Manipulating genomes

Manipulating genomes

1. DNA sequencing

   • Fred Sangers DNA- 1981
   • Pyrosequencing
     • Involves synthesising a single strand of DNA, complementary to strand to be sequenced
     • Long length of DNA cut
       • Lengths degraded into single stranded DNA - immobilised
         • Sequencing primer added - DNA incubated with DNA polymerase
           • One activated nucleotide incorporated into a comp strand of DNA
   • Allows genes to be isolated & read
   • Cloning DNA
     • Gene sequence was isolated using restriction enzymes from bacterium
     • DNA inserted into vectors and then Ecoli host
       • Divided many times, allowing plasmid to be copied

2. Applications

   • Human genome project - launched03 learned only 24,000 - not many more than a mouse
   • Comparisons between species
     • 99% with chimpanzees
     • Some genes have altered and now work in subtly dif ways
   • Help confirm evolutionary relationships

3. DNA profiling

   • Alec Jeffreys 1978
   • Procedure
     • 1) DNA obtained
       • 2) DNA digested with restriction enzymes - cut at specific sites
         • 3) Fragments separated by gel electrophoresis & stained
           • 4) Banding pattern can be seen
   • Application
     • Forensic science
       • Identify suspects
     • Maternity
       • Compare DNA profiles
     • Analysis of disease
       • Look at certain sequences

4. Polymerase chain reaction

   • Amplify amount of DNA
   • Relies on:
     • Antiparallel
     • 5' 3'
     • Grows from 3' end
     • Complementary base pairing
   • Steps
     • 1) DNA mixed with DNA nucleotides, primes and mg ions
       • 2) Mixture heated 95c, break H bonds between pairs = single strands
         • 3) Cooled to 68c, primers can bind (H) to end of DNA = small bit of 2strand
           • 4) DNA polymerase bin to double strand - Polymerase obtained from high temp
             • 5) Temp raised to 72c keep single strands
               • 6) Polymerase catalyses addition of DNA nucleotides to single strand
                 • 7) When polymerase reaches other end = double strand generated
   • Applications
     • Detection of oncogenes
     • Detecting mutations
     • Identify viral infections
     • Forensic science
     • Research

5. Electrophoresis

   • Used to separate different sized DNA fragments
   • Uses agarose gel plate covered by a buffer solution
     • Electrodes places @ each end of tank- when connected to electric - flow through gel
   • Separating proteins
     • Add a charged detergent
     • =ises surface charge - separate as move through gel
   • Using DNA probes
     • Single stranded comp DNA to that being investigated
     • Help locate specific DNA

6. Genetic engineering

   • Involves combining DNA from dif organisms
   • Needs
     • Required gene
     • Copy of gene
     • Vector
     • Recipient express gene
   • Obtaining required gene
     • DNA probe used to locate, gene cut out using restriction enzymes
     • Use PCR
   • Playing gene into a vector
     • Plasmid had exposed unpaired nucleotide bases = sticky ends
       • DNA ligases catalyses bind to the gene of sticky ends
   • Getting vector to recipient
     • Heat shock treatment 42c-0c membrane & wall more porous
     • Electrofusion - introduce DNA into cells

7. Issues with genetic manipulation

   • Microorganisms
     • + make human insulin to treat all diabetics, produce growth hormone
     • - Transfer genes to others = resistance , can't live outside lab
   • Mice
     • + bread for medical research for cancer & learn functions of genes
     • - Object to uses of animals
   • Silk
     • + can be used for sutures, cables etc - genes inserted into goats - produced in milk
     • - Concerns for welfare for GM goats
   • Pathogens
     • + Used to make vaccines, still have antigens present and can also be used as vectors in gene therapy
     • - Risk of cancer and interfere with regulation
   • Playing GOD

8. Gene therapy

   • Insert a functional allele of a particular gene into cells that contain only mutated and non-functioning alleles of gene
   • Somatic cell
     • Alliterations made to genome often two cells
     • Not passes to offspring