Genome projects & Gene technology

• a genome is the entire set of genes in an organism.
• a proteome is all the proteins an organism can produce.
• It is easier to determine the proteome of bacteria because they do not contain introns in their DNA.
• More complex organisms have large sections of introns / non-coding DNA.

  • They also contain regulatory genes which determines when genes that code for a particular protein are switched on or off.
• recombinant DNA technology involves transferring fragments of DNA from one organism to another.
• genetic code is universal meaning the same DNA triplets code for the same amino acids in all living things.
• what are the three different ways of making DNA fragments?

  reverse transcriptase

  restriction endonuclease

  gene machine
• DNA frags from reverse transcriptase:

  1. mRNA is extracted from the required cells.
  2. the enzyme reverse transcriptase is added with free DNA nucleotides and used to make a single strand of DNA.
  3. This strand of DNA is called cDNA.
  4. the enzyme DNA polymerase the converts the cDNA to double stranded DNA.
  5. this new DNA can then be inserted into a vector.
• DNA frags from restriction endonuclease:

  1. Restriction endonuclease enzyme recognises specific palindromic base sequences.
  2. the DNA is cut producing sticky ends.
  3. If there is a suitable sequence on either side of the DNA fragment then the DNA is incubated with restriction endonuclease.
  4. This cuts out the requires fragment of DNA by hydrolysis reaction.
• DNA frags from the gene machine

  1. Determine the amino acid sequence of a required protein

  2. Use this to determine the mRNA and DNA sequences

  3. Check sequence of nucleotides for biosafety to international standards

  4. Fix the first nucleotide on a support e.g a bead.

  5. Add more nucleotides in the correct order

  6. Produces small sections of DNA called oligonucleotides

  7. Puts together oligonucleotides to form the gene without any introns

  8. Makes double stranded with complementary sticky ends and replicates using PCR

  9. Inserts into the plasmid
• once DNA fragments are isolated they need to be amplified (make lots of copies), using in vivo cloning.
• In vivo cloning is where copies of DNA fragments are made inside a living organism
• in vivo amplification / cloning:

  1. the DNA fragment is inserted into a vector e.g plasmids.
  2. the same restriction endonuclease is used to cut open the vector creating complementary sticky ends to the DNA fragment.
  3. the vector DNA, DNA fragment & DNA ligase enzyme are mixed together.
  4. DNA ligase joins the sticky ends together.
  5. producing recombinant DNA.
• what are the three stages of in vivo gene cloning?

  insertion

  transformation

  identification
• during insertion:

  1. the DNA fragment is inserted into a plasmid vector.
  2. if the same restriction endonuclease is used to cut the DNA fragment and the plasmid it leaves complementary sticky ends.
  3. the fragment and the plasmid then pair up and join using DNA ligase enzyme.
  4. producing recombinant DNA.
• Transformation:

  1. this involves reintroducing the recombinant plasmids into the host cell.
  2. calcium chloride and heat shock is used to make the cell membrane more likely to allow plasmids through and into the cell.
  3. not all cells will take up the recombinant plasmids.
• Identification:

  1. marker genes are used which are present in the plasmid e.g antibiotic resistance
  2. the bacteria are grown on a plate containing an antibiotic, any bacteria without the plasmid will die so only bacteria that took up the recombinant plasmid will grow.
• what are the reactants mixed in the PCR vial?

  • DNA to be amplified.
  • DNA polymerase enzyme
  • small primer sequences of DNA
  • a supply of nucleotide bases (A, T, C & G)
• PCR:

  1. the PCR reactants in the vial are placed in the PCR machine.
  2. it is heated to 90-95 degrees.
  3. this causes the DNA strands to seperate by breaking hydrogen bonds.
  4. it is then cooled down to 55-60 degrees, allowing primers to bind to the single DNA strands.
  5. it is heated again to 75 degrees which is the optimum temperature for DNA polymerase action.
  6. the enzyme adds the free nucleotides to the complementary strands of DNA identical to the origional molecule.
  7. the last three steps are repeated to get many copies of the origional DNA.
• what are the advantages of in vivo cloning?

  • useful if gene needs to be introduced into an organism.
  • no risk of contamination.
  • very accurate.
  • cuts out specific genes
  • produces transformed bacteria which can be used to produce large quantities of a gene product e.g insulin.
• what are the advantages of in vitro cloning?

  • its very fast.
  • does not require living cells.
• what are the three main uses of recombinant DNA technology?

  Agriculture

  Industry

  Medicine
• In agriculture crops can be transformed so they give higher yields or are more nutritious.

  • crops can be transformed to have pest resistance so less pesticides are needed, lowering cost.
• In Industrial processes enzymes are used to catalyse reactions.

  • these enzymes can be produced by transformed organisms in large quantities for less money, reducing costs.
  • for example enzymes involved in cheese making.
• In medicine many drugs & vaccines are produced by transformed organisms.

  • they can be made quickly & cheaply in large quantities
  • for example insulin.
• there are ethical, financial & social issues sometimes with using recombinant DNA technology.
• issues in agriculture:

  • the farmer might plant only one type of crop making it vulnerable to the same disease because all the plants are genetically identical.
  • superweeds might occur due to resistance to herbicides being transferred by interbreeding with wild plants.
• issues in industry:

  • some people wont like consuming food that has been genetically engineered.
• issues in medicine:

  • some people worry that DNA technology could be used to make designer babies. This is currently illegal.
• recombinant DNA has many humanitarian benefits:

  • crops could be produced to reduce risk of famine & malnutrition. e.g drought resistant crops
  • crops that produce pharmaceutical products, making them more available to areas where they arent as available.
  • medicine can br reproduced more cheaply so more people can afford them.
  • the potential to treat human diseases by gene therapy.
• what are the two types of gene therapy?

  somatic

  germ line
• somatic therapy involves altering the alleles in body cells, particularly cells that are most affected by the disorder.
• germ line therapy involves altering the alleles in the sex cells / gametes.

  • this means that every cell of any offspring produced will be affected by this gene therapy & they wont suffer from the disease.
• DNA probes can be used to locate specific alleles of genes or to see if a person has a mutated allele that causes a genetic disorder.
• DNA probes are short single strands of DNA.

  • They have a specific base sequence that is complementary to the base sequence of the target allele.
  • This means they will bind to the target allele if its present in the DNA.
  • A DNA probe has a label attached so it can be detected.
• what are the two main types of DNA probe labels?

  radioactive - detected using X-ray.

  fluorescent - detected using UV light.
• gel electrophoresis;

  1. A sample of DNA is cut into fragments using restriction endonuclease enzyme & is loaded into wells.
  2. A direct current is passed through the gel causing DNA fragments to separate.
  3. DNA moves towards the anode (positive end).
  4. The smaller the DNA fragment the faster they move through the gel.
  5. When the current is turned off the DNA fragments will have moves through the gel which can then be transferred onto absorbent paper by southern blotting.
• detecting different alleles;

  • a sample of DNA is cut into fragments by restriction endonuclease and separated with gel electrophoresis.
  • The separated fragments are transferred onto a nylon membrane & a probe is added.
  • If the specific allele is present the DNA probe will hybridise / bind to it.
  • The membrane is then exposed to UV light to detect any alleles present.
• electrophoresis separates DNA fragments to make a genetic fingerprint.
• DNA is slightly negatively charged.