1. GENETIC ENGINEERING Akinniyi A. Osuntoki,Ph.D. 13/07/20181

2. WHAT IS GENETICS ? The study of heredity and variations. The study of how characteristics are passed from parents to offspring. Also studies how new traits arise. 13/07/20182

3. Genetic engineering or gene manipulation is a term applicable to various in-vivo and in-vitro techniques. In most Western countries, there are legal definitions due to Government legislation aimed at regulating the field. A key feature of genetic engineering is the capability to surmount natural species barriers and transfer genes from organisms to unrelated species. 13/07/20183

4. THE CHROMOSOME A nucleoprotein complex Contains the genetic material Polygenic Consists of DNA and basic proteins The protein kind and types may vary in different cell types Twenty three (23) pairs in humans 13/07/20184

5. THE GENE Sequence of DNA capable of independent replication (a replicon). Finite sequence of nucleotides. Contained on the chromosome. The unit of heredity. In eukaryotes, each cell contains the full genetic complement (exception: mature erythrocytes). In most life forms DNA is the molecule of heredity. Contains information on the blueprint for the organism. The major function is to specify the sequence of amino acids in a protein. Determines the structure of a protein. Must be expressed to perform its biological roles. 13/07/20185

6. THE GENETIC CODE Genetic information is organized into genes or cistrons. The message is encoded in a triplet of bases called codons. Codons specify the amino acids to be incorporated into a growing protein chain. Codons also specify chain termination. There are 64 codons 61 codons specify amino acids. 3 codons specify chain termination. The code is degenerate. The code is universal. 13/07/20186

7. WHY GENETIC MANIPULATION? 13/07/20187

8. THE BASIS OF GENETIC ENGINEERING The genetic code Enabling Technologies: Recombinant DNA techniques Polynucleotide sequencing Nucleic acid amplification (PCR) 13/07/20188

9. FUNDAMENTALS OF GENE CLONING Transfer of a gene from one cell to the other. Method for purifying, propagating and amplifying a specific DNA sequence or gene. The cloned fragment is placed under the genetic expression machinery of a suitable host cell. The extraneous DNA is faithfully copied. The cloned DNA is inherited by the clone cell line. Possible because of the universality of the genetic code. 13/07/20189

10. KEY FEATURES OF CLONING 1.A method for generating the DNA fragment or gene to be cloned. - Enzymatic or mechanical means can be used 2. Vectors - Vehicles for introducing the foreign DNA fragment into the host cell. 3. A means of fusing the DNA fragment to the vector. 4. An appropriate host cell and transformation protocol. 5. A recombinant selection method. 13/07/201810

11. VECTORS Various types Choice depends on the size of the DNA to be cloned Choice may also be informed by the screening protocol to be utilized Must have at least one selectable marker Relatively small molecules Must be able to replicate in the host cell If the protein product of the gene to be cloned is required, it must be capable of expression in the host cell. 13/07/201811

12. POLYMERASE CHAIN REACTION (PCR) PCR is a primer directed enzymatic amplification process It enables the amplification of specific DNA sequences The target sequence is defined by the primers The sequences of the flanking regions to the target site must be known The primers are complementary to the flanking regions 13/07/201812

13. “The invention and successful practice of the polymerase chain reaction (PCR) by Kary Mullis and colleagues in 1983 set the stage for a scientific revolution. PCR established a base technology from which many specific and diverse applications have grown. PCR has played a crucial underlying technological role in many aspects of the genomic age that we experience today. “Mickey Williams 13/07/201813

14. PCR: A POWERFUL TOOL PCR is one of the most powerful laboratory tools available It possesses a unique combination of attributes It can be carried out with comparative ease It is sequence specific It is sensitive It is flexible It is relatively cheap Permits rapid amplification of template DNA 13/07/201814

15. REQUIREMENTS Thermal cycler (thermocycler) PCR Amplification mix The amplification mix contains : ds DNA Thermostable DNA polymerase Two (or more) oligonucleotide primers dNTPs reaction buffer (Mg 2+ and reagents) 13/07/201815

16. STEPS IN PCR PCR serves to copy DNA It involves repeated cycles each consisting of three steps STEP 1 :Denaturation (thermal dissociation of ds DNA) STEP 2: Primer annealing STEP 3: Primer extension After each cycle, the number of copied molecules doubles. Thus, after 20 cycles about a million copies are produced. 13/07/201816

17. THE PCR CYCLE 13/07/201817

18. POLYMERASE CHAIN REACTION 13/07/201818

19. ANALYSIS OF PCR PRODUCTS The products of PCR amplification (amplicons) can be analyzed by various methods The analysis depends on the PCR technique used and what is being investigated. Methods that are employed include sequencing Electrophoresis To determine size Number of bands Changes in mobility DNA melting and hybridization kinetics Restriction enzyme digests 13/07/201819

20. REAL TIME PCR Real time PCR provides direct measurement of amplicon accumulation during the reaction Incorporates data collection during the amplification process Conventional PCR products are analyzed after the reaction has finished (end point analysis) End point analysis usually requires a separation technique (e.g. agarose gel electrophoresis) Real time PCR allows amplicon quantification and analysis during amplification 13/07/201820

21. ADVANTAGES OF REAL TIME PCR Much faster than conventional methods More reliable quantification results due to more sensitive detection systems Contamination is minimized The closed system, reagents used and end point melting analysis simplifies detection, quantification and genotyping 13/07/201821

22. OTHER TECHNIQUES IN GENETIC ENGINEERING GENE MAPPING This is the location of a gene relative to other genes on the chromosome. GENE PROBE A short sequence of nucleic acids, labeled with a tag, used to search for the presence of specific nucleotide sequences. BLOTS OR TRANSFERS Transferring genetic materials or products onto filters or membranes for screening purposes is called blotting. The followings are examples. Southern blot: This is the transfer of DNA, usually ssDNA, onto nitrocellulose or nylon filters for hybridization. Northern blot: This is the transfer of RNA onto a filter or membrane for screening purposes.

23. SOME OTHER IMPORTANT TECHNIQUES Gene therapy Microarrays