PV92 PCR

In this lab, you’ll use the polymerase chain reaction (PCR) to copy a particular segment of your DNA, then run a gel to compare your amplified DNA to that of other students. The target sequence comes in two versions, or alleles: one containing an Alu transposon and one without. Thus, this lab is both an introduction to PCR and an introduction to transposons.

Alu is a transposon

Transposons, or transposable elements, are regions of DNA that can be copied and moved from one region of the genome to another. Because transposons tend to get duplicated, they can occur many times in an organism's genome, and because they tend to get inserted at new locations, these repeated DNA sequences can be scattered throughout the genome. Transposons exist in all kinds of organisms, but the Alu transposon is unique to primates. The Alu transposon is a short sequence (300 nucleotides, compared to the 3 billion nucleotides of the haploid human genome) that has been duplicated and inserted in the genome over a million times, making up over 10% of the human genome.

All humans have the Alu transposon, and most of those Alu elements are in the exact same locations in every human genome. In other words, most of these Alu insertions are conserved. This indicates that these Alu insertions existed at the very beginning of the evolution of humans. On the other hand, there are a few Alu elements that are polymorphic, meaning that they are present in some individuals but not others. This suggests that these copies of Alu have been inserted in their new locations fairly recently.

PV92 PCR strategy

PV92 alleles

The DNA region, or locus, that you’ll copy is called PV92; it's part of an intron on chromosome 16. Everybody has the PV92 locus, but there are two common alleles: with or without an Alu transposon inserted.

PV92 Sequence diagram, with and without Alu transposon

There are only two common PV92 versions, or alleles, in the human population – the short one, with no Alu repeat, and the long one, with the Alu repeat. As shown in the diagram above, you'll use PCR primers that target a conserved region of the PV92 locus. The result is that for any copy of human chromosome 16, you should be able to amplify either a short (no Alu) or a long (+Alu) PV92 PCR product. The sizes of the expected PCR products for the two alleles are:

  • –Alu: 641 bp
  • +Alu: 941 bp

PV92 genotypes

An allele is a particular version of a nucleotide sequence; a PV92 genotype is the set of alleles found in one individual. Since everybody has two copies of chromosome 16 (one from mom and one from dad), everybody has two copies of the PV92 locus. Therefore, you could possibly get both a short and a long PV92 PCR product from one person. Today you can collect DNA from each person in your lab group and test it to find out whether that person has the short PV92, the long one, or both. The possible genotypes for this experiment are:

  • Homozygous +/+
  • Homozygous –/–
  • Heterozygous +/–

Gel results

Since the sizes of the PCR products for the two different alleles are different, you should be able to use electrophoresis to identify the sizes of the products from each person's DNA template, and therefore identify each person's genotype at this locus. Here's an example of a typical Bio 6B gel:

PV92 PCR gel results

  • Lane 1: Negative control. No band, as expected, indicating that the PCR master mix was not contaminated with template DNA. That's particularly important in this type of experiment, because there's plenty of human DNA floating around in the lab.
  • Lanes 2–4: Positive controls. These all produced products, as expected; the genotypes are labeled on the gel.
  • Lane 5: Molecular weight marker. We buy this marker, and the manufacturer tells us the sizes of the bands. You might have a different marker when you do your experiment.
  • Lanes 6–8: Student DNA samples. You should be able to determine each student's genotype by comparison to the positive controls and the marker. Lane 6 shows a primer dimer, a small product that is produced when the polymerase copies the primers, without any template. Primer dimers are a common PCR artifact, and we can usually ignore them. However, sometimes the primer dimers can look like real bands, so it's important to have a marker to help you distinguish the correct products by size.

Insertion polymorphism

The PV92 lab is based on a polymorphism, or a DNA region that varies from one individual to another. In this case, the polymorphism is due to an insertion, which means that an extra piece of DNA (the ALU transposon) is inserted into the genomes of some people, but not others. Since the polymorphism allows us to get PCR products of different sizes for different people, it's easy to see on the gel. You can contrast this with the substitution polymorphism that we will invesigate in the PTC PCR lab. A substitution simply means that one or more nucleotides in a sequence has been mutated, without changing the length of the sequence. In that lab, we won't be able to distinguish the alleles simply by looking at the length of the PCR products.

PCR background

How PCR works

See the Polymerase Chain Reaction page for some background on PCR.  I will assume that you've already had an introduction to PCR.

Purifying DNA for PCR

You’ll need to obtain some of your own DNA for this lab. Luckily, PCR is very efficient and you don’t need much DNA. You can get what you need by swishing a few detached cells from the lining of your cheek. DNA doesn’t need to be very pure for PCR, but you need to get rid of two things: enzymes that might damage DNA, and cations like Mg2+ that would interfere with the PCR enzyme. You can achieve these two goals by simply boiling your cells with a product called Chelex. Boiling kills the enzymes and lyses the cells. Chelex is a solid resin that is negatively charged, so it binds cations. Chelex isn’t water soluble, so it will be a powder in the bottom of your micro tube, pulling away harmful ions and leaving you with ready-to-use DNA. You’ll be using a version of Chelex called Insta-Gene Matrix, made by the Bio-Rad Corporation.

Procedures

Preparing DNA sample

Materials

  • Pipetmen and tips
  • Beaker for waste tips
  • Styrofoam cup with ice
  • For each person in your lab group, obtain the following:
  • 1 small Dixie® cup
  • Sterile saline solution
  • 1 2 ml micro tube. (1.5 ml will work, if we don't have 2 ml)
  • 1 small (500 µl) microfuge tube for each sample
  • 1 500 µl microcentrifuge tube containing Instagene® Chelex resin (Note: the chelex is a solid, mixed in with the buffer in the tube. Look at your chelex tubes and make sure each one contains a substantial pellet of chelex. It's difficult to pipet the suspended chelex, so sometimes some of the tubes come up short.)

Procedure for DNA sample preparation

Note: this procedure is written as if you’re going to have only one tube. However, you should have a tube for each person; you can incubate them all together.

  1. Put approximately 10 ml of sterile saline into the cup. Pour the saline into your mouth and swish it vigorously in your cheeks for 30–60 seconds. Vigorous swishing frees loose epithelial cells from the inside of your mouth. Expel the saline back into the cup.
  2. Fill the 2 ml  tube with 2 ml of the saline rinse suspension. Use the same size tube for all your samples, so you can balance the centrifuge. If you have an odd number of people, make a balance tube with saline. Centrifuge at high speed for 2 minutes. You should observe a match-head size white pellet at the bottom of the tube with your exfoliated cheek epithelial cells. If the pellet isn't white, it isn't your epithelial cells; it's something else that was in your mouth. Your pellet should be clearly visible and at least several millimeters across, but bigger isn’t necessarily better. If you don’t have enough cells, carefully decant or pipet off the supernatant, add another 2 ml of oral rinse suspension, and centrifuge again. Repeat until a sufficient pellet is observed.
  3. After pelleting your cells, pipet off most of the supernatant, taking care not to lose the pellet. It's ok if a small amount of liquid remains with the pellet.
  4. Label 1 tube of Chelex (Instagene®) for your DNA sample.
  5. Using the P-200 micropipettor, pipet up and down the liquid in with your oral pellet to evenly resuspend your cells. Transfer the entire volume to your tube with the Chelex resin. Cap and vortex thoroughly.
  6. Incubate the tubes at 100°C for 10 minutes.
  7. Vortex the tube again and spin for 5 minutes with the microfuge on low speed.
  8. Be sure that all the Chelex resin has pelleted to the bottom of the tube. You can use the liquid directly in the PCR. When you transfer some of it to your PCR tube, be sure not to transfer any of the Chelex-containing pellet; it will interfere with PCR.

Your DNA is now ready to use in PCR. Keep it on ice until you start the PCR. (If PCR cannot be run immediately, store it frozen.)

PCR procedure

For today’s PCR reactions, you’ve got it easy: the master mix is already made for you. For each reaction, all you need to do is add the DNA templates to the master mix in the reaction tube. You should have one PCR template for each person in your group. In addition, you should have two controls:

  • Negative control: a PCR reaction tube that contains the master mix, but no template DNA (add 20 μl sterile water instead of the DNA template). This negative control tube should produce no PCR product and no band on your gel. If you do get a product, it means that the master mix somehow became contaminated by template DNA. The purpose of the negative control is to test for this contamination.
  • Positive control: PCR reaction tube that contains the master mix and a known, high-quality template for the PCR reaction. The positive control should reliably produce a product. If it doesn’t, there may be problem with the master mix or the PCR machine. On the other hand, if your experimental PCRs don’t work and your positive control does work, you know that the problem is with your experimental DNA templates and not the master mix or the machine. For this lab, there are several possible positive controls, corresponding to the genotypes described in the introduction to this chapter (+/+, +/-, or -/-). These are all positive controls, even if they are negative for the ALU insert!

The PCR tubes we're using for this experiment usually come in a strip of eight connected tubes, but you should only use seven. In the next lab, you'll analyze your results by running a gel, and our DNA electrophoresis units have eight lanes. You'll need to use one of those lanes for a molecular weight marker. If there are four people in your group, you can do one negative control and two different positive controls, for a total of 7 tubes. You can adjust the number of positive controls depending on the number of people in your group, but make sure you do at least one positive and one negative control.

Program the PCR machine

The machine needs to be programmed to take your samples through the proper temperature steps. This has already been done, according to the program below.  Each set of denature, anneal, and extend steps is called a cycle. Before starting the cycles, there may be a one-time initial denaturation step. After completing the cycles, there may be a final extension step. The program for today’s reactions should be:

  • Denature 94° 1:00
  • Anneal 60° 1:00
  • Extend 72° 2:00
  • Repeat for a total of 30 cycles
  • Final extension 72° 10 min
  • Final hold 4° 00:00

The whole program takes about 3.5 hours to run, so you'll have to wait until next lab period to see the results.

Set up your PCR tubes

Now you’re ready to start pipetting. You need the following materials:

  • The DNA samples (templates) you just prepared.
  • Sterile water.
  • Positive control DNA templates. As described above, there are three different positive controls. Make sure you know which controls you're using.
  • PCR master mix (also called a PCR cocktail). It’s already dispensed into the PCR tubes, 20 μl per tube.For this experiment, the tubes normally come as a strip of eight connected tubes.

Keep all the master mix tubes and your DNA samples on ice. Don’t perform the following steps until the instructor tells you; it’s important that everyone adds their templates to their reaction tubes at about the same time. If you add your DNA too early and don't start PCR immediately, the primers may bind in the wrong locations on the template, causing your PCR to fail.

  1. Get a strip of 8 PCR tubes. Don't separate the tubes. Label the tubes, including your group name and the template DNA for each tube. Label by writing on the sides of the tubes; don’t use tape. Any writing on the top of the tube may get smeared by the lid of the PCR machine.
  2. When the instructor tells you to start, add 20 µl of DNA template to each PCR tube. For the negative control, use 20 µl sterile water. When you add the template to the master mix, mix by swishing gently up and down with your pipet tip.
  3. After setting up your reaction tubes, make sure other groups are ready to go before you put your tubes into the thermal cycler. All the tubes need to go in at the same time. Your tubes need to be on ice until the reaction starts. When everyone is ready, put the tubes in the thermal cycler, close the lid, and start the program.

The machine will take more than an hour to go through all the cycles. After the PCR is complete, the DNA is fairly stable; it can sit at room temperature for a couple of days. You don’t need to wait around until the PCR is done; you can leave it in the machine. In the next lab period you’ll use electrophoresis to look at your PCR products.

Before you leave

You should have:

  • PCR tubes in the thermal cycler.
  • DNA templates in a rack in the freezer.
  • Dump your mouth rinse solutions into the sink; throw the cups in the regular trash.
  • Chelex tubes, other microfuge tubes and pipet tips go in the biohazard trash.
  • Beakers used for waste tips: dump the tips into the biohazard trash and put the beaker into the dirty-glassware tub on the cart (if there’s not a tub, check with the instructor).
  • Gloves, paper towels, etc. go in the regular trash.

Review

Terms and concepts

  • Allele
  • Alu
  • PCR (=cocktail)
  • Conserved sequences
  • Controls in PCR, and in this experiment specifically.
  • Genotype (vs. genome or allele)
  • Homozygous & heterozygous
  • Insertion vs. substitution
  • Locus
  • Template
  • Transposon
  • Polymorphism
  • Primer dimer

Review questions

  1. What is the Alu repeat? How many Alu repeats are there in the human genome? How many are you trying to copy in this lab? Why don't the primers used in this lab copy all the Alu elements in the genome?
  2. What is a polymorphism, and how do you see it in the results for this lab?
  3. What results do you expect to see on the gel? Why would different individuals give different results? Why would some individuals show one band, while some show two? Diagram the possible results.
  4. Why do you sometimes see a third band in heterozygous samples, between the main upper and lower bands? (I'll explain this if we see it in our results; sometimes we do, and sometimes we don't.)
  5. Explain the possible results in terms of: locus and allele; genotype; homozygous and heterozygous.
  6. Suppose you aligned the DNA sequences of the +Alu allele and the -Alu allele. Would any parts of these sequences be the same?
  7. Compare and contrast: Chelex, HIC columns, spin columns for plasmid DNA purification.
  8. Why do you need a negative control? Why do you need a positive control?
  9. What is a molecular weight marker? Why is it important in this experiment? When would it be OK to not use a molecular weight marker?

References & further reading

Bio 6B flickr site. This is where I'll post the gel images.

Video & animation

Alu PV92 Detection by PCR Bio-Rad. We're using the Bio-Rad kit for this lab, so our procedures are more or less the same as shown in this video.

How Alu jumps from DNA Learnng Center. Animation explaining how Alu works.

Background

Alu elements: know the SINEs. Deininger, 2011. Genome Biology. Good general review of Alu elements in the human genome; not specifically relevant to the PV92 lab, but gives an overview of the biological context.

Recent Insertion of an Alu Element Within a Polymorphic Human-Specific Alu Insertion. Comas et al., 2001.

African origin of human-specific polymorphic Alu insertions. Batzer et al., 1994. This early article described how Alu insertion locations can be used to reconstruct evolutionary events. I think it's the first publication describing a PCR for PV92 Alu.

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