THE CAVEAT
A preface

DNA FINGERPRINTING OR (ALTOGETHER NOW) THE POLYMERASE CHAIN REACTION!
A quick introduction

THE GIST AND HOW IT MORE OR LESS WORKS
A primer on the polymerase chain reaction

YOUR TURN... LOOKING FOR AN ALU INSERTION.[1]
Conceptual synopsis of a DNA fingerprint experiment for you to try.

THE EXPERIMENTAL DETAILS
Run down of things needed before starting the experiment.

O.K. READY TO START THE PROCEDURE
Experimental protocol. Setting up the reaction.

LINK TO TEMPLATE CODE
An example of code required for themal cycling.

RESULTS
Some data obtained

SOME TIPS FOR TROUBLESHOOTING
A guide to problems that can arise.

FOOTNOTES

ETHICS: GENETIC TESTS AND TOYS
So what exactly can you do with this: an ethical perspective

DNA FINGERPRINTING OR (ALTOGETHER NOW) THE POLYMERASE CHAIN REACTION!

The polymerase chain reaction, or PCR, is a powerful genetic technique that allows researchers to amplify DNA sequences of interest. This amplification allows better and easier genetic characterization and can be used for a variety of tasks including those in the context of genetic disease diagnosis, maternity/paternity tests, DNA forensics, or simply in the production of large amounts of DNA fragments for further study.

In nature, cells make more DNA by undergoing a process known as replication. This is where the amount of DNA is essentially doubled so that a cell dividing into two new cells can give each of these two cells a complete copy of the DNA. However, replication is a relatively complex process, requiring the regulated actions of over half a dozen different enzymes - technically challenging to pull off in the laboratory. PCR is therefore an experiment that simplifies the process of replication for greater efficiency in test tube settings.

THE GIST AND HOW IT MORE OR LESS WORKS:

So how does PCR work? And how, exactly do you simplify something like replication?

Essentially, this entails the collection of a few components needed for a successful DNA amplification reaction. Although, we’ll later highlight some specific places to get them, for now, let’s quickly summarize what they are. They include:

1. A sample of DNA to start with: there are many ways to get this, but you can use the cheek cell procedure outlined in the print article.

2. Two primers: These are short pieces of DNA that have been specifically designed to complement the region of your DNA that you wish to make more of. They can be ordered from places that do something called oligonucleotide synthesis (fancy talk for making small pieces of DNA synthetically).

3. A solution containing the 4 different nucleotides (the A’s,T’s, C’s, and G’s): necessary for use as the building blocks for DNA replication.

4. A heat stable DNA polymerase: This is an enzyme that is actually responsible for making a complementary DNA copy (i.e. replicating). The cheapest ones are usually those derived from the Thermophilus Aquaticus bacteria, and are called Taq polymerases. These polymerases will often come with a buffer solution which is a specific recipe that allows the polymerase to function at its best.

5. Equipment setup to allow a PCR reaction to cycle through three different temperatures for certain lengths of time, over and over again. This can take the form of the MAKE thermal cycler, or in a bind can be as low tech as a person moving a test tube from three different pots of water set at three different temperatures for about 3 to 4 hours!

(A good animation of PCR)

To start the PCR reaction, you’ll need to mix in a plastic tube, your DNA sample, the primers, the nucleotides, and the heat stable DNA polymerase.

First up, is a high temperature step usually at about 95C for about 1 minute, and often called the denaturation step. This is all about allowing your DNA to open up, since it naturally exists as a tight helical coil. The high temperature step will effectively melt the double stranded DNA strands into single strands. This step whilst elegant in its simplicity is the reason for the need of heat stable DNA polymerases, since the enzyme needs to be able to withstand the heat.

With the DNA open, we move to the second temperature step, referred to as the annealing step. Here, the primers, which have been designed to interact with the desired region of amplification, can now “get in.” This binding is mediated by choosing a second temperature which ensures a specific interaction, usually around 60C or so, and usually for about a minute.

With the primers bound, we can now move on to the third and final temperature step – the elongation step. By switching to a temperature where our enzyme works best, the heat stable DNA polymerase can extend the production of DNA from the two primers. This is the actual “replication” part, and consequently also involves uptake of the nucleotides. Note that at the end of this step you have two copies of the original piece of DNA of interest.

And there you have it! You’ve essentially made more of your DNA of interest (doubled it actually). However, what is cool is that you can now repeat the process over and over in the same experiment. In other words, take the two copies you’ve produced and subject them to the same three temperature steps. You’ll hopefully see that this allow your reaction to go from two copies to four. In essense, going through these three temperatures (which is called a single cycle) will result in the doubling of your DNA of interest. The thermal cycler simply allows you to do this automatically and in a programmable fashion. Most PCR experiments are done with a total of 30 or more cycles (or 30 rounds of doubling), so that you can effectively increase your DNA interest by billion times or more. Factor in the fact that a single cycle is often less than 5 minutes, and what you have is an incredibly powerful, yet simple, procedure to amplify out your DNA in a relatively short period of time.

YOUR TURN... LOOKING FOR AN ALU INSERTION.[1]

This suggested experiment will allow an individual to look within their own genome for a genetic element called an Alu insertion. Basically, the Alu insertion is a small 300 base pair DNA sequence that is often classified as junk DNA. A person will actually have approximately 500,000 copies of them scattered throughout their own DNA code, but this experiment will focus on looking for the presence or absence of one Alu insertion in particular – specifically at the TPA 25 loci on chromosome 8 (address lingo for being around a gene called Tissue Plasminogen Activator). Furthermore, since humans received genetic instructions from both their mother and father, each individual will actually have two different chromosome 8’s to query. Therefore, possible genotypic outcomes may be +/+, -/-, or +/- for this Alu insertion.

It’s also very important to note that this Alu insertion in found within a junk region of the TPA 25 loci, so in effect represents junk DNA within junk DNA. Consequently, the results obtained will be phenotypically neutral, meaning that they do not mean anything and have no consequences (positive or negative) on your wellbeing. However, this experiment done on multiple family members can possibly result in paternity/maternity discrepancy, so it is advised not to perform this particular DNA fingerprint without first contemplating this issue.

Two primers, called the Alu Forward and the Alu Reverse, have been designed to flank the point of insertion where this Alu insertion may or may not exist. This means that they will be amplifying the region of DNA where your Alu insertion may exist. Consequently, data readout will be based on whether this amplified DNA has the extra 300 base pairs of your Alu insertion or not. Since the readout is dependant on observing a discrepancy in the size of the amplified product, you will visualize your data by doing a gel electrophoresis. The figure below summarizes this:

Figure Legend: The TPA 25 locus contains a dimorphism reflecting the presence or absence of a 300bp Alu sequence. When the Alu sequence is absent, amplification by PCR yields a 100bp product. If the Alu sequence is present, the size of the PCR product is increased to 400bp.

THE EXPERIMENTAL DETAILS

This is what you’ll need to start the experiment:

1. Your cheek cell DNA isolated as described in the print article. This involves obtaining a sample up to and including the step where you dissolve the snot pellet in 3 drops of running buffer overnight.

2. Two primers of the following sequence:

Alu Forward 5’-GTAAGAGTTCCGTAACAGGACAGCT-3’
Alu Reverse 5’-CCCCACCCTAGGAGAACTTCTCTTT-3’’

These can be ordered and shipped from a variety of oligonucleotide synthesis services (try typing that in google), which are fairly commonplace throughout the United States and Canada. Almost all vendors will accept credit card payment or invoice clients for a cheque. Here is a place in my neck of the woods that sells the primers for instance. Cost of both primers will run about $50, and likely good for well over 200 reactions.

3. A Heat Stable DNA Polymerase. This is where it can get confusing as well as needlessly expensive. There are many varieties of heat stable DNA polymerases out there, but some are quite sophisticated, capable of working better, working faster, working in odd conditions, etc. To add to the confusion, many polymerases will not come with all the reagents needed. These include the nucleotides, and often a MgCl₂ solution (the polymerase needs Mg²⁺ to function properly). As well, most current enzymes are capable of something called a “hot start.” This is where, your enzyme is effectively turned off until the first high temperature step starts. This is an attempt to prevent non-specific amplification which likely starts the moment you begin you mix all together the various PCR reagents in your test tube.

In light of this complexity, we suggest using something like Invitrogen’s Platinum Blue PCR Supermix [2] (click on the reagent with the cat no 12580-015), because it is a solution that comes bundled with everything needed, except for the primers and the actual starting DNA sample. It even includes loading buffer in the reaction, so that it is ready for immediate loading onto the gel. Note that this reagent costs about $125 for 100 reactions, although you may be able to request for a smaller sample size if you can convince the vendor that this test run could lead to the purchase of more in the future (i.e. for high school programs, etc)

4. Plastic tubes: Essentially, you would want to obtain some sterile microcentrifuge tubes, or more specifically PCR tubes. Both are small plastic tubes that are generally designed to contain a maximum of either 1.7ml, 0.65ml or 0.2ml of fluid. PCR tubes are particular in that they have thinner walls so that more efficient heat transfer can occur during the cycling steps. The tubes available at Fisher Scientific would work well (a pack of 1000 tubes will run about $100). Note that ideally, the tubes you choose to purchase should physically match the hole sizes in the thermal cycler.

5. Mineral oil. When your reaction is heated at high temperatures on the thermal cycler, some of the fluid may evaporate and therefore significantly affect the PCR reaction. Consequently, an old trick would be to put some mineral oil into the reaction which results in a thin overlayer of oil that acts as a liquid lid. In addition, the mineral oil can be used to fill the tube holes in your thermal cycler heat block, to additionally ensure good heat transfer from the heat block to the reaction tube.