DNA RECOMBINANT Introduction To DNA recombinant Introduction To DNA recombinant methodologies Uses...
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Transcript of DNA RECOMBINANT Introduction To DNA recombinant Introduction To DNA recombinant methodologies Uses...
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
DNArecombinant
PCR
Sequencing
HybridisationSynthetic
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 - 2n- • 230 - 268,435,456 dsDNA molecule• These steps were repeated for 30-40
times:-Denaturation, 94o C-Annealing 45-72o C-Elongation/synthesis 72o 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 enzymeEcoRI from E.coliG A A T T C
C T T A A G
A A T T C
C T T A AG G
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 fromE.coli
CloningFour 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’
OHP
POH
5’-AATCGATCGTCC- -TTAGCTAGCAGG-5’
ATP
Ligase
- DNA ligase does not need specific sequences
CloningFour 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 palntsFlowers with new colour and patternProtein producing plants
Delayedripening of fruit
Tomato-Flavr savr
Anti-sense technology
Enviromental biotechnology• Detoxification of heavy metals • Degradation to less toxic molecules
e.g. CuS04 and PbSO4
at mines or contaminated water• Waste disposal management
-Detoxification of organic compounds
Industrial biotechnology
• EnzymeBeverage/food processes
Detergent (protease)• Flavouring, colouring • Organic acids• Amino acids• Vitamins
Forensic TechnologyBlood or tissue
Fresh samples (if available)Not unique
DNA Unique, more certainRFLP analysis and Southern blotting DNA of suspect and victim were comparedRestriction enzyme analysis and
electrophoresisPCR to amplify DNA copies
Forensic Technology DNA fingerprinting -
crime, accident, inheritance DNA repeats as marker
-microsatellite/“short tandem repeat”
Forensic Technology
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