polymerase chain reaction (idea)

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.

         ----------------------------------------
          | | | | | | | | | | | | | | | | | | | 
          | | | | | | | | | | | | | | | | | | |  
         ----------------------------------------
       Double-stranded DNA at rest.


         ----------------------------------------
          | | | | | | | | | | | | | | | | | | | 


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

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

         ---------------------------------------
          | | | | | | | | | | | | | | | | | | | 



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

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

3) Then, elongation occurs.

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


         ---------------------------------------
          | | | | | | | | | | | | | | | | | | | 
          | | | | | | | | |
         -----------------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.