Bio 135 Summer 2009 Name_________________________
Introduction
Every human cell has a "second" genome, found in the cell's energy-generating organelle, the mitochondrion. In fact, each mitochondrion has several copies of its own genome, and there are several hundred to several thousand mitochondria per cell. This means that the mitochondrial
(mt) genome is highly amplified. While each cell contains only two copies of a given nuclear gene
(one on each of the paired chromosomes), there are thousands of copies of a given mt gene per cell. Because of this high copy number, it is possible to obtain a mt DNA type from the equivalent of a single cell's worth of mt DNA. Thus, mt DNA is the genetic system of choice in cases where tissue samples are very old, very small, or badly degraded by heat and humidity.
Under good circumstances - working from fresh cell samples - mt DNA is the easiest human DNA to amplify by PCR. This experiment examines a 568-nucleotide sequence from the noncoding region of mt genome called HVR1, or hypervariable region 1. The complete HVR1 locus is located within the control region (see picture below), and extends from position 16,001 to position
16,568. (The entire genome is about 16,600 base pairs long.) HVR1 is the primary locus used in many genealogical tests. We’re actually amplifying from position 15,926 to position 639, a region of 1283 base pairs (15,926 is on one side of the origin of replication (position 1) and 639 is on the other side). Only HVR1 will be sequenced.
Comparison of control region sequences reveals that most people have a unique pattern of single nucleotide polymorphisms (SNPs). These sequence differences, in turn, are the basis for farranging investigations on human DNA diversity and the evolution of hominids.
15,926
639
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Bio 135 Summer 2009 Name_________________________
Part I: DNA Isolation by Saline Mouthwash
Reagents
Saline solution (0.9% NaCl),10 ml
10% Chelex®, 100 µl
Equipment & Supplies
15 ml disposable test tube
Paper cup
1.5 ml test tubes, polypropylene
1 ml transfer pipet or
1001,000 µl micropipet and tip
Cup of ice (optional)
Shared Items microcentrifuge heat block
Procedure
1. Pour the saline solution from a purple capped tube labeled (0.9% NaCl) into mouth and vigorously swish for 30 seconds.
2. Expel saline solution into a paper cup.
3. Swirl to mix cells in the cup and transfer 1 mL of the liquid to a 1.5 mL tube, using a sterile transfer pipet. You can also use the transfer pipet to be sure your cells are in a well mixed suspension by pipetting up and down a number of times before transferring the liquid. There are markings on the side of the 1.5 mL tube that you can use for measuring 1 mL.
4. Place your sample tube, together with other student samples, in a balanced configuration in a microcentrifuge, and spin for 1 minute at the highest speed. Each tube must be opposite another tube with similar volume of liquid in it. All tubes should have the same volume, 1 mL, but please double check.
5. Carefully p ipet off supernatant into paper cup and dispose by putting into the “disinfected” waste container. Be careful not to disturb the cell pellet at the bottom of the test tube. A small amount of saline will remain in the tube.
6. Resuspend cells in remaining sali ne by pipetting in and out. (If needed, 30 µl of saline solution may
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Bio 135 Summer 2009 Name_________________________ be added to facilitate resuspension.)
7. Using a micropipet, withdraw 30 µl of cell suspension, and add to 1.5 mL tube containing 100 µl of
Chelex. These are on a labeled tube holder, and each tube is marked with a blue dot. Vortex for about 3- 5 seconds to mix. (Chelex helps remove metal ions that can inhibit PCR.)
8. Boil your cell sample for 10 minutes using the heat block. Then, cool tube briefly on ice for 3-5 minutes.
9. Using a Vortex, mix the tube at high speed for about 5 seconds. Place in a balanced configuration in a microcentrifuge, and spin for 1 minute at the highest speed. (Again, together with other student samples.)
10. Transfer 30 µl of supernatant (containing the DNA) to clean 1.5 ml tube. Label the top of the tube with a number assigned to you by your instructor. Avoid cell debris and Chelex material. This sample will be used for setting up your PCR reaction. Keep this sample even after you have set up your PCR reaction. Your instructor will put all the DNA samples together on a rack.
11. Store your sample on ice or put into the rack provided by your instructor until ready to begin Part II.
If the tubes will be stored, be sure to put your tube into the position number on the rack that matches your assigned number.
Part II: DNA quantification gel
Reagents
2% agarose gel
1X electrophoresis buffer
DNA quantification markers
1 µg/ml ethidium bromide or 0.05% methylene blue staining solution
Equipment & Supplies
15 ml test tube, polypropylene
120 µl micropipet and tips
Staining tray or weigh boat
Shared Items
Electrophoresis chamber
Electrophoresis power supply
Procedure
1. Use a micropipet with a fresh tip to add 10µl of your extracted DNA to a 1.5 mL tube.
Save the remaining DNA carefully in a tube labeled with your name.
2. Using a fresh tip, add 2 ul of loading dye to the 10 ul of your product. Gently pipet up and down to mix. If you introduce air bubble, spin your sample in the microcentrifuge for about 10 seconds. Remember to balance your tube and share the microcentrifuge with classmates that may also need it.
3. Pipet your entire sample into your assigned well of a 2% agarose gel. Expel any air from the tip before loading, and be careful not to push the tip of the pipet through the bottom of the sample well.
4. Load 12 µl of each of the DNA quantification markers into the gel, each in a different lane.
5. Electrophorese at 130 volts for 20-30 minutes.
6. Gel will be stained by your instructor. The gel is placed in 1 µg/ml ethidium bromide for
10 minutes, followed by 20-30 minutes destaining with water. Ethidium bromide is a potential carcinogen, so you will not be handling the gel after staining.
7. The gels will be viewed using an ultraviolet light box called a transilluminator. Ultraviolet light can damage your retina, so you must be sure only to observe your gel when the protective Plexiglas shield is in place.
8. Compare the intensity of your DNA to that of the standard.
9. If your DNA is approximately as bright or less bright, do nothing.
10. If your DNA is brighter than the standard, estimate how many times brighter it is and multiply that factor by 10 to get the concentration of your DNA.
11. In a NEW TUBE, m ake 25 μl of a 10ng/μl solution of your DNA.
12. Write your name on the tube, and write 10 ng/μl.
Part III: DNA Amplification by PCR
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Bio 135 Summer 2009 Name_________________________
Reagents
Master mix, 23.5 µl
Human DNA, 1.5 µl
Equipment & Supplies
120 µl micropipet and tips
0.2 mL PCR tubes
Shared Items
Thermal Cycler
Procedure
1. Use a micropipet with a fresh tip to add 23.5 µl of the Master mix to a PCR tube.
2. Use fresh tip to add 1.5 µl of human DNA (your DNA from Part I) to reaction tube, and tap to mix.
Do NOT throw your DNA sample away. Please return it to your instructor.
3. Label the cap of your tube with your name.
4. Store all samples on ice until ready to amplify according to the following profile. Your instructor will program thermal cycler for 30 cycles according to the following cycle profiles.
1. Initial denature: 3 min at 94°C
2. Denaturing time and temperature 30 sec - 94°C
3. Annealing time and temperature 60 sec - 56°C
4. Extending time and temperature 2 minutes - 72°C
5. Final extension 6 min at 72°C
Review questions, day 1
1. What do you think was the purpose of boiling the cheek cells?
2. What was in the pellet the first time we centrifuged the sample, and how did this differ from what was in the next pellet we formed?
3. Why did we pour off the supernatant the first time but save it the second time?
4. PCR is an exponential process such that the number of DNA molecules doubles with e ach cycle. If your original 2.5 μl contained 10,000 target DNA sequences, how many copies would there be after 30 PCR cycles?
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Bio 135 Summer 2009 Name_________________________
Part IV: PCR product (DNA) visualization by Gel Electrophoresis
Reagents
2% agarose gel
1X electrophoresis buffer
DNA size marker
1 µg/ml ethidium bromide or 0.05% methylene blue staining solution
Equipment & Supplies
15 ml test tube, polypropylene
120 µl micropipet and tips
Staining tray or weigh boat
Shared Items
Electrophoresis chamber
Electrophoresis power supply
Procedure
1. Use a micropipet with a fresh tip to add 12µl of your PCR product to a 1.5 mL tube. Give the remaining DNA to your instructor so that it can be sent off for sequencing.
2. Using a fresh tip, add 2 ul of loading dye to the 12 ul of your product. Gently pipet up and down to mix. If you introduce air bubble, spin your sample in the microcentrifuge for about 10 seconds. Remember to balance your tube and share the microcentrifuge with classmates that may also need it.
3. Pipet your sample (DNA + dye) into your assigned well of a 2% agarose gel. Expel any air from the tip before loading, and be careful not to push the tip of the pipet through the bottom of the sample well.
4. Load 5 µl of the DNA size markers into one lane of the gel.
5. Electrophorese at 130 volts for 20-30 minutes.
6. Gel will be stained by your instructor. The gel is placed in 1 µg/ml ethidium bromide for
10 minutes, followed by 20-30 minutes destaining with water. Ethidium bromide is a potential carcinogen, so you will not be handling the gel after staining.
7. The gels will be viewed using an ultraviolet light box called a transilluminator. Ultraviolet light can damage your retina, so you must be sure only to observe your gel when the protective Plexiglas shield is in place.
8. If the amplification of your DNA was successful, you should see a band of DNA at about
400 bp.
Part V: DNA sequence ‘clean-up’
Reagents Equipment & Supplies
ExoSAP-IT enzyme (1/32 strength) 120 μl micropipette and tips
Shared Items
Thermal Cycler
Unused primers and dNTPs from the PCR master mix can interfere with the sequencing reaction.
ExoSAP-IT contains two enzymes, an exonuclease and a phosphatase, that selectively digest short, single-stranded DNA and dNTPs.
Procedure
1. Pipette 2.0 μL of ExoSAP-IT solution into your PCR product (note: you should have about
10μl product left).
2. Place your tube in the thermocycler. Your instructor will set the thermocycler to perform a fifteen minute incubation at 37°C followed by a fifteen minute denaturation at 80°C.
3. Your sample will be taken to the Genomic Analysis and Technology Core (GATC) Facility at the University of Arizona for sequencing.
4. Electronic files of your sequence will be downloaded from their facility for you to analyze in a later lab.
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Bio 135 Summer 2009 Name_________________________
Review questions, day 2
1. Why did the quantification gel look like a smear, whereas the gel with the PCR product showed a discrete band?
2. Why were all students’ PCR product bands the same size if we expect to see variation among students’ sequences?
3. To which end of the gel did the DNA move during electrophoresis (the positive or the negative)? Why did it move in this direction?
4. The four main components of a PCR reaction are dNTPs and DNA polymerase (in master mix), primers, and DNA template. What role does each of these play in the PCR reaction?
5. Why did we choose to sequence a ‘hypervariable’ region of DNA?
6. Why is it often easier to amplify a mitochondrial region than it would be to amplify a nuclear region?
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