polymerase chain reaction

Method of rapidly amplifying a given DNA sequence. Uses short primers which anneal to both ends of the sequence, acting as a template for DNA polymerase to fill in the intervening sequence. Uses a thermocycler to raise the temperature, causing the DNA strands to melt (separate). The temperature then drops, new primers anneal, and the process repeats. Every cycle doubles the amount of DNA present, allowing infinite amplification of a given sequence. This has allowed techniques like DNA fingerprinting to become useful, as a very small sample of DNA from a crime scene can be amplified and used to identify a suspect. Invented by Kary B. Mullis, a breakthrough for which he won the Nobel Prize in Chemistry.

How to Perform PCR at Home

Yes! Now you, too, can know the joys of making lots and lots of copies of your own DNA in a test tube - in the privacy of your own home! You never know when you'll need some extra DNA, so stock up now while you have a chance. It's great fun for kids, too! (Okay, I admit it's not an incredibly useful thing to have, but hey, it would make an impressive science project.) Here's what you'll need:

• A stove
• Three pots with water at 94°, 60°, and 72° C
• A fourth pot with boiling water (temp doesn't matter)
• Three thermometers to monitor water temperature
• Several Pyrex test tubes or plastic microcentrifuge tubes
• Bleach
• Ice
• Latex gloves (or plastic, if you're allergic)
• Plastic coffee stirrers (clear)²
• Cotton swab
• Distilled water
• Buffer¹
• Magnesium chloride¹
• The four deoxynucleotide triphosphates, or dNTPs¹
• Β-globin primers 1 and 2¹
• Polymerase enzyme¹
• Mineral oil
• Some DNA
• A stopwatch

¹ Contact the Sustainable Sciences Institute (www.ssilink.org) to find out how you can purchase these in small, inexpensive quantities.
² The plastic coffee stirrers are used to measure tiny quantities of the reagents you'll be using. You could order a handy dandy micropipette from Fisher, but that's pretty darn expensive. Instead, measure quantities visually by dipping your coffee stirrer into whatever reagent you're measuring, put your finger over the tip, and pull it out. Exact measurements are unnecessary; what's important are the relative quantities of reagents.

Okay! Now that you've gathered all of your supplies, wipe down your glassware and kitchen stove and countertop with bleach to get your workspace as sterile as possible.

To collect your DNA sample: Put a bit of the DI water in one of your test tubes. Take the cotton swab and gently scrape the inside of your cheek (or your kid's or your dog's or your arch nemesis's cheek) with it; then, slosh the tip of the swab in the tube with the DI water. Stopper the tube and put it in the boiling water for a couple of minutes to rip open the cells and let your DNA out into the solution. You should use a centrifuge at this point to separate the DNA from the rest of the cellular debris, but since we're going low-tech, we're not going to worry about that. Tap your tube gently with your finger to evenly distribute the DNA in the solution and use a plastic coffee stirrer to measure out a tiny amount; dilute this in DI water by a factor of 10, and then dilute that solution by a factor of 100. Put your samples on ice until you're ready to use them.

Recipe for PCR Soup:

• 100 units DI water
• 15 units buffer
• 8 units magnesium chloride
• 5 units each of the four dNTPs
• 3 units each of Β-globin primer 1 and 2
• 1 unit polymerase
• 1 drop mineral oil (for freezing your sample)

Add this solution to several of your sterile test tubes. Add DNA to all of the tubes but one (you'll want a negative control.) Put your tubes in the 94° water for one minute; transfer them to the 60° water for 90 seconds; transfer them to the 72° water for another 90 seconds. There! You've just gone through one cycle of PCR! Don't pat yourself on the back and call it a day just yet, though: you'll need to repeat this cycle over and over and over (30+ times) to get a good amount of DNA copies.

Well, now that you're done, what do you do with your DNA? Unless you plan on doing something immediately, you should put your samples in the freezer so they won't decay. If you just like the satisfaction of knowing that you're got some extra DNA just in case you ever need it (and even I can't think of a good scenario for that one), then pat yourself on the back and call it a day. If you want to take a look at your DNA, you'll need to run a gel electrophoresis. This will show you whether you succeeded in creating copies of your DNA, and it's also something you can do at home. That, however, is another node for another time.

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I can't take credit for this idea. Shawn Carlson of Scientific American put forth this procedure in an "Amateur Scientist" article last year. You can see the whole thing here: http://www.sciam.com/2000/0700issue/0700amsci.html

What happens during a polymerase chain reaction

During the polymerase chain reaction (PCR), a certain portion of DNA is amplified many times.

The essential ingredients for a successful PCR are:

1) DNA polymerase enzyme. Usually isolated from Thermus aquaticus, a bacteria that can withstand high temperatures. Thus, the enzyme can endure temperatures up to 94°C without being denatured, unlike most other enzymes.

2) Buffer and Magnesium Chloride. These solutions serve to optimise the conditions in which the enzyme functions, as enzymes are very sensitive to pH and salt concentrations.

3) Four basic deoxynucleotides, also known as dNTPs. The four basic nucleotides are the building blocks of DNA. In order to generate build copies of a house, you need bricks. These act like bricks to build the new copy of the DNA.

4) Primers. This are short pieces of DNA which act as starter material from which the DNA polymerase will begin.

5) DNA. This is the original copy of the DNA, also known as the template.

6) A PCR machine. A typical program on the PCR machine will read like this:

Stage 1
• 94°C for 2 minutes
Continue to Stage 2

Stage 2
• 94°C for 30 seconds
• 60°C for 15 seconds
• 72°C for 1 min 20 seconds
Repeat 30 times; then continue to stage 3

Stage 3
• 72°C for 6 minutes
End.

As you will notice 3 temperatures are used in PCR. These temperatures have their purposes:
-- 94°C is known as the denaturing temperature. It causes the double-stranded DNA to separate from each other.
-- 60°C is known as the annealing temperature. It allows the primers to anneal to the template.
-- 72°C is the elongation temperature, enabling the polymerase to finish building the copy it is making.

The amplification occurs during Stage 2. Stage 1 is just to denature the DNA; Stage 3 allows for complete elongation of all the PCR fragments.

What happens in a PCR machine during Stage 2

1) Denaturation occurs first.

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       Double-stranded DNA at rest.


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      The DNA strands separate from each other. This is denaturation.

2) Next, the primers anneal (stick) to the DNA.

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      Denatured DNA.   
      
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          | | | 
         ------- {Upper Primer

                                         ------- {Lower  
                                          | | |   Primer
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      The primers anneal to the denatured DNA.   

3) Then, elongation occurs.

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          | | | 
 Primer} -------
 
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          | | | 
         -------P
      The polymerase (P) attaches.


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         -----------------P        |    |
                          |    |            |
              |               
                    
     The polymerase "builds" the second strand of DNA using the "bricks" (|) present. 
The "bricks" are the dNTPs.

After 1 round of amplification, 2¹ copies of the DNA fragment would be present. After 2 rounds, it would increase to 2²=4 copies. After 30 cycles, 2³⁰ copies, i.e. 1073741824 copies would be present. This is the reason why PCR can be used even if very few copies of the DNA is available. It is now a common technique used in almost every biological laboratory.