factories & are now cultured in vats where they produce insulin that can be used to treat diabetes

  • Insulin produced by bacteria is identical to human insulin as the human gene was engineered into bacteria

    • Frequently performed on yeast cells as the growth medium

    • Reduced side effects from this insulin compared with animal variety

• hGH

  • Production of hGH by genetically engineered E.coli bacteria has dramatically increased supply of the hormone

  • Used to enhance athletic performance & delay physical deterioration associated with ageing

  • Little evidence that hGH has any benefit & it can produce serious side effects

    • Technology has resulted in production of GH for dairy cattle

    • Administration has increased milk production, and current research indicates this milk does not pose a risk to human health

• Factor VIII

  • Haemophilia A (Classic haemophilia) is an inherited disorder where blood-clotting protein (Factor VIII) is in poor supply or missing

  • Condition results in people unable to form blood clots adequately & therefore at risk of life-threatening bleeding from small injuries

    • To treat condition, injections of Factor VIII concentrates made from human plasma

    • To obtain sufficient quantities of Factor VIII, plasma from thousands of donors was required

  • With large numbers of donors, there was constant risk of transmission of viral disease

    • 2 diseases that caused  deaths of many haemophiliacs all over the world were: HIV & hepatitis C

  • Production of Factor VIII by recombinant DNA overcame this problem

    • Another advantage: free of other plasma proteins that could cause an immune response or allergic reaction

  • Recombinant Factor VIII is one of the largest molecules synthesised to date, is cultured in mammalian cells & is highly effective in control of excess bleeding

• Vaccines

  • First vaccine for human use produced using recombinant DNA technology was hepatitis B vaccine, introduced 1986 and still being used currently

  • Disadvantages:

    • Very expensive, as genes for desired antigens must be located, cloned & expressed efficiently in a new vector

    • Those involved in vaccine research must be conservative because vaccines are used on large numbers of healthy people, including children, so safety of product is paramount. Therefore, if a conventional vaccine is known to be safe, there is little incentive to develop a new one using genetic engineering

    • Currently vaccine for Hepatitis B is produced using recombinant technology

    • Current technology: Gene for a surface antigen on the virus is isolated & added to plasmid

    • Human Papilloma Virus (HPV), produced in a very similar way to Hepatitis B vaccine

• DNA/RNA Vaccines

  • Another area of research is DNA vaccines:

    • DNA / RNA for the antigen is introduced in the vaccine instead of the antigen itself.

    • DNA / RNA is incorporated into the host’s cells, which will produce the antigen. The thought is that the antigen will then be expressed by host cells, in a similar way to what happens during a viral infection