Fascinating genetics # The Polymerase Chain Reaction
PCR
Today i would like to present the mechanism behind DNA replication and one involved protein, DNA polymerase, responsible for putting new base-pairs, A, T, G, C, the building blocks of DNA, where they are supposed to be. The replication of DNA must generate 100% exact copies of our DNA or mutations will cause severe problems for the affected cells.
DNA replication is a complex procedure which we use in laboratories, in a simplified way, with a procedure called PCR or polymerase chain reaction. This is done to copy DNA fragments into billions of copies and using them for different purposes. If I were to express Insulin in E.coli bacteria i would only need a few genes coding for insulin, and i could copy that gene into billions. This is done in small test tubes, so its cheap to run, and it mimics the mechanism used by our body in a few ways.
With the copies, I can cut and paste the gene for insulin into a cloning vector, or a plasmid, as we call them. The plasmid is then mixed with E.coli, enters through the membrane of the bacteria, and lastly, Insulin is produced. By copying the gene of interest we save a lot of money and receive essentially unlimited amounts of insulin coding DNA.
"Schematic picture of the PCR procedure resulting in 10^9 fragments or 2^30 after only 30 quick cycles"
The polymerase chain reaction is carried out by an enzyme called DNA polymerase and this happens continuously in our bodys. The procedures are very different in the body and a test tube, since we simply use temperature to split the DNA in to two strands, while the body is dependent on many extra proteins that perform complex functions in order to do this.
After heating our DNA sample, the DNA helix with its two combined strands will unwind and separate into two single strands of DNA, this step is called denaturation.
"Denaturation,Annealing and Elongation with their respective temperature intervals".
Then we add copies of the start sequence according to one of the strands and the complementary sequence of the other, seen in yellow above, in a step called Annealing. These sequences is called primers, reverse and forward primers, and will allow for DNA-polymerase to bind to and start making the copy of the DNA stand. This results in new double stranded DNA since it is synthesized directly upon the old one in the last step, elongation or extension/hybridization step.
To make this happen we must of course also throw in all the ingredients needed such as the DNA base pairs called dNTP, the primers, DNA polymerase and the gene coding for insulin that we want to copy.
The hardest parts is to make the primer bind to the single stranded DNA since this requires an exact temperature. When designing the primers it is important to know that they will bind to the right spot under temperatures of 75 degrees or there will be problems with stability. If it´s to cold the primer loses specificity while it cant bind if its to hot. The temperature can be calculated precisely as well as the required time frames and if you are interested in the details I made a long post on the exact procedure behind cloning and expressing a protein in E.coli here:
To heat the sample and and keep the temperature stable during the different steps, we use something called an PCR machine. This is really nothing more than an glorified oven with pre programmed cycles and and an very usable interface.
They can be a bit pricey compared to a simple oven but under 10,000$ for newer models which is nothing in this area of science. For example I´ve bought 1 gram of a rare compound that we needed for a study for OVER 10,000$ and that´s after some SERIOUS haggling.
Ah, The glorious oven, this particular one takes two products simultaneously for the cost of 3 machines, no just joking, but almost :)
They are super easy to use, when all calculations are done just specify the temperature, time duration for each step and the number of cycles. The PCR reaction can be used to verify cloning success as well by simply lysing the bacteria in a test tube, followed by a PCR run with the same primers used prior to cloning. If the insertion of the desired gene was success full we will revive millions of copies which can be measured and verified easily with nano drop, an absorbance measuring machine, that works with minimal sample amounts.
I must say that the polymerase chain reaction simply is a fantastic tool to use when working with genetics and i don´t know what we would do without it!
FIG 1 thermofisher
FIG 2 thermofisher
FIG 3 laboratoryequipment
Thanks to Thermo Fischer Scientific for your great products and manuals.
https://www.thermofisher.com/se/en/home.html
And thanks to every one else at #steemit for checking my posts out, it is much appreciated.