DNA RECOMBINANT Introduction To DNA recombinant Introduction To DNA recombinant methodologies Uses of genetic engineering

Introduction DNA recombinant is the linking of two DNA molecules It is a product of cloning technology The technique required for the technology is so called genetic engineering

DNA recombinant methodologies DNA recombinant PCR Sequencing Hybridisation Synthetic Oligonucleotide DNA restriction by endonuclease Cloning

Hybridisation DNA transferred to nylon membrane or nitroselulose Addition of probes labelled with radioactive/enzyme that will hybridise to the DNA sequences of interest Identification of DNA of interest on the DNA recombinant/clone Figure 10.8

Sequencing Determination of nucleotide sequences for a particular DNA fragment, new recombinant DNA molecule, gene or chromosome Chemical degradation method by Maxam and Gilbert (1977) -Figure 5-3 Sanger DNA-sequencing procedure (Sanger et al. (1977) - Figure 5-4 Now  automated procedure

Output of a DNA sequence from an automated sequencer

Polymerase Chain Reaction The production/amplification of target DNA fragment exponentially - 2 n ,435,456 dsDNA molecule These steps were repeated for times: -Denaturation, 94 o C -Annealing o C -Elongation/synthesis 72 o C Figure 10.11

PCR Components needed for PCR –Template DNA (small quantity) –Primers (of known sequences) –Themostable polymerase enzyme (Taq polimerase) –dNTP (addition of nucleotides) –Mg++ (salt) –Thermocycler (an equipment that changes temperature instantly)

PCR-Thermocycler

DNA digest Restriction enzyme cuts DNA to fragments at palindromic sequences The enzymes recognises specific sequences of 4-8 bp long (palindrom) Cuts double helix DNA specifically In nature, the enzymes destroys foreign DNA that enters the bacteria

Restriction enzyme EcoRI from E.coli G A A T T C C T T A A G A A T T C C T T A A GG Sticky ends

Resriction enzymes mode of action G A A T T C C T T A A G PALINDROMIC sequences A A G C T T Hind III from T T C G A A Haemophilus influenza EcoRI from E.coli

Cloning Four processes are involved (Figure 10.2): 1) Vector and DNA fragment containing gene of interest were digested (cut) using restriction endonucleases 2) Vector and DNA fragment containing gene of interest were linked together to form recombinant DNA with the aid of DNA ligase

Ligation enzyme - DNA Ligase To form phosphodiester bond The mode of action for DNA ligase is the reversed of restriction enzyme needs ATP and 5’ phosphate and 3’ hydroxyl Both DNA molecules with sticky ends and blunt ends can be ligated Links more efficiently sticky ends DNA molecules — OH (P)— 5’-AATCGATCGTCC- -TTAGCTAGCAGG-5’ OH P P 5’-AATCGATCGTCC- -TTAGCTAGCAGG-5’ ATP Ligase - DNA ligase does not need specific sequences

Cloning Four processes are involved (continued): 3) The recombinant DNA molecule were forced into the host by a procedure called transformation. The recombinant DNA will replicate in the new host. 4) Identfying clones bearing recombinant DNA by hybridisation/restriction enzyme analysis/PCR Examples of cloning vectors (Figure 10.4 and 10.9)

Synthetic Oligonucleotide Single stranded DNA can be syntesised in the laboratory (Figure 10.10) The sequences can be determined by the researcher according to their needs Can be used for (as) - probe or label in hybridisation - primer in PCR - site-directed mutagenesis (Figure 10.15)

Uses of genetic engineering Human health: various health products Agriculture and livestock (breeding): Transgenic plant and animals Enviromental biotechnology: degradation of pollutants Forensic Industrial biotechnology

Therapy products from DNA recombinant technology Blood protein, Human hormones, Immune modulator, Vaccine (Table 10.1) Gene therapy : drug delivery, abnormal gene replacement (Cystic fibrosis, Duchenne Muscular Dystrophy, Adenosine deaminase deficiency) Genetic disease diagnosis:Sickle cell anemia, Thalassemia, cancer Gene regulation: Anti-sense RNA

Agriculture by means of Agrobacterium tumefaciens (Figure 10.19) Transgenic plant benefits: Delayed fruit ripeness/spoil Disease resistant plants Herbicide resistant palnts Flowers with new colour and pattern Protein producing plants

Delayed ripening of fruit Tomato- Flavr savr Anti-sense technology

Enviromental biotechnology Detoxification of heavy metals Degradation to less toxic molecules e.g. CuS0 4 and PbSO 4 at mines or contaminated water Waste disposal management -Detoxification of organic compounds

Industrial biotechnology Enzyme Beverage/food processes Detergent (protease ) Flavouring, colouring Organic acids Amino acids Vitamins

Forensic Technology Blood or tissue Fresh samples (if available) Not unique DNA Unique, more certain RFLP analysis and Southern blotting DNA of suspect and victim were compared Restriction enzyme analysis and electrophoresis PCR to amplify DNA copies

Forensic Technology DNA fingerprinting - crime, accident, inheritance DNA repeats as marker -microsatellite/“short tandem repeat”

Forensic Technology

Processes involved in genetic engineering All the techniques needed in genetic engineering were related to each other Figure 10.23

DNA, gen & kromosom