DNAEXTRACTIONGrade 10 Science / Life and Living: DNACompiled by Madre’ Nortje
SCIENCE @ WORK EXPERIMENT 2.1 DNA EXTRACTION A CONTEXTUAL APPROACH SCIENCE 10 (HEINEMANN): P1252 GRADE 10: Life and Living DATE of Practical: 17 March 2011
QUESTION: WHAT DOES DNA STAND FOR? DEOXYRIBONUCLEIC ACID THE COMPLEX CHEMICAL COMPOUND FOUND INN CHROMOSOMES THAT CONTAINS THE GENETIC CODE3 TODAY WE WILL CONNECT DNA WITH:......extraction......
DO ONLY LIVING ORGANISMSCONTAIN DNA?What do you think?Discuss in pairs for 1 minuteGive reasons.
EXTRACTING DNA FROM ANY LIVING THING Just follow these 3 easy steps: DetergenteNzymes Alcohol 5 http://learn.genetics.utah.edu/content/labs/extraction/howto/
•All living organisms contain DNA in their cells – FOUND in nucleus. DNA - Deoxyribonucleic acid! The nucleus is a membrane bound YOU MEAN I CAN SEE IT? HOW? structure that contains the cells hereditary information and controls the cells growth and reproduction. It is commonly the most prominent organelle in the cell. www..kamibudaksains.blogspot.com •DNA - The complex chemicalcompound found in chromosomes that 6 contains the genetic code.
FIGURE: :// HTTP . . / / / 331/ EMPLOYEES CSBSJU EDU HJAKUBOWSKI CLASSES CH DNA/OLDNASTRUCTURE.HTMLPACKAGING OF DNA IN THE NUCLEUS 7
DNA extraction refers to the process that scientists use to break down a piece of evidence, such as a piece of hair or a drop of blood, in order to determine the individuals DNA, which is unique to each person. Understand the process of DNA extraction with information from a biology teacher in this free video on science. Expert: Janice Crenetti Contact: WeAreHDTV.com Bio: Janice Creneti has a Bachelor of Science in secondary science education and a Bachelor of Art in biology from Boston University. Filmmaker: Christopher Rokosz Category: Entertainment Tags: science dna dna testing dna structure dna model dna replication dna molecule 9
REVIEWING OF INFORMATION ON: LABORATORY SAFETY RULES AND RISK ASSESSMENTS Do not enter the laboratory unless you are with a teacher. Never touch equipment in the laboratory unless you are told to use it. Don’t eat or drink in the laboratory. Always walk—never run. Wear protective clothing— a laboratory coat or apron and, when appropriate, safety glasses. Never taste anything. Tie up long hair. Always point test tubes away from people. Check with your teacher on how to dispose of waste liquids and solids. Broken glass should be cleaned up using gloves, a brush and dustpan, and placed in a special bin. If you spill something on your skin or clothes, wash it immediately with lots of water. Tell your teacher. Report all accidents and breakages to your teacher. After heating equipment, let it cool on a heatproof mat before picking it up. This will avoid burns. Clean all equipment after use and put it back where you got it from. 10 Clean and dry your work bench.
RISK ASSESSMENT ON EXPERIMENT AND INVESTIGATION – KIWI FRUIT Eyewear and protective clothing No eat or drinking Cover the MSDS’ e on Isopropanol and Methylene blue – hard copy handouts (health and safety regulation) Any broken glassware must be reported Care must be taken when using and 11 washing the blender – sharp blades
ISOPROPANOL – MSDS HAND OUTSCHEMWATCH ISSUE DATE: 15/05/2010 ISOPROPANOL - FLAMMABLE Dangerous Good 3 SAFETY-DATA(tm) Ratings (Provided here for your convenience) ----------------------------------------------------------------------------------------------------------- Health Rating: 2 - Moderate Flammability Rating: 3 - Severe (Flammable) Reactivity Rating: 2 - Moderate Contact Rating: 3 - Severe Lab Protective Equip: GOGGLES & SHIELD; LAB COAT & APRON; VENT HOOD; PROPER GLOVES; CLASS B EXTINGUISHER Storage Colour Code: Red (Flammable) Potential Health Effects ---------------------------------- Inhalation: Inhalation of vapours irritates the respiratory tract. Exposure to high concentrations has a narcotic effect, producing symptoms of dizziness, drowsiness, headache, staggering, unconsciousness and possibly death. Ingestion: Can cause drowsiness, unconsciousness, and death. Gastrointestinal pain, cramps, nausea, vomiting, and diarrhoea may also result. The single lethal dose for a human adult = about 250 mls (8 ounces). Skin Contact: May cause irritation with redness and pain. May be absorbed through the skin with possible systemic effects. Eye Contact: Vapours cause eye irritation. Splashes cause severe irritation, possible corneal burns and eye damage. Chronic Exposure: Chronic exposure may cause skin effects. Aggravation of Pre-existing Conditions: Persons with pre-existing skin disorders or impaired liver, kidney, or pulmonary function may be more susceptible to the effects of this agent 12
METHYLENE BLUE – MSDS HAND OUTSCHEMWATCH ISSUE DATE: 15/05/2010 METHYLENE BLUE SAFETY-DATA(tm) Ratings (Provided here for your convenience) ----------------------------------------------------------------------------------------------------------- Health Rating: 2 - Moderate Flammability Rating: 1 - Slight Reactivity Rating: 1 - Slight Contact Rating: 1 - Slight Lab Protective Equip: GOGGLES; LAB COAT; VENT HOOD; PROPER GLOVES Storage Colour Code: Green (General Storage) ----------------------------------------------------------------------------------------------------------- Potential Health Effects ---------------------------------- This material is relatively nonhazardous in routine industrial situations. Inhalation: No adverse health effects expected from inhalation. May cause a short period of rapid or difficult breathing. Ingestion: A burning sensation of the mouth may be noted following ingestion of methylene blue. May cause nausea, vomiting, diarrhea, and gastritis. Large doses may cause abdominal and chest pain, headache, profuse sweating, mental confusion, painful maturation, and methemoglobinemia. Skin Contact: Not expected to be a health hazard from skin exposure. Methylene blue may colour the skin a bluish colour. May cause photosensitization. Eye Contact: No adverse effects expected. May cause mechanical irritation. Chronic Exposure: No information found. Aggravation of Pre-existing Conditions: No information found. 13
NEW WORDS TO WORD BANKADD THESE TO YOUR EXCEL ALPHA TABLEWORDS MEANING: Precipitate Spooling Denaturising Lysing Homogenized Aggregate Lipid Centrifuging PCR 14
•DO YOU THINK YOU HAVE VERY MUCH IN COMMON WITH A KIWI FRUIT? BELIEVE IT OR NOT, A KIWIS GENETIC MATERIAL IS VERY SIMILAR TO YOUR OWN! SEE AND TOUCH THE GENETIC MATERIAL THAT YOULL EXTRACT FROM THE CELLS OF A KIWI FRUIT.15 Activity Overview Extract DNA from kiwi fruit using simple household chemicals
16FRUITDNA EXTRACTION USING KIWIHTTP://LEARN.GENETICS.UTAH.EDU/CONTENT/LABS/EXTRACTION/HOWTO/
MATERIALS / EQUIPMENT www.exploratorium.edu TO EXTRACT DNA FROM THE CELLS OF KIWI FRUIT.! AIM (per student group takes 30 minutes) ➤ ½ cup Kiwi fruit ➤ 200 mL water ➤ dishwashing detergent ➤ dropping pipette ➤ fine mesh kitchen strainer & cheese cloth ➤ glass rod ➤ large beaker ➤ large test tube ➤ light microscope ➤ meat tenderiser ➤ methylene blue ➤ microscope lamp ➤inoculation needle ➤ microscope slide and cover slip ➤ paper towelling ➤ small beaker of alcohol (Isopropanol) ➤ spatula 17 ➤ test-tube rack ➤ mortar and pestle
PART A: EXTRACTING THE DNATAKE NOTES – NO TEXTBOOKS ALLOWEDWHEN PERFORMING EXPERIMENT –MUST RECALL PROCEDURES / METHOD METHOD: 1. Peal and Place the kiwi and water in the beaker and mush it with a mortar and pestle. DNA SOURCE About 125 ml / 1/2 cup Twice as much water as DNA source (250 ml/ 1 cup) Table salt, large pinch 1g / 1/4 teaspoon Stir until the mixture is of a thin, soupy 18 consistency.
WHAT MUST I DO NOW? DISHWASHINGSTRAIN THE DNA MIXTURE DETERGENT 19
2.Pour the thin cell mixture through the kitchen strainer (cheesecloth) into another large beaker. 3.Measure 12 ml of the soup into a small beaker Add 2 ml of liquid detergent Swirl to mix stir thoroughly using a glass rod Let mixture sit in container with hot tap water (60 - 65°C) for 5 - 10 minutes 20
WHAT MUST I DO NOW?MEAT TENDERIZER ALCOHOL SEPARATIONENZYME POWDER www.forums.overclockers.com.au 21
4. Add a pinch of enzyme (meat tenderizer) to mixture. (Using pineapple juice or contact lens cleaning solution will do the same as tenderizer.) Gently stir with toothpick/ skewer for 5 minutes, continue stirring, not too vigorously. Be careful! If you stir too hard, youll break up the DNA, making it harder to see. 5. Quarter-fill a large test tube with the 22 mixture.
SLOWLY pour the same amount ice - cold (70 - 95% isopropyl or ethyl alcohol) into test tube pour alcohol down the side of the test tube. Tilting the test tube will make this easier to do. It forms a layer on top of the cell mixture. DO NOT MIX THE TWO LAYERS TOGETHER. Amount of alcohol and mixture should be the same. 23
7. Observe the mixture for a few minutes. You will see a white, threadlike substance rise from the mixture to rest above in the alcohol layer. This is the DNA that you have extracted from the cells of the kiwi fruit. 8. You can get more DNA to precipitate from the solution using a DNA collecting tool (glass or paper clip hook or cut inoculation needle) Gently lift the water solution up into the alcohol layer (this allows more DNA to get in 25 contact with the alcohol and precipitate).
INFORMATION: DNA precipitates as a white stringy / ―snotty‖ film at the water - alcohol interface and eventually will rise into the alcohol layer from the mixture layer. Allow test tube to sit for several minutes. The clearer the DNA is the fewer impurities you have. Ifyou have an acceptable amount of DNA, it can be "spooled" by rotating your collecting tool and then transferred into a clean tube / container. 26
WOW – SPOOLING THE DNA!!! If you are careful you may be able wind up the DNA around a glass rod or a skewer. Position the tip of the glass rod or skewer where you can see the threads of DNA. Steadily twist the rod or skewer as if you were making candy floss. Alternatively use a straw to pull it out by suction – be careful not to get in you mouth. Don’t go too quickly. You should be able to pull the strands of DNA out of the mixture. ASK STUDENTS TO TAKE MOBILE PHONE PICTURES OF THEIR OWN EXTRACTED DNA AND COMPARE APPEARANCE WITH OTHERS Photos of steps can be inserted in flow diagram.(ICT) 27
Ifyou want to save your DNA, you can transfer it to a small container filled with alcohol Leave tube / container uncapped until the ethanol has evaporated. DNA can be stored in the fridge (dry or water /buffer can be added). You can now investigate the property. 29
WHAT IS THAT STRINGY STUFF? DNA IS A LONG, STRINGY MOLECULE. THE SALT THAT YOU ADDED IN STEP ONE HELPS IT STICK TOGETHER. SO WHAT YOU SEE ARE CLUMPS OF TANGLED DNA MOLECULES! DNA NORMALLY STAYS DISSOLVED IN WATER, BUT WHEN SALTY DNA COMES IN CONTACT WITH ALCOHOL IT BECOMES UNDISSOLVED. THIS IS30 CALLED PRECIPITATION. THE PHYSICAL FORCE OF THE DNA CLUMPING TOGETHER AS IT PRECIPITATES PULLS MORE STRANDS ALONG WITH IT AS IT RISES INTO THE ALCOHOL.
THE WHY’S!!!! Blending separated the pea cells. In order to extract DNA from a cell, the associated membranes and proteins must first be removed (break apart the cells) and then physically separated (loosen the tough cell wall) from the DNA. To see the DNA, we have to break open these two sacks. We do this with detergent and salt( Sodium can be involved in several of the steps ). Sodium is an element. Its chemical symbol is Na for Natrium, the Latin word for sodium. It is a positive ion and often associates with negative ions as part of useful compounds. Salt help precipitate protein and carbohydrates away from the DNA. Salt helps strip away the proteins associated with DNA. + and – charge 31
THE WHY’S!!!! Salt and Detergents are used to break down cell walls and nuclear membranes to release the DNA. They work by chemically poking holes in the cell membranes or walls. Once holes are poked in the membranes, the membranes can be further disrupted mechanically, as with a blender. After that, it is easier to get the contents of the cell out, including the DNA. LETS HAVE A LOOK AT THE CELL! 32
!http://learn.genetics.utah.edu/content/labs/extraction/howto/HOW TO EXTRACT DNA FROM ANYTHING LIVINGCELL TO DNA 33
THE WHY’S!!!! WHY DETERGENT? Each cell is surrounded by a sack (the cell membrane) DNA is found inside the second sack (nucleus) within each cell. To see the DNA we have to break it open. A cell membrane has 2 layers of lipid(fat) molecules with protein going through them. When the lysis buffer (detergent) comes close to the cell, it captures the lipids and the proteins - breaks open the cell destroying the fatty membrane - DNA now released into the solution. 34
A CELLS MEMBRANES HAVE TWO LAYERS OFLIPID (FAT) MOLECULES WITH PROTEINS GOINGTHROUGH THEM. 35
Why detergent? How does detergent work? Think about why you use soap to wash dishes or your hands. To remove grease and dirt, right? Soap molecules and grease molecules are made of two parts: 1.(Blue) Heads, which like water. 2.(Green) Tails, which hate water. 36
AFTER ADDING THE DETERGENT, WHAT DOYOU HAVE IN YOUR SOUP? 37
THE WHY’S!!!! ENZYME (MEAT TENDERIZER) The DNA in the nucleus of the cell is molded, folded, and protected by proteins. The tenderizer cut the proteins away from the DNA In this experiment, meat tenderizer acts as an enzyme to cut proteins just like a pair of scissors. The meat tenderizer cuts the proteins away from the DNA 38
THE WHY’S!!!!CUTTING PROTEIN AND DNA The DNA in the nucleus of the cell is moulded, folded, and protected by proteins 39
THE WHY’S!!!!ALCOHOL Alcohol is less dense than water, so it floats on top. Look for clumps of white stringy stuff where the water and alcohol layers meet. DNA is not soluble in alcohol - other cell parts are. By adding alcohol DNA precipitates out of the solution and collect at the interface of the alcohol and soap layer. The colder the alcohol the less soluble the DNA will be in it. 40
COLD ALCOHOL DNA dissolves in water but precipitates in alcohol. Cold alcohol is used to separate DNA out of water-based solutions. This allows the DNA to be purified for subsequent genetic testing. Adding alcohol to a solution containing DNA is a simple way to obtain the pure DNA required, and colder temperatures slow down enzymes that can break down DNA, giving better extraction results. 41
WHY IS DNA EXTRACTION IMPORTANT? DNA extraction is an important molecular biology procedure. By definition, extraction is taking DNA out of any type of cell for the purpose of analysis. See Handouts for more information on the use of DNA extracted (extension only) 42
BREAK DOWN CELL WALLS Cells walls have to be destroyed to reach the DNA within cells. Extraction procedures to obtain pure DNA have to get rid of all the molecular and chemical components of the tissue from which the DNA is being extracted. First steps involve lysing or destroying the cell walls. This can be done with a variety of chemical agents that are caustic to cell membranes but do not harm the DNA. Cells can also be sonicated, homogenized, or ground up to destroy membranes. 44
REMOVING Removing Lipids Once the cell walls have been destroyed, a detergent is added to get rid of the fats and oils that make up the cell membranes. Detergents cause the fats and oils to dissolve into the solution. Remove Proteins Proteins and enzymes can be digested by adding a protease to the solution. Proteases break down proteins into small peptides and amino acids. 45
PRECIPITATING AND PURIFYING Precipitate DNA Adding cold alcohol to a solution will cause DNA to precipitate and aggregate. The DNA can be collected by centrifuging the sample and pouring off the liquid layer. The DNA should exist as a small pellet in the bottom of the centrifuge tube. Purify DNA The DNA can be washed by re-suspending it in a cold alcohol solution and re-centrifuging it several times to obtain a very pure DNA sample. Typical alcohols used to precipitate DNA include ethanol and Isopropanol. This process leaves a very pure sample for stringent DNA testing. 46
PART B: OBSERVING EXTRACTED DNA UNDER THE MICROSCOPE47
PART B: A CLOSER LOOK 8. Use a dropping pipette to carefully remove some of the threadlike substance from the top of your preparation. 9. Place one or two drops onto the middle of a microscope slide. 10. Add two drops of methylene blue. Wait 3 or 4 minutes to allow the methylene blue to be absorbed by the DNA. 11. Carefully place a cover slip on the slide. Gently press a folded piece of paper towelling over the top of the prepared slide to soak up any excess liquid. 12. Observe the DNA under low power, then high power. 48
DISCUSSIONANSWER IN GROUP RELATED WORKED. 1 Write a detailed description of the material floating at the top of the test tube after the alcohol was added. 2 Describe the DNA as it appears under the microscope under high power. 3 Prepare a diagram of your DNA specimen. 4 What was the reason for using methylene blue? 49
HOMEWORK:CONSTRUCT A FLOW CHART / DRAWING/ MIND MAPOF THE PROCESS YOU USED EXTRACTING THE DNAFROM KIWI’S Next to each step explain how the method you used was important. (Hint: What substances make up the membranes of cells and cell organelles? How do detergents work? What is the active ingredient in meat tenderiser? 50
TIPS:STEPS FOR FLOW CHART In order to release the DNA from the nuclei of the pea cells you first separated the cells from one another. Then the cell and nuclear membranes needed to be ruptured to release the cell contents and the contents of the nucleus. Once removed from the nuclear membrane the DNA had to be untangled into the visible, threadlike structures you ended up with. 51
HOMEWORK:EXTENSION GROUP QUESTIONS Where can DNA be found in the cell? Discuss the action of the soap (detergent) on the cell. What is the purpose of the soap in this activity? What was the purpose of the Sodium Chloride? Include a discussion of polarity and charged particles. Why was the cold ethanol added to the soap and salt mixture? Describe the appearance of your final product? Draw a diagram of DNA containing 5 sets of nucleotide bases labelling the hydrogen bonds between the bases. References and Resources: Adapted from Berry Full of DNA by Diane Sweeney for Biology: Exploring Life to be published by Prentice Hall. Websites: http://www.carlinvilleschools.net/linke/Biology/DNA.htm http://carnegieinstitution.org/first_light_case/horn/DNA/dnaind ex.html 52
EXTRA:CHROMOSOMES EXIST IN PAIRS Because our chromosomes exist in pairs (and consequently we have 2 alleles of each gene), we are a diploid species. This is why our somatic cells are represented as "2n". Our gametes (sperm and ova), on the other hand, are haploid, and are represented as "n". Other species may have different ploidy, for example: triploid (3n): seedless watermelons www.cyberus.ca tetaploid (4n): salmonidae fish pentaploid (5n): Kenai birch hexaploid (6n): some types of wheat, kiwi fruit octaploid (8n): acipenser (a genus of sturgeon fish, strawberies) decaploid (10n): some strawberries dodecaploid (12n): some types of amphibians, e.g. Xenopus 54 ruwenzoriensis
YOUR EXTRACTED DNA UNDER THE MICROSCOPE LOOKED LIKE .........! Collaboration-Brainstorming on what to look at under the - DNA extraction- DNA is too small for even a microscope to see and after the extraction it just appears like a blob. True or false? Could you any DNA strand? www.employees.csbsju.edu 55
HANDOUT ON DNA EXTRACTION: BACKGROUND EVERY DAY LIFE EXTRA READING MATERIAL (EXTENSION ) DISCOVERING DNA DNA ON THE INSIDE USE OF DNA EXTRACTION INN EVERYDAY LIFE WHY IS DNA TESTING GOOD? MOLECULAR BIOLOGY PCR-based diagnostics of genomic DNA 56
REFERENCES: http://www.squidoo.com/how-to-get-dna-from-a-kiwi-fruit Why Is DNA Testing Good? | eHow.com http://www.ehow.com/about_6684410_dna- testing-good_.html#ixzz1MkbcIJs5 Why Is DNA Extraction Important? | eHow.com http://www.ehow.com/list_5839095_dna- extraction-important_.html#ixzz1MkZDrqyY Why Is Sodium Used in DNA Extraction? | eHow.com http://www.ehow.com/about_6504902_sodium-used-dna- extraction_.html#ixzz1MkaGWg57 Why Is Cold Alcohol Used in DNA Tests? | eHow.com http://www.ehow.com/about_6399349_cold-alcohol-used-dna- tests_.html#ixzz1MkbEmEdu http://learn.genetics.utah.edu/content/labs/extraction/howto/ www.. kamibudaksains.blogspot.com http://www.chemwatch gold.com References and Resources: Adapted from Berry Full of DNA by Diane Sweeney for Biology: Exploring Life to be published by Prentice Hall. Websites: http://www.carlinvilleschools.net/linke/Biology/DNA.htm 57 http://carnegieinstitution.org/first_light_case/horn/DNA/dnaindex.html