Genetics Practical Revision PDF 24-25

Genetics practical revision Medical Biochemistry department 24-25 Safety Rules and Laboratory Instruments Safety Rules in the lab -Complete the missing parts -List the general lab safety rules Appearance General work...

Genetics practical revision Medical Biochemistry department 24-25 Safety Rules and Laboratory Instruments Safety Rules in the lab -Complete the missing parts -List the general lab safety rules Appearance General work Hygiene Emergency and attitude procedures practice procedures Clean , dry, non-broken Clean white coat glassware First aid materials are available. Place personal items in Cautious to hazardous cabinet chemicals and flame Do not touch your face ,eyes Hold hot glassware with or clothes while using Stay in proper position/don’t solutions Any accident → inform your holder supervisor sit on benches When heating point test Listen to instructor tubes away from your face and others If any accident, such as burns from acid or alkali Follow guide Wash the affected area → Never carry, taste, touch wash immediately with chemicals plenty of running water. Tie up hair/veil-jewels inside coat Don’t “sniff directly” the chemicals (Gently wave the In case of eye injury, Flush chemical towards your face) eyes immediately with plenty No inflammables Always clean your work area of water for at least 15 and wash your hands before minutes. Never pour back-never mix leaving he lab. No drinks/food chemicals-never keep bottles open In case of Fainting Provide fresh air and keep the head Respect supervisor-colleagues Use suction device-wear lower than the rest of the -time gloves-safety goggles body 1. Which of the following is NOT a laboratory safety rule? A. You should never mix unknown acids with bases B. You should tie back your long hair C. You should never add water to acid D. All of the above are valid safety rules 2. Students are using heat and chemicals in a laboratory experiment. Which of these is the best method of eye protection? A. Safety Goggles B. Prescription Glasses C. Contact Lenses D. Eye Protection is not needed. Identify three wrong actions not following lab safety rules Answer: 1-Never taste anything 2-Gently wave the chemical towards your face. 3-Don’t sniff directly the chemicals Never mix chemicals without your instructor permission. Laboratory Instruments  Categorize the different instruments with their uses in practice 1. Liquid volume measurements. 2. Transfer, mixing, boiling and storage of chemicals. 3. Handling of chemicals. 4. Filtration.  Complete the missing parts in the presented figure Volume measurements From 1ml to More than Less than 1ml 10ml 10ml Glass Automatic Graduated Volumetric Graduated pipette cylinder flask pipette  Liquid volume measurements Pipe es → Measure and deliver small volumes of liquid Automatic pipettes Glass graduated pipettes Pasteur pipettes Micro volume Small volume Small Non- accurately(up to → accurate volumes 1ml=1000µl) 1 ml- 10 ml  Liquid volume measurements Measure and deliver large volumes of liquid (more than 10ml) Graduated cylinder Volumetric flask Draw backs: Measure different Measure different One volume volumes of larger volumes And only liquids concentrations More accurate  Beaker, Graduated cylinder and volumetric flask as regard Precision and Cost Beaker Graduated cylinder Volumetric flask Accurate Not accurate More accurate Expensive Cheap 12  Transfer, mixing, boiling and storage of chemicals Glass rod Test tubes Chemical bottles Performing chemical Storage of different Manually stir experiments in lab. chemicals, reagents, solutions e.g Holding liquid and even strong samples for heating, corrosives. dissolution and Always carry an others informative label. Crucible It is made of porcelain or ceramic to withstand heat. used for heating certain metals, to very high temperatures. 1. Liquid volume measurements. 2. Transfer, mixing, boiling and storage of chemicals. 3. Handling of chemicals. 4. Filtration.  Handling of chemicals Pipette pump Spatula Droppers It is inserted into It resembles small They are used the end of the paddles or teaspoons. to transfer glass pipette to It resists corrosions. small amounts help the delivery It is used to handle or drops of of liquid without solid powdered liquid solutions. suction by mouth.. chemicals.  Handling of chemicals Test tube racks Test tube holder Holding hot test Test tube racks tube. are for holding and organizing test tubes on the laboratory counter. Don’t put hot test tubes in Plastic racks.  Handling of chemicals Test tube brushes Bunsen burner Used to clean Forcing a large Heating non- test tubes brush into a volatile liquids and graduated small test tube and solids cylinders. will often break the tube.  DNA Extraction Applied DNA extraction from commercial DNA extraction kit  Complete the missing items in DNA extraction methods Chemical Physical Combined method method method Lysis Magnetic Spin particle reagents column purification We are going to use the: Spin column-based nucleic acid purification method. (Combined method)  What are Spin Columns? They are small plastic tubes which comprise a silica resin that selectively binds DNA or RNA depending on various factors. Principles of DNA extraction... The basic steps of DNA extraction are Lysis Precipitation Purification First, we obtained a buccal mucosa sample.  Cell lysis using a lysis buffer AIM: The cell membrane is disrupted chemically using a lysis buffer to get a fluid containing all the cell components including DNA.  Centrifugation by centrifuge centrifuge AIM: Centrifugation is a method of separating molecules having different densities at high speed. Centrifugation is used to collect cells and to precipitate DNA. Wash of the membrane from impurities AIM: The wash steps remove impurities.  Discard the flow through What is the aim of the elution step? It is the process of extracting one material (DNA) from the loaded spin column (attached to its silica membrane) by washing with a solvent to release it in a soluble form. What is the function of elution buffer? Elution buffer is the major solvent in spin column. Elution buffer is used to wash away unbound impurities at first and it releases the desired nucleic acid (DNA). Basic Molecular Biology Techniques, Restriction Digest & DNA Electrophoresis 31 Do you know what are the basic steps of analyzing DNA??? DNA Extraction DNA Restriction DNA Electrophoresis What are Restriction enzymes?? Molecular Scissors Restriction enzymes are proteins act as molecular scissors that recognize and bind to specific DNA sequences and cut the DNA at or near the recognition site.  Types of cuts by Restriction enzymes 1- 2- Sticky ends Blunt ends Activity: write the expected results of cuts produced by the 2 RE in the shown 2 examples answer 1 2 G AATTC GAT ATC CTTAA G CTA TAG DNA ELECTROPHORESIS Parts of DNA gel Electrophoresis apparatus: Power supply Cover with leads Cover Gel tank  Casting tray Gel combs Casting tray for vertical Casting tray for horizontal electrophoresis electrophoresis Identify the provided part of electrophoresis equipment and mention its use. Name use It is the place for the gel, it is filled with the Gel tank buffer allowing the current to pass through the gel The electrode leads attached to the cover Cover with leads are connected to the power supply allowing the current to pass To cast the gel where it provides the shape Casting tray of the gel To form the sample wells in the gel Gel combs To supply the electric current Power supply To visualize ethidium bromide-stained DNA Transilluminator in gels How to Perform Standard Agarose DNA Gel Electrophoresis Run?? Gel preparation Casting the gel Setting up electrophoretic chamber Sample preparation & loading Running the gel Gel staining Visualizing DNA & Analysis - Visualizing the DNA by an Ultraviolet light Transilluminator 40 Molecular Biology Techniques Blotting Techniques DNA Fingerprinting 42 Blotting steps 43 Probe Specific labelled sequence short piece Antibody to single-stranded certain protein oligonucleotides hybridize (join) to the biological labelled with a molecules (DNA, Marker RNA or protein) on the membrane 44 Examples of labelling molecules: 1- Radioisotope, such as 32P, 2- Nonradioactive molecule, such as biotin or a fluorescent dye.  Types of blotting techniques Southern blot Northern blot Western blot protein DNA fragments are RNA molecules are molecules are transferred transferred transferred 46 A police officer is investigating a crime scene. A DNA test was performed on dried blood spot at the crime scene (felon) as shown in figure above. Which of the suspects is the murderer? A.Suspect 1 B.Suspect 2 C.Suspect 3 Introduction to Clinical Bioinformatics (1) Biochemistry Department - 48  Define the following terms: Bioinformatics. The use of computers to collect, analyze, and interpret biological information at the molecular level. Database. Organized collection of biological data. They contain information from research areas including genes, protein, metabolic pathway, , gene expression and diseases  Flowchart for using clinical bionformatics to analyze the provided case DNA of Osteogenesis imperfecta Gene finding genes in the sequence prediction Comparing Once identified, the sequence can be compared to the known genome sequence of similar or closely related organisms in order to genome identify any key similarities or differences. Translation Translate sequence into protein Determine 3D structure of the Protein shape protein   Comparison between different databases: Blast program Expasy program Cn3d program Tool used for rapid Tool that allows Tool that searching of the translation of predict the 3D nucleotide and protein sequences a nucleotide structure of a  Blastn: for (DNA/RNA) protein. nucleotide sequence to a sequence protein alignment  Blastp: for amino sequence. acid sequence alignment What is the name of the tested gene in this figure? aldo-keto reductase family 7 This previous alignment of nucleotides is performed in ---------- by----------: A. Human, BLASTp B. Human, BLASTn C. Mice, BLASTn D. Guiana pig,BLASTn E. Horses, BLASTp What is the name of this program? Expasy Which frame is the most propable to be the expected protein& why? Frame 1 the longest genetic message starting with methionine and ending with stop Choose the frame with the longest genetic message starting with methionine and ending with stop The above presented printscreen shows single sequence alignment that can be performed using: A. BLASTn B. BLASTp C. Expasy D.Cn3D program E. BLASTx The number of deleted bases in the mutated sequence is: A. 0 B. 2 C. 3 D. 4 What is the name of the tested gene in this figure? How much is it similar to normal sequence ? Cystic fibrosis transmembrane conductance regulator(CFTR) gene 99% This alignment can be performed by using: A. BLASTn B. BLASTp C. Expasy D. Cn3D program E. BLASTx Presented printscreen shows single sequence alignment that can be performed using: A. BLASTn B. BLASTp C. Expasy D. Cn3D program E. BLASTx Percentage of difference between mutated gene and matched sequence in the following data base is: a) 9 % b) 1 % c) 99 % d) 100% e) 0 % Match up the program in column A with its proper use in column B 1. BLASTp D 2. Expasy C 3. BLASTn A A.compare nucleotide sequences B.predict 3D structure of the protein C.translate DNA sequence into protein D.compare amino acid sequences E.drug docking