Extension - This is where the exciting thing happens where copies of the snippet are made. I suggest you be patient and go through each of these steps carefully yo understand whats going on. You will find some stuff repeated from before for completeness.
Here is the separated strands of a denatured DNA with the 5' and 3' ends marked and the snippet we want to copy is indicated.
Here is where I show the primers paired with the single strands of the denatured DNA. This association happens during the annealing step.
In the extension step, polymerase goes to work. In short, DNA polymerase looks for 3' end of a single stranded fragment of a DNA and adds complimentary nucleotide to make double stranded DNA. This is important to understand. For one, you need to have a single stranded potion of the DNA and secondly you need a 3' end open for the extension. As you see in the diagram below, this is exactly what the primer addition provides for both of the separated strands of the original DNA. Secondly, you need a tons of nucleotide for polymerase to add. So, in this step, the scientists add a ton of nucleotide. The temperature where this takes place is also very important it is typically in the mid 70's. DNA Polymerase is a generic term and there are a lot of different polymerases. The one that fits the bill here becaue of the temperature is a naturally occuring polymerase called Taq Polymerase. So, the original DNA, primers, LOTS of nucelotides, and Taq polymerase constitute the mixture. For reasons of stability and efficiency etc. additional chemicals such as a buffer are added. Scientists have figured out that this step should be carried out in the 72 - 76°C.
When done, you will get this.
That completes the first cycle! At the end of this, you have two double stranded DNA with some portions remaining single stranded in one of the two strands. This whole step of mixing everything, denaturing, annealing and extending all takes a few minutes.
In the next post we will look at the subsequent steps.
No comments:
Post a Comment