mekentosj.com | 4Peaks - The Science Behind

Introduction: The blueprint of life is stored in our genome, the complete DNA content of every cell in our body. By deciphering the information encoded by the DNA, scientists try to find out how the cells in our body work for example, or how diseases develop, why we age, and what goes wrong in the event of cancer. But before that can be started, the "book of life" has to be read first. Scientist do this with a technique called "sequencing" that determines the type and order of the different bases DNA is made of.

Sequencing: In order to understand why we are talking about 4 peaks all the time, we have to start by telling how sequencing is done. For this scientists use a modified PCR reaction (see 'Chain Reaction'). The trick is to include in the reaction not only normal nucleotides, but also a type of nucleotides that can not be coupled to the next one. As a result these, when incorporated, terminate the reaction in a stochastic manner, producing fragments of different lengths. If the terminating nucleotide is an adenosine for instance, all these fragments will end with an A. By doing this for all four bases, and separating the fragments on size, a ladder pattern is created that allows us to read the sequence (see figure).
Why 4Peaks?: But that still doesn't tell where the peaks come from. Scientists originally used radioactive nucleotides to visualize the DNA fragments, generating these beautiful ladders which you often still see in movies and photos. Nowadays this is hardly used anymore, the radioactive nucleotides have been replaced for fluorescently labelled ones. The figure shows an example of such a gel. Another advantage besides the reduction of radioactive waste is that the reaction for all four bases can now be done in one reaction as four differently coloured nucleotides are used. Finally, the gel is quantified, resulting in the famous peaks; one peak for each band on the gel, one coloured trace for each type of nucleotides. For peaks, four peaks.

The ABC of DNA: DNA stands for deoxy-ribonucleic acid, and point to the chemical building blocks it is made of: nucleotides (nitrogen-containing bases), ribose (a sugar), and phosphoric acid. DNA itself is a long polymer where the backbone is formed by the ribose and phosphoric acid, and with the nucleotides (also known as bases) as sidechains. These nucleotides exist in the DNA in four types: adenosine (A), cytidine (C), guanosine (G), and thymidine (T), which together for the alphabet of DNA. By forming "strings" of these "letters", DNA can store the genetic information it carries.

In the early 1950s, Watson and Crick introduced the characteristic double helix structure that DNA forms: it consists of two complementary strands together spiralled like a helical staircase. In the complementary strands, an A always sits opposite to a T, and a G always opposite of a C. This unique feature allows both strands to separate and serve as a template for duplication, a prerequisite for transmission of genetic information from one generation to the next.

Chain Reaction: PCR, or Polymerase Chain Reaction, is a technique that generates many copies of DNA from an initially small sample. The DNA that has to be amplified is put in a test tube and subsequently heated to separate the two complementary strands. Next, as the solution of DNA halves cools, short pieces of DNA called primers are added into the test tubes. These are designed such that they bind to a specific region in the DNA, and form the start from where to begin. Finally, an enzyme called DNA polymerase is added to the reaction in combination with single nucleotides. The polymerase enzyme will now find the site where the primer had bound the DNA, and starts duplicating it, thereby producing millions of copies in a matter of hours.