Techniques in biotechnology Principles, Methods AND Applications of Biotechnology

BIOTECHNOLOGY TEST ONE DATE: 28 JULY 2017 TIME: 8:00 AM VENUE: LECTURE ROOM

I. Introduction A. What is Biotechnology, Genetic Engineering? Biotechnology is the use of living cell or parts thereof of the benefit of mankind. Remember that the interpretation of benefits’ is contextual. For example in bioterrorism, who is deriving benefits? Genetic Engineering is the deliberate (artificial) manipulation of the genome of a organism to cause an altered gene expression pattern from the wild type. You will realize any biotechnology related book that you will read will have a slight different version of these definition, this should not worry you as this is one of the still evolving disciplines of biology. B. Why is this subject important? This subject is important in the sense that man is continuous using it to improve his/her quality of life in agriculture, health and industry. This technology is being used to narrow the productivity/efficiency gap. Under a textbook scenario on would expect productivity of agricultural systems of efficiency of medical health delivery system to 100% but this is not the case in real life, so Genetic engineering and biotechnology tries to get this 100% rate.

II. Recombinant DNA A. Goal: to move gene (1-2 Kbases) from a complex gene pool into a simple vector and transfer to another complex gene pool  B. Cloning Steps 1. Isolate and fragment of source DNA 2. Join source DNA to cloning vector 3. Incorporate DNA into host 4. Detect and purify desired clone 5. Replicate clone to high numbers C. Restriction and Modification Enzymes 1. Restriction Enzymes definition: enzymes that cut DNA in specific places function: inactivate foreign DNA which can derange metabolism breaks only palindrom sequences, i.e. those exhibiting two-fold symmetry mechanism examples Important in DNA research, i.e. sequencing, hybridization Companies purify and market restriction enzymes

2. Modification Enzymes   protect destruction of native DNA by native restriction enzymes mechanism A nucleotide can be a substrate for a particular restriction enzyme or modification enzyme but not both D. Gene Amplification: polymerase chain reaction (PCR)  1. Objective: to make multiple copies of a specific gene 2. Method DNA is removed and purified DNA is heated to denature (separate strands) Specific primer are added to locate desired sequences along with DNA polymerase; primer initiates replication of desired fragments during cooling cycle Cycle is repeated resulting in logarithmic increase in desired fragments Amplified DNA is separated using electrophoresis. Amplified DNA may now be sequenced or used for other purposes E. Cloning Vectors 1. Plasmids example of plasmid cutting plasmid with restriction enzymes (a) (b) (c) DNA modification using plasmids advantages 2. Phage DNA modification using phage Steps in cloning with phage 3. Detection of Cloned Gene

F. Mechanical Injection of DNA 1. Gene Gun: DNA-coated pellets are injected into recipient cells  2. Microinjection method (injection into mouse sperm pronucleus; nucleus has entered egg but has not combined with it) example: salmon eggs injected with gene for growth hormone (14 month-old fish) III. Other Tools and Methods A. Gels and Micropipettes B. DNA Sequencing Gel C. DNA Fingerprints D. DNA Synthesis E. Reverse Transcription F. Gene Probes IV. Genetically Engineered Organisms A. Insulin (two active proteins(A and B) separated by a third (C) that must be removed B. Genetically Engineering Disease Resistance in Tobacco Plant C. Therapeutic Use of Antisense DNA for Clogged Arteries (gene blocks healing which can harden or block arteries after an angioplasty; may be applied using a virus vector) D. Human Genome Project humans have about 3.2 billion base pairs in 23 pairs of chromosomes 99.9% of the nucleotide sequence is the same will be useful in detecting genetic diseases and gene therapy

V. Biotechnology Protein Sequencing DNA Sequencing DNA Synthesis Protoplast Fusion Electron Microscopy Antibody Probes Biological Computing Education and Training VI. Methods in Biotechnology Isolation of DNA Gel Electrophoresis Apparatus Gel Results UV Absorption Spectrum of DNA DNA Labeling with Dyes Cesium Gradient Centrifugation DNA Melting Heating and Cooling of DNA (Thermocycler) DNA Hybridization Blot Plot From DNA Analyzer