With the advent of gene technology, it is important to understand not only the phenotype of the organism but also the genotype. Previously, you should have learnt the analysis of genetic traits and the various ways where they can be transmitted from parents to children (by phenotype analysis). Each chromosome is divided into different sections called genes. Genes are the basis of inheritance where traits like hair colour and blood type are controlled by the production of proteins by these genes. Genes contain coded instructions that the body uses to assemble hundreds of different types of proteins that make an individual unique!
These amazing trait controllers (genes) are made up of molecules called deoxyribonucleic acid (DNA). DNA is a double-helical polymer bound together by hydrogen bonds between complementary base pairing nucleotides (A to T, G to C). A particular gene is a set of coded instructions made up of a particular order of nucleotides. The variation of which allows the myriad of codes to exist in an organism for it to be unique. This is what controls the genotype of an organism and henceforth, the extraction and isolation of an organisms DNA is imperative, in order to allow further insight into the organism using different molecular-based methods.
In this experiment, you will be taking a closer look at this DNA molecule. You will be extracting your own DNA using buccal/cheek cells as the starting material.
15 ml centrifuge tube Paper cup
Drinking water Vortex Centrifuge 10% SDS
Bromelain protease (50mg/mL) Ice cold isopropanol Graduated pipettes
IMPORTANT NOTE: Ensure that you have not eaten in the past 1 hour before conducting this experiment (if you are the DNA donor). Ensure that gloves are worn at all times in the experiment.
1. Swish you mouth with about 100 mL drinking water, for about 20 seconds, to remove any food particles. Discard this wash into the sink.
2. Using a permanent marker pen, label your group name onto the paper cup and 15 mL centrifuge tube containing 10mL saline.
3. Pour all the 10mL saline solution into your mouth and vigorously swish for 60s. Do not discard the centrifuge tube.
4. Expel the saline mouthwash into the labelled paper cup.
5. Carefully, pour the saline mouthwash from the paper cup, back into the 15 mL centrifuge tube from step 2. Tightly cap the tube.
6. Pass the capped tubes to the laboratory technician in order to be centrifuged (4500 rpm, 5 min).
7. Upon centrifuging, you should be able to see your buccal cell pellet (the whitish lower solid layer at the bottom of the tube). Gently, pour away the supernatant (the liquid upper layer).
8. Place the tube on ice.
9. Add 2 mL saline into the tube and vortex for 5-10 seconds.
10. Add 1 mL 10% (w/v) sodium dodecylsulphate (SDS) solution (active component in detergents).
11. Gently tap the tubes several times (~8 times) to gently mix the contents. You may invert the tube twice if needed.
12. On ice, add 2 – 3 drops of the lab supplied bromelain protease enzyme into the tube.
13. Gently tap the tubes several times (~8 times) to gently mix the contents. You may invert the tube twice if needed.
14. Cap the tube and place it is a 50oC for 10 minutes.
15. With a clean pipette, gently pipette in 10 mL ice cold isopropanol (95% v/v) slowly into the tube. Tip: Place the filled pipette with its tip against the inside wall of the test tube. Slowly allow the isopropanol to dribble down the inside of the tube.
16. Cap and place the tube in a test tube rack at room temperature for 10 minutes. DO NOT mix, shake, or bump the test tube during this period.
17. The isopropanol is lighter than the contents of the tube. When added according to the directions, the isopropanol will form a clear layer ABOVE the suspension.
18. Observe the test tube for 5 minutes. The DNA will gradually separate from the suspension and rise into the isopropanol layer. Describe the appearance of the DNA.
19. Take a photo as proof of your observation.
20. To remove the accumulated DNA from the tube, follow the directions for DNA spooling as below:-
a. Gently insert the glass rod through the isopropanol layer into the clumped/accumulated DNA.
b. Carefully, twirl the rod between your fingers, winding the DNA strands onto the rod.
c. Slowly remove the rod. Describe the appearance of the spooled DNA.
d. Take a photo as proof of your observation.
1. Which one of the following do you think will contain DNA? Explain your reasoning.
Bananas; concrete; fossils; meat; metal; spinach; strawberries.
2. What effect would the SDS have on the cell membranes and cold ethanol on DNA?
3. What type of enzyme would be needed to separate the DNA into smaller pieces?
4. Is the DNA extracted pure enough for further applications (i.e. PCR)?
5. If you were to repeat the experiment with an equal number of red blood cells, the amount of DNA collected would either: increase / decrease / stay the same (choose one). Explain your answer.
Bres, M., Weisshaar, A., 2008. Thinking about Biology: An Introductory Laboratory Manual. 3rd Ed. Pearson Prentice Hall: New Jersey, USA. Pg. 333 – 338.
Teaching AS Biology Practical Skills. University of Cambridge: International Examination. Pg. 74 – 78.
43 Practical 10
Digestion of Lambda (λ) DNA with a Restriction Enzyme (EcoR I endonuclease)
Restriction enzymes (nucleases) are enzymes that cleave the phosphodiester bonds on the sides of deoxyribonucleic acid (DNA). These nucleases recognize specific DNA sequences in the double-stranded DNA, which is usually a four to six base pair sequence of nucleotides, and digests the DNA at these sites, resulting in the DNA becoming fragmented into various lengths. Some restriction enzymes cut cleanly through the DNA double helix while some produce uneven or sticky ends. By using the same restriction enzyme to cut DNA from different organisms, the sticky ends produced will be complementary and the DNA from the two different sources can be recombined. In humans, no two individuals have the exact same restriction enzyme pattern in the DNA except for identical twins.
Restriction enzymes are named based on a system of nomenclature where the first letters represents the genus name of the organism whereas the next two letters come from the species name. If there is a fourth letter, it stands for the strain of the organism. Finally, if there are Roman numerals, it represents whether that particular enzyme was the first or second etc. isolated in that category.
FIGURE 10.1 Cartoon of how EcoR I recognises the restriction site and cleaves the DNA.
The second technique used in this practical is the separation and analysis of DNA fragments. Agarose gels are commonly used for this where the gels that have been prepared with a suitable nucleic acid stain in it, have wells for the samples of DNA to go into. The agarose gel is covered in a suitable buffer so that the DNA is in a neutral pH solution. That way, the DNA moves one direction because of its charge. Since the phosphate groups on the skeleton of DNA are negatively charged, the whole molecule takes on the negative charge.
Hence, when the DNA is placed inside the gel and the electricity is turned on, the poles are drawing the DNA toward the positive side, where it will then move through the gel and separate according to the size of the fragments. This technique is called electrophoresis. Results are obtained with the help of UV light that is refracted by the nucleic acid stain that sticks onto the DNA fragments.
In this experiment, you will be using the EcoR I restriction endonuclease to digest a known DNA called phage lambda (λ) and analysing your sample using agarose gel electrophoresis.
Pre-laboratory work Computer/laptop
LambdaDNA.docx (Word document file)
Laboratory work Micropipette Sterile pipette tips
Microcentrifuge tube (1.5 mL capacity) EcoR I (20 U/µL) and buffer
Distilled deionised sterilised water Agarose gel (prestained with EtBr) 1x TAE buffer
Agarose gel electrophoresis set UV Transilluminator