The Polymerase Chain Reaction The polymerase chain reaction, PCR, can produce many copies of a specific target segment of DNA A three-step cycle brings about a chain reaction that produces an exponentially growing population of identical DNA molecules The key to PCR is an unusual, heat-stable DNA polymerase called Taq polymerase. 1

Technique Target sequence Genomic DNA Denaturation Annealing Cycle 1 Figure 13.25 Technique 5 3 Target sequence Genomic DNA 3 5 1 Denaturation 5 3 3 5 2 Annealing Cycle 1 yields 2 molecules Primers 3 Extension New nucleotides Figure 13.25 Research method: the polymerase chain reaction (PCR) Cycle 2 yields 4 molecules Cycle 3 yields 8 molecules; 2 molecules (in white boxes) match target sequence 2

Target sequence (TAS2R38) Figure 13.25a 5 3 Target sequence (TAS2R38) Genomic DNA 3 5 Figure 13.25a Research method: the polymerase chain reaction (PCR) (part 1) 3

Cycle 1 yields 2 molecules Figure 13.25b-1 1 Denaturation 5 3 3 5 Cycle 1 yields 2 molecules Figure 13.25b-1 Research method: the polymerase chain reaction (PCR) (part 2, step 1) 4

Cycle 1 yields 2 molecules Figure 13.25b-2 1 Denaturation 5 3 3 5 2 Annealing Cycle 1 yields 2 molecules Primers Figure 13.25b-2 Research method: the polymerase chain reaction (PCR) (part 2, step 2) 5

Cycle 1 yields 2 molecules Figure 13.25b-3 1 Denaturation 5 3 3 5 2 Annealing Cycle 1 yields 2 molecules Primers 3 Extension Figure 13.25b-3 Research method: the polymerase chain reaction (PCR) (part 2, step 3) New nucleotides 6

After 30 more cycles, over 1 billion (109) molecules Figure 13.25c Cycle 2 yields 4 molecules Cycle 3 yields 8 molecules; 2 molecules (in white boxes) match target sequence Figure 13.25c Research method: the polymerase chain reaction (PCR) (part 3) Results After 30 more cycles, over 1 billion (109) molecules match the target sequence. 7

So what are we doing with this technique??? We will use PCR + restriction digests to determine your genotype for PTC tasting (TAS2R38) Calendar 05.18.16 Isolate DNA from your cheek cells 05.20.16 Set up PCR using your DNA 05.24.16 Set up restriction digests of your PCR product 05.26.16 Run gels and analyze What you’ll need to turn in on 06.01.16 Completed lab worksheet (5 pt) Picture of your gel (5 pt) Hand-drawn standard curve of your DNA marker (10 pt) This will go into the main labs scoring (20 pt total)

Day 1: Isolating your DNA Label two microtubes and a cup of saline solution with your identity number given to you by your teacher.   Pour saline solution in your mouth and vigorously rinse your cheeks for 1 minute. Expel the saline solution back into the cup. Carefully, over the garbage or a sink, pour your spit and saline solution into your microtube. Close the microtube tightly and put it into the high powered microcentrifuge and spin it for 5 minutes at 10,000 rpm. Look for a cell pellet at the bottom of the tube and carefully pour the liquid above the pellet (called the supernatant) into a waste container and keep the cell pellet in the tube. Pipette 500 λ of 10% chelex into your microtube and resuspend the cell pellet by gently mixing the solution up and down in the pipette. Once resuspended fully, close the tube lid tightly and poke a hole in the top of the lid using a tack. Place the tube in the 99o hot block for 10 minutes. Resuspend the solution one more time and then spin in the high-speed centrifuge for 5 minutes so the Chelex beads fall to the bottom of the tube. Without disturbing the beads at the bottom, pipette about 50 λ of the solution into second a tube labeled with your name or identity number and the date. This is your extracted DNA. Your teacher will freeze your tube for you to use in your next experiment.

Day 2: Performing PCR Keep PCR tubes on ice. Label a PCR tube with your identity number and then put it back on ice. Obtain your DNA tube from your teacher and carefully thaw it on the countertop. Pipette 5 λ of your DNA into your PCR tube. Come up to front of room with your 20 λ pipette set at 20 λ and with a fresh tip. Pipette 20 λ of Master Mix from the teacher’s stock tube into your PCR tube. Return to your bench and quickly cap and vortex your PCR tube and then spin it down in the low-speed minicentrifuge. Bring your PCR tube to the PCR machine and put your tube in the ice container (if your teacher is waiting for other student tubes) or put your tube directly in the PCR machine. Your teacher will run the PCR machine once all tubes are in the PCR machine.

Day 3: Restriction Digest of PCR Product Get your “D” microtube from your teacher and label it with your name. This tube has 2 λ of HaeIII enzyme for the restriction digest. Label another microtube with your name and “U” for your undigested PCR product. Obtain your PCR product. Pipette 10 λ of PCR product into “U” tube. Pipette 12 λ of your PCR product into your “D” tube. Add 4 λ of 2.5 x Restriction Buffer to both the “U” and “D” tubes. Cap your tubes, mix them, and then spin them quickly in the microcentrifuge Put your “D” tube in the 37o C waterbath for 1 hour. Bring your “U” tube to your teacher who will pull your “D” tube out of the waterbath and put both tubes into the refrigerator.

Obtain your “U” and “D” tubes. Day 4: Analyzing your Restriction Digests using Agarose Gel Electrophoresis Obtain your “U” and “D” tubes. Set up your electrophoresis box with 1X TAE buffer and a 2% agarose gel. Be sure to put the wells of the gel on the correct side of chamber (near the power supply). Pipette 2 λ of loading dye into your “U” tube and 2 λ of dye into your “D” tube. Load 10 λ of PCR Marker into the first well of your gel. Load 12 λ of “U” tube into the next lane. Load 12 λ of “D” tube into the next lane. Record which lanes your samples are in. When all lanes are loaded, close the electrophoresis box and run your gel at 125V for 35-45 minutes. Carefully remove your gel from the gel box when finished running. Take a picture of your gel using the LED camera. Analyze your results. Create a standard curve from the DNA ladder, then use it to identify the weights of the different bands in your sample. Note your genotype based on the band pattern.