The Molecular Dilema:
 
 
 

You've finally gotten to travel to the beautiful island of the Dominican Republic, and you decide to explore its tropical forests. As you venture into the savage jungle, machete in hand to cut yourself a path through the shrubbery, you come across an old amber mine. Curiosity leads you into the mine, and suddenly you stumble on a chunk of amber. You clear the dust off this fossil resin, and you can hardly believe your eyes, the amber rock has a preserved insect inside! Instinctively the molecular biologist awakens within you -as well as scenes from Jurassic Park- and you are curious as to whether this insect's DNA is preserved too. You realize the rarity of actually finding an insect in an amber piece and get an intuitive feeling that you should check this out. You fly home to the molecular lab you have secretly kept in your basement , inspired by the frustration and determination to fully understand one of Dr. Malcolm Campbell's molecular biology assignments. You discover you indeed have prehistoric DNA, but only one strand! Caught between the excitement of your discovery and the fear that you may lose it while experimenting on it, you try to recall a method to amplify this rare piece of DNA. Aha!

The Solution:

You smile as you submerge yourself in this magnificent method to replicate your DNA....

ADVANTAGES

Now why did you chose The PCR Method to replicate DNA rather than other methods such as the use of plasmids or phage DNA?

-You wanted a method that would replicate your DNA the fastest as well as one you could do in vitro. Plasmid and phage DNA take weeks, way too long! In a matter of hours you will have millions of copies of this DNA strand!

-These other methods are not as specific as PCR. You can use primers to target exactly what you want to copy.

-You don't even need a purified sample of DNA nor a DNA piece that is in perfect shape. It can be partially degraded.

-Most importantly, you don't need large amounts of the DNA for this method to work.

-PCR can be used to replicate DNA that is millions of years old!

THE RECIPE

You must first collect your materials:

Target ds DNA + DNA polymerase + nucleotides + the primers + buffer +DMSO

THE METHOD

Before you start the PCR reaction, it is important to come up with a table with the measurements in microliters (uL) of the reagents you are going to mix. PCR is usually performed with a total volume of 100 uL. Make sure that in determining the amounts to be added of each reagent, you consider the concentration of the stock solutions you are provided with, and adapt your volumes accordingly.

A sample table follows:
 

Now that you know the amount of each reagent needed, you are ready to begin the reaction. In a test tube, combine the water, the buffer, the DMSO, the template dsDNA, the nucleotides (dNTPs), the primers, and lastly, the polymerase. The DMSO works with the primers to enhance their specificity to the targeted DNA. It is important that the reagents be added in this order, especially the enzymes. Otherwise, the enzyme may denature. Thus, the polymerase is kept on a 'cold block' to keep it from denaturing. The rest of the reagents should also be kept on ice.

You are now ready to begin the first cycle. The first step in the cycle involves the heating up the mixture up to 95 degrees celcius for five minutes. At this high temperature, the double-stranded DNA denatures into two single DNA strands. The mixture is then cooled to 55 degrees celcius where the primers bind to the separate DNA strands. Primers, which target the desired DNA sequence, bind through hydrogen bonds to each strand. The DNA polymerase then works to elongate new strands by adding on nucleotides unto the original template strand. The number of double-stranded DNA has now been doubed. This completes one cycle.

However, we want many copies of this DNA so we repeat this cycle multiple times. The dsDNA strands are heated up again to separate the double strands, primers bind, DNA polymerase (such as Taq polymerase) extends the strands, new strands are synthesized, and so forth. Usually, the cycle is run about thirty times or as many times necessary to produce the number of clones needed.

 
  Figure 1 Demonstrates the cycles involved in a PCR reaction resulting in multiple copies of a target DNA. Image was taken from http://apollo.co.uk/a/pcr/

SUMMARY OF WHAT HAPPENS DURING A CYCLE

sample cycle:

95 C for 5min ( dsDNA template denatures so polymerization begins)

95 C for 1min (DNA template denatures)

55 C for 1min (primers base pair to target DNA)

72 C for 1min (DNA polymerase elongates strand) (return to 95C for 1min and repeats these steps for the specified number of cycles. Then it proceeds to the last temperature below.)

20 C hold
 
 
 

REFERENCES

Campbell, A. Malcolm. Molecular Biology (BIO304) Lab Manual. Davidson College; Spring 1998. p.7

Campbell, Neil A. Biology. 4th edition. California: The Benjamin/Cummings Publishing Company, Inc; 1996. p.379-380.
 

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