Name: ______________________________________________________ Date: ___________ Section:______
Chromosome 16- PV92 Alu Insert Amplification: Extraction of DNA and PCR Reactions
Prelab Questions:
1 What is the purpose of PCR?
2 List all chemicals/ reagents that are needed in a PCR reaction tube.
3. What are the temperatures necessary for a successful PCR reaction? What occurs at each temperature?
4. Draw a labeled diagram that includes: DNA to be amplified (or template DNA), primers, Taq polymerase, and free nucleotides, and newly synthesized strands.
5. What is the role of gel electrophoresis used with PCR?
6. Why would the presence of InstaGene Matrix in your PCR reaction inhibit PCR?
7. Imagine you successfully extracted DNA, successfully ran PCR, but your gel show no DNA. What could have happened?
8. Imagine you successfully extracted DNA but forgot to add primers to the PCR reaction. Explain the outcome of the gel electrophoresis.
Background
The Polymerase Chain Reaction (PCR) is a technique used to produce many copies of a specific DNA sequence. In order to perform PCR, you must know something about the sequence you want to amplify (called the target or template sequence) to make primers. A primer is a short (18-40 bp) piece of single-stranded DNA manufactured in a laboratory. You need 2 primers for each PCR reaction. One primer (primer 1) will hybridize to a sequence at the 5’ end of the target sequence, at the 5’ end of one strand of the genomic DNA, and the other primer (primer 2) will hybridize to the 3’ end of the target sequence, at the 5’ end of the complementary strand of DNA.
In the first step of PCR the genomic DNA is denatured by heating . Then the temperature is lowered to allow the primers to “anneal or hybridize” to their complementary sequences. The temperature for the hybridization step of PCR depends on the sequence and length of the primers being used. The hybridization step may be carried out between 42oC to 62oC, depending on primers. The last step of PCR is extension step. In this step DNA is synthesized from the primers, using the target DNA as a template. DNA synthesis is performed using a special DNA polymerase called Taq, that is active at very high temperatures. Taq polymerase is isolated from the bacterium Thermophilus aquaticus that lives in hot springs. The temperature optimum for Taq polymerase is 72oC, but it is also stable at higher temperatures (95oC). DNA synthesis by Taq polymerase, as for all DNA polymerases, requires a primer, a DNA template, the nucleotides dATP, dGTP, dCTP, and dTTP (usually called dNTPs), and Mg++. The DNA synthesis step of a PCR reaction is called extension, since the length of the primer is being extended by addition of nucleotides during DNA synthesis. Since DNA is being synthesized from both strands of DNA, two different primers are needed, one for each strand.
PCR reactions are performed in a machine called a thermocycler, that rapidly heats and cools samples to specific temperatures by cycling. A cycle consists of a denaturation step (94oC), a hybridization step (42-62oC) and an extension step (68-74oC). At the end of each cycle the amount of target DNA present is doubled, so after 1 cycle there are 2 copies of the target sequence for every molecule of target DNA present at the start, after 2 cycles there will be 4 copies of DNA and so on. The number of copies of DNA produced at the end of a PCR reaction is determined by the equation: # copies of DNA = 2n, where n = # of cycles performed. In our experiment we will be performing 40 cycles of PCR, so we will produce 240 copies = 1.1 X 1012 copies. At the end of our PCR reaction we will have so many copies of our target sequence that it will be visible as a band of DNA on an agarose gel.
In this lab you will isolate your own genomic DNA from cheek cells and determine whether you are homozygous or heterozygous for a particular sequence. The human genome contains large amounts of DNA that does not code for proteins. In humans 10-15% of the genomic DNA is tandemly repeated DNA. In tandemly repeated DNA a short sequence of bases is repeated numerous times, one sequence (or repeat unit) located immediately behind the first. Telomeres and centromeres contain tandemly repeated sequences. Another important set of tandemly repeated sequences is the VNTRs. VNTR stands for Variable Number of Tandem Repeats. All individuals have the same VNTR sequence. However, the number of times that sequence is found on a particular chromosome may vary dramatically, from a few copies to many copies. VNTRs are used in one type of DNA fingerprinting, which we will discuss later in lecture.
The type of repeated DNA sequence we will be studying today is called Interspersed Repeated DNA belonging to the Alu family. In this type of repeated DNA the repeated sequence occurs only once at any one location, but there are numerous copies of the sequence on different chromosomes or in other regions of the same chromosome. Interspersed repeated DNA makes up 25-40% of the human genome. The Alu sequence is 300 bp long. On chromosome 16 there is a region called PV92 where the Alu sequence may be inserted into the intron of a gene. A chromosome that lacks the Alu sequence will produce a 641 bp DNA fragment after PCR, while a chromosome that has a copy of the Alu sequence inserted into the intron of the gene will produce a 941 bp fragment of DNA after PCR. Individuals who are homozygous for the absence of the Alu sequence (-/-) will produce only the 641 bp fragment after PCR amplification. Individuals who are heterozygous for the presence of the Alu sequence (+/-) will have one chromosome that has the Alu sequence, producing a 941 bp fragment after PCR, as well as one chromosome that lacks the Alu sequence, producing a 641 bp DNA fragment. Individuals who are homozygous for the presence of the Alu sequence (+/+) will produce only the 941 bp DNA fragment, after amplification by PCR. These fragments will be visualized as bands on a gel.
Procedure (reproduced with modification from the Biorad PV92 PCR manual)
Extract Genomic DNA from Cheek Cells (do individually)
*Be sure to mix the tube containing the InstaGene Matrix thoroughly before pipeting, or the beads will settle out of solution at the bottom of the tube. *
1 Label one microfuge tube and one screwcap tubes with your initials on lid and side.
2 Add 200ul InstaGene Matrix to the screwcap tube.
The InstaGene Matrix contains negatively charged microbeads. These beads will bind to positively charged ions in the solution. Many enzymes that degrade DNA (DNAses) require positively charged ions such as Mg++ as cofactors. Removing or stripping the positively charged ions from solution will inhibit the activity of DNAses, and help maintain the integrity of the genomic DNA.
3. Obtain a cup containing 10 ml solution from your instructor. Pour into your mouth and swish vigorously for 1 minute. Expel the saline back into the cup.
4. Transfer 1 ml = 1000 ul of your saline rinse into clean, labeled microfuge tube (not the screw cap tube). Spin your tube at 14,000 for 2 minutes. Make sure the tubes are balanced before turning on the microcentrifuge.
You should see a match-head sized pellet of cells at the bottom of the tube.
If you don’t see a visible cell pellet, discard the supernatant (pour off liquid)
and add another 1 ml of saline rinse to the microfuge tube, and spin again.
4 After pelleting the cells, carefully remove the supernatant with a pipetman set at 1000l, and discard the supernatant. There will probably be a small amount (10-20) of saline on your cell pellet.
Or pour off the liquid and try not to disturb the pellet.
5 With a P-200 pipetman set to 20ul, resuspend (or vortex) the cells in the remaining saline solution by pipeting up and down. If there is no saline solution in your tube, add 20 of buffer from the tube containing InstaGene Matrix and resuspend your cells in the buffer.
No chunks in liquid. Should appear yellowish and cloudy.
6 Transfer the entire contents of the microfuge tube (cells and saline or buffer) to the screwcap tube containing InstaGene matrix. Screw the cap tightly on the tube. Vortex to thoroughly mix the contents.
Take your resuspended cells from microtube to screwcap tube
7 Place the tubes in the foam holder and incubate in the 56oC waterbath for 10 minutes. After 5 minutes, vortex the tube and then place back in the waterbath for the remaining 5 minutes.
Waterbath 5 minutes vortex waterbath 5 minutes
8 Remove the tubes, vortex, and place the tubes in a 100oC heat block for 5 minutes.
HOT!!! Do not touch Heat block!!!
9 Remove the tubes from the heatblock and vortex or vigorously mix the tubes. Spin the tubes at 14,000 rcf for 5 minutes. Store your screwcap tube on ice until you are ready to set up the PCR reaction.
Your DNA is in the supernatant (liquid). KEEP!!!!
The pellet contains InstaGene Matrix, cell debris and denatured proteins.
Set Up PCR Reactions of Control DNA Teacher!
1 There are 3 PCR tubes in your ice bucket labeled: +/+, +/-, -/-. The +/+ tube contains the homozygous dominant control DNA, the +/- tube contains the heterozygous control DNA, and the -/- tube contains the homozygous recessive DNA. To each tube transfer 14 l of the yellow Master Mix. Mix thoroughly by pipeting up and down. Cap the tubes tightly and keep them on ice until your instructor is ready to load the thermocycler.
Part 5: PCR of Genomic and Control DNA (all reactions from a lab section will be run at the same time)
*The lid on the thermocycler has been heated to 105oC to prevent evaporation. Be careful not to touch the lid while loading your samples.
1 Keep your samples on ice until they are loaded into the thermocycler. Place the tubes in the row corresponding to your group number.
2 The following program will be used:
step 1 94C X 2 min Initial Denaturation step
step 2 94oC X 1 min Denaturation
step 3 60oC X 1 min Hybridization
step 4 72oC X 2 min Extension
Repeat steps 2-4 a total of 40 times
step 5 72oC X 10 min Final Extension
step 6 Hold at 4oC
This program takes approximately 3 hours to run.
3 Next week your instructor will return your samples to you and you will analyze the results of your PCR by horizontal gel electrophoresis.
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