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Genome Projects

Genome Projects

  • Human genome consists of >3 billion base pairs
  • Organised into 20,000 genes
  • Took approximately 13 years to sequence the entire genome
  • It was accomplished using bioinformatics (the science of collecting and analysing complex biological data i.e. genetic codes)

Project aims:

  • Identify all the genes of the human genome
  • Determine locus of genes
  • Determine sequence of base pairs that make up human DNA
  • Functions of different genes
  • Publish results for free use

DNA Sequencing

  • Determining the complete DNA base sequence utilises Whole-Genome Shotgun (WGS) sequencing.
  • DNA is cut into small pieces
  • Each smaller section is easily sequenced
  • Computer algorithms align overlapping segments to assemble entire genome

Single Nucleotides Polymorphisms (SNPs)

  • Most common type of genetic variation among people
  • Each SNP represents a difference in a nucleotide
  • The location of these SNPs on the DNA sequence allows for genetic screenings of these known areas so if a person is a carrier it can be easily determined (without having to sequence the entire human genome again)

The Proteome

  • The entire set of proteins expressed by a genome at a given type of cell or organism at a given time under specified conditions
  • b. not all genes, and therefore not all proteins are produced, and are therefore excluded

Human Genome Map

  • All the DNA found in the human is located on the

Human Microbiome Project

  • Aimed to map and identifying the microorganisms which are found in association with both healthy and diseased humans
Genetic Fingerprinting

Genetic Fingerprinting

Genetic Fingerprinting

  • The genome of any organism contains many repetitive non-coding bases of DNA
    • Approximately 95% of human DNA does not code for any characteristics
    • Non-coding sequences – introns
  • For every individual the number and length of core sequences has a unique pattern
    • Except for identical twins
  • The more closely related two individuals are the more similar the core sequence will be

Stage 1: Extraction

  • Small samples (e.g. drop of blood, hair root etc.) is adequate
  • To extract the DNA, it is separated from the rest of the cell
  • As the amount of DNA is usually small the quantity must be increased through PCR

Stage 2: Digestion

  • DNA is cut into fragments using restriction endonucleases
  • Endonucleases are chosen for their ability to cut close to groups of core sequences

Stage 3: Separation

  • Fragments of DNA are next separated according to size by gel electrophoresis under the influence of an electrical voltage
  • Gel is then immersed in alkali in order to separate the double strands into single strands
  • Single strands are ten transferred onto a nylon membrane through Southern Blotting:
    1. Thin nylon membrane is laid over the gel
    2. Membrane is covered with several sheets of absorbent paper (which draws up the liquid contains the DNA by capillary action)
    3. This transfers the DNA fragments to the nylon membrane in precisely the same relative positions that they occupied on the gel
    4. DNA fragment are then fixed to the membrane using UV light

Stage 4: Hybridisation

  • DNA probes (i.e. radioactive/fluorescent) are now used to bind with the core sequences
  • Probes have bas sequences which are complementary to the core sequences and bind to them under specific conditions (temperature and pH)
  • Process is carried out with different probes with each binding to a different core sequence

Stage 5: Development

  • An x-ray film is placed over the nylon membrane
  • Film is exposed by radiation from the radioactive probes (or if fluorescent proves then the position is located visually)
  • As these points correspond to the position of the DNA fragments as separated during electrophoresis, a series of bars can be seen
  • The pattern of these bands is unique to every individual
Gene Therapy

Gene Therapy

  • Gene Therapy is the introduction of DNA into a patient to treat a genetic disease
  • Using a vector, typically a bacteriophage, a gene is deliver to the target cell
  • The cell reads the gene and uses the information in the gene to build RNA and protein molecules
  • The proteins (or RNA) can then carry out their job in the cells.

Viral Vectors

  • Viruses can be used to deliver DNA to cells for gene therapy
  • The utilisation of viruses uses their own biological mechanisms and requires not further processing
  • Transduction: Delivery of genes by a virus
  • Transduced: the infected cells

Advantages of viral vectors:

  • Highly accurate
  • Easily embeds DNA into cell
  • Some target specific types of cells.
  • They can be modified so that they can’t replicate and destroy cells.

Disadvantages of viral vectors:

  • Carries a limited amount of genetic material. Some genes may be too big to fit into some viruses.
  • May result in immune responses in patients, leading to:
    1. Patients deteriorates further.
    2. Patient’s immune system may block the virus from delivering the gene to the patient’s cells, or it may kill the cells once the gene has been delivered.

Non-Viral Vectors

  • Some of the limitations of viral vectors can be overcome through the use of non-viral vectors
  • A common non-viral vector is the use of plasmids
  • Bacteria typically use plasmids to transfer genes among each other
  • Gene therapy plasmids can be packaged into liposomes (small membrane-wrapped packets)
  • Liposomes deliver the plasmids by fusing with cell membranes

Advantages of non-viral vectors

  • carry larger genes,
  • Most don’t trigger an immune response.

Disadvantages of non-viral vectors

  • much less efficient than viruses at getting genes into cells.
  • Virosomes are liposomes covered with viral surface proteins, they can a high carrying capacity and immune advantages of plasmids with the efficiency and specificity of viruses#
  • Viral proteins interact with proteins on the target-cell surface, helping the virosome fuse with the cell membrane and dump its contents into the cell

Somatic Therapy

  • Copies of corrected gene are inserted directly into the somatic (body cells) of the carrier
  • It does not allow disease to be passed onto future generations
  • Therapy must occur frequently (due to short term effectiveness)

Germline Therapy

  • The targeting of genes towards the egg and sperm cells (germ cells), however, which would allow the inserted geneto be passed to future generations.
  • Corrected gene is inserted into fertilised egg (via IVF)
  • IF successful, all cells of the embryo will contain the corrected gene when cells divide by mitosis
  • Germ cell therapy is permanent and also ensures offspring inherited corrected gene
Gene Markers

Gene Markers

  • Gene markers are used to identify whether a gene has been take up b y a bacterial cell
  • They all require a secondary separate gene to be used

Antibiotic Resistance Marker

  • Replica Plating refers to the identification of cells with plasmids that have take up the new gene
  • Replica plating is the process which utilises the other antibiotic resistance gene in the plasmid which cuts in order to incorporate the required gene
  • Bacterial cells which survived treatment with ampicillin (antibiotic) are known to have take up the plasmid
  • These cells are cultured by spreading them on a nutrient agar plate
  • Each sperate cell on the plate will grow into a genetically identical colony
  • A small sample of each colony is transferred onto a secondary (replica) plate in the same position as the colonies on the original plate
  • The replica plate contains a different antibiotic (tetracycline) against which the antibiotic-resistance gene will have not been disabled if the new gene has been take up
  • The colonies killed by tetracycline must be ones that have take up the required gene
  • Colonies in the same position on the original plate are the ones that posses the required gene these colonies are therefore, made up of bacteria that have been genetically modified and have been transformed

Fluorescent Markers

  • A gene taken from a jelly fish results in a green fluorescent protein (GFP)
  • The gene is cloned and inserted into the centre of the GFP gene
  • Any bacterial cell that has take up the plasmid with the gene that is to be cloned will not be able to produce GFP
  • The cells that have taken up the gene will not fluoresce
  • As the bacterial cells with the desired gene are not killed, there is no need for replica plating
  • Results can be obtained by viewing the ceclls under a microscope and retaining those which do not fluoresce
  • Process is more rapid

Enzyme Marker

  • Lactase will turn a colourless solution substrate into blue
  • The required gene is transplanted into a gene which produces lactase
  • If a plasmid with a required gene is present in a bacterial cells the colonies grown from it will not produce lactase
  • When these bacterial cells are grown in the colourless substrate solution they will be unable to change the solution to blue
  • The bacteria which did not obtain the gene will turn the substrate blue.
Epigenetics

Epigenetics

Epigenetic Control

  • Epigenetic control is determined by whether a gene is being expressed or not
  • It is the attachment or removal of epigenetic marks to or from the DNA/histone proteins
  • Epigenetic marks do not alter the DNA sequence
  • Epigenetic marks do alter the accessibility for enzymes and other proteins needed for transcription to interact with and transcribe the DNA
  • Epigenetic changes to gene expression is vital in many normal cellular processes
  • Epigenetic change can be the result of environmental factors

Inheritance

  • As organisms inherit the DNA sequences from parental most of the epigenetic markers on the DNA are removed between generations
  • However, some epigenetic markers are left on (accidently) and therefore express of these genes can be affected by environmental changes that had affected the parents/grandparents

Controlling Gene Expression

  • Increased Methylation of DNA
    • Methylation is the addition of a methyl group to the DNA for a gene
      • Methyl groups are an epigenetic marker
    • Methyl group is always attached to CpG site
      • CpG site is where cytosine and guanine bases are next to each other in the DNA. They are linked by a phosphodiester bond
    • Increased methylation results in a alteration to the DNA structure
    • Therefore, transcriptional enzymes cannot interact with the gene – the gene is not expressed
  • Decreased acetylation of histones
    • Histone proteins wrap DNA around to form chromatin
      • Chromatin can be highly condensed or relatively less condensed
      • It is how condensed the chromatin is does it affect the accessibility of the DNA and whether or not it can be expressed
    • Histones can be epigenetically modified by the addition/removal of acetyl groups
      • Aceytl groups are an epigenetic marker
    • When histone acetylation occurs the chromatin is less condensed – which allows genes to be expressed
    • When acetyl groups are removed from histones the chromatin becomes highly condensed – which prevents genes to be expressed
    • Histone deacetylase is responsible for the addition/removal of the acetyl from the histone

Treating Disease

  • Epigenetic changes are reversible – therefore it makes them more suitable for drugs which target the disease. These types of drugs are designed to counteract the epigenetic change
    • g. increased methylation is an epigenetic change that can lead to a gene being switched off
    • Drugs that stop DNA methylation can sometimes be used to treat the disease
  • Decreased acetylation of histones could also lead to genes being not expressed
  • Issues faced is developing a drug to counteract an epigenetic change:
    • All cells will be affected due to the nature of targeting epigenetic sites so drugs have to be highly specific
Cancer

Cancer

Cancer

  • Acquired Mutations: A mutation that occurs in individual cells after fertilisation
  • A mutation in genes linked to the rate of cell division can lead to uncontrolled cell division.
  • Tumour: A mass of abnormal cells brought about by uncontrolled cell division
  • Malignant (Cancer): Tumour that invades and destroy surrounding tissues

Tumour Suppressor Gene

  • Normal: Slows cell division through production of proteins that stop cells dividing or apoptosis
  • Mutation: Gene will become inactivated so protein is not produced. Cell divides uncontrolled

Proto-oncogene

  • Normal: stimulates cell division through production of proteins which results in cell division
  • Mutation: the gene can become overactive and stimulates cells to divide uncontrollably
    • A mutated proto-oncogene is called an oncogene

Types of Tumour:

  • Malignant:
    • Cancer
    • Rapid growth
    • Invasive of surrounding tissue
    • Metastasis: tumour cells can spread via blood/lymphatic systems
  • Benign
    • Non-cancerous
    • Slower growth
    • Covered in fibrous tissue that prevents invading other tissue
    • Greatly harmless, however can cause blockage and pressures and may develop into malignant

Tumour Cells

Tumour Cells differ from normal cells as:

  • Nucleus is larger and darker
  • Some cells have more than 1 nucleus
  • Irregular shapes
  • Not all proteins needed to function are produced
  • Different antigens
  • No response to growth regulating processes
  • More frequent mitosis

Causes of Tumour Growth

  • Abnormal Methylation
    • Addition of -CH3 onto DNA is used to regulate gene expression as it can determine whether a gene is transcribed and translated
    • Hypermethylation (too much methylation)
    • Hypomethylation (too little methylation)
  • Oestrogen in Breath Cancer