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Genetic engineering

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To describe DNA extraction To explain and demonstrate DNA cloning To explain the process of PCR and its uses. To explain DNA fingerprinting and its uses

Text of Genetic engineering

  • 1.Aim: to introduce DNA technology techniquesand its applications Objectives: To describe DNA extraction To explain and demonstrate DNA cloning To explain the process of PCR and its uses. To explain DNA fingerprinting and its uses

2. The structure of nucleic acidsThere are two types of nucleic acids:1. Deoxyribonucleic acid (DNA)2. Ribonucleic acid (RNA)Both are polymers made up of sub-units called nucleotides 3. Each nucleotide is made up of three parts: A Phosphate group A pentose sugar (either ribose or deoxyribose) A base which contains nitrogen 4. DNA molecule 5. RNA molecule 6. DNA extraction 7. centrifuge Break down Precipitate the cell wallthe DNA andusing membranesethanolAdd somebuffered detergentto break down the Centrifuge tocell membranesisolate theDNAvideoDissolve DNA 8. Gene cloning 9. Restriction enzymes Restriction enzymes are enzymes that cut DNA intosmall fragments. This allows individual genes to beisolated. Restrictions enzymes recognize and cleave at specificDNA sequences. E.g. EcoRI 10. Vectors Vector - A DNA molecule that carries the foreign DNA fragment into a host cellSeveral types of vectors are used including: Plasmids Bateriophages (i.e. the DNA of the bacteriophage genome) Viruses 11. Plasmids vectorsEssential features of a plasmidvector: A origin of replication A selectable marker that allowscells that carry the plasmid to bedistinguished from cells that donot. This is usually an antibioticresistance gene. One or more unique restrictionenzyme sites into which DNAcan be ligated. 12. Hosts Host cells include: E. coli Other types of bacterium Yeast 13. Basic steps in genetic engineering1. Isolate the gene2. Insert it in a host using a vector3. Produce as many copies of the host as possible4. Separate and purify the product of the gene 14. Step 1: Isolating the gene The action of a restriction enzyme 15. Step 2: Inserting gene into vector Plasmid removed frombacterial cell and are cutwith the samerestriction enzyme A chunk of DNA canthus be inserted intothe plasmid DNA toform a recombinant DNA ligase attacheshuman gene to plasmidat sticky ends 16. Step 3: inserting vector into host The recombinantplasmids are thenmixed with bacteria. Vector plasmid takenup by treatedbacterium This insertion is calledtransformation 17. Step 4: Multiplication of the host cells bycloning In these conditions,the bacteria multiplyrapidly, makingmany copies of thehuman gene. 18. Recombinants Vs non-recombinantsThe plasmids have naturally occuring genes for antibiotic resistance An agar plate containingAmpicillin is used to allowonly those cells which havetaken up a suitable plasmidto survive and divide. Thesecells must have resistanceto Ampicillin 19. Video 20. What is PCR? PCR involves the targeted amplification of aspecific DNA sequence. Using PCR the amount of a chosen DNAfragment can be increased 10-10 fold in 2-3hours 21. Performing a PCR reactionA PCR reaction contains:1. A small amount of DNA fragment to be amplified.2. The primers which act as signals to the DNA polymerase enzyme to start copying.3. The different types of nucleotide containing the bases adenine, guanine, cytosine and thymine.4. Taq DNA polymerase 22. ReactionPCR tube Thermocycler 23. Performing a PCR reaction Strands of sample DNAseparated by heating to95oC Mixture cooled to 55oCto allow primers to bind. Mixture heated to 70oCfor replication(optimum temp of DNApolymerase)AnimationVideo 24. PCR AmplificationExponential Amplification of template DNA 25. DNA fingerpriting DNA profiling allows individuals to be unambiguously identified. It relies on differences between the genomes of different individuals. The DNA of every human being is 99.9% the same. It is the 0.1% that makes all the difference 26. Short Tandem Repeats (STR) An STR is a sequence of 2-5 bp (e.g. TCAT) repeated from 1-50 times. STRs occur at many sites in the genome of humans andother animals. The number of copies of the repeat in a particular STRvaries enormously between different individuals. It is thenumber of times that these blocks of STRs are repeatedthat produces the variation in individuals. 27. Obtaining a DNA profile The DNA is extracted from the sample and cut into millions of small fragments using resctriction enzymes, aimed at a specific base pair sequence, called a restriction site. 28. DNA fragments are separated using electrophoresis. The DNA samples to be analysed are each added to a well. The fragments are then subjected to an electric field The smaller fragments move faster, the larger ones move slower 29. The DNA fragments are transfered to a nylon membrane by aprocess called Southern blotting. Radioactive probes are used to attach to specific parts of thefragments. The nylon sheet with DNA fragments attached is placed underX-ray film. Patterns of bands on DNA profile 30. This produces a visiblepattern of light and darkbands (where the radioactiveprobe is present) rather like abar-code. Everyones bar-code is different 31. Animation 32. DNA fingerprinting in Forensics DNA fingerprints can be used as biological evidence Strands of DNA can be found on hair, blood or semen Useful in solving crimes like murder and rape DNA Fingerprinting has exonorated people who were falsely convicted 33. DNA profiling in forensic science DNA from a victim (e.g. in a bloodspot) may be left on the clothing ofan attacker. Or attacker may leave their DNA on acrime victim (e.g. in blood, hair orsemen). In this case the DNA profilefrom the attacker is compared to thatof suspects. A large database of DNA profiles ofknown offenders has beenassembled. 34. In paternity testingFor each STR a child inherits oneallele (band on gel) from the mother,one from the father. In the example shown on the right, which shown the inheritance of a single STR, who is the father of the child? 35. Detection of pathogens by PCR Infections by certain pathogens can be detected byPCR using primers that recognise the pathogengenome. E.g. rabies can be diagnosed by amplifying rivalnucleic acid present in viruses in saliva. PCR is also valuable in the diagnosis of HIV it can detect infection very soon after exposure and also newborn babies