I have talked about studying the function of genes by RNAi before. However, to study the function of a gene we need to have access to enough copies of the gene to work with. In every genome there are 2 copies of each gene, 1 is on the chromosome from the mother and 1 is on the chromosome from the father (see DNA, RNA and protein). Unfortunately, this is not enough to study the function of the gene. That is why we want to copy the gene of interest many times until we have enough to work with. Although it is possible to get DNA commercially, the number of mistakes in the sequence increases with length. So in general we cannot get sequences of much more than 30 bases from companies. Luckily we can use a bacterial enzyme that is used by bacteria to make DNA (Taq polymerase). This method to increase the copy number of a certain gene is called the polymerase chain reaction (PCR). When we want to amplify the DNA we need some starting material. We get this starting material from a tissue of interest, which in my case is the beetle egg. So I extract DNA from beetle eggs, but DNA can be extracted from any tissue. The taq DNA polymerase needs this DNA as a template to start copying from. Next to a template, polymerase also needs a place where it can start copying. This place is provided by something we call primers.




Primers

Primers are short strands of DNA often around 20 bases long. We select the sequence of primers in such a way that they fit on the gene we want to study. If we heat the DNA to 95 degrees, the double stranded DNA will fall apart into one 5’ strand and one 3’strand of single stranded DNA (denaturation). To amplify the DNA we need 2 primers, one for the 5’strand and one for the 3’strand. These primers bind to the single stranded DNA, this makes a small part double stranded and this serves as a starting point for the polymerase (see figure below). The primers are important for the specificity of our reaction; if we design them properly the primers will fit on only the gene of interest and on no other genes.

Primer
The chain reaction

To initiate a chain reaction we need to make sure that we have all the right ingredients. First we need our DNA, second we need to have enough bases in the solution to make the DNA from (A,T,C and G), third we need to add primers to the reaction and fourth we need the polymerase. The amplification of the DNA requires 3 steps.

Step 1) The double stranded DNA is split into 2 single stranded DNAs at approximately 95 degrees (Denaturation).

Step 2) Cool down to 60 degrees, at approximately 60 degrees the primers will bind the single stranded DNA (Hybridization).

Step 3) At approximately 72 degrees the DNA will be extended from the primers by the polymerase (Elongation).
This reaction doubles the number of copies we have of the gene of interest. By repeating these 3 steps we double the number of copies in our solution every round (see figure below).

PCR
So by using the PCR we amplify the DNA exponentially. Usually these steps are repeated approximately 30 times which leads to a lot of copies of our gene of interest and very little other DNA. Eventually the ingredients for the reaction will run out (bases and primers), this causes a decline in the exponential amplification and when we look at the amount of DNA in a graph we will find an s-shaped curve. In the graph below you see such a curve, the number of cycles is on the x-ax and the amount of DNA on the y-ax.

Amplification
We can check whether we amplified the right fragment on an agarose gel and we can use this DNA in combination with other techniques like RNAi to assess the function of this gene.

Leave a Reply