The molecular sexing of birds is a simple process.  We take a biological sample, is analyzed in the laboratory and the sex of the bird is determined. This description, although it is right, is similar to be contented with reading the summary of a book instead of the original text. If we open that book, we would see that it has been necessary millions of years of evolution, decades of research, two Nobel prizes and a work team to fill in that gap between the shipment of a sample and the sex determination of our bird. In this blog entry we will show some curiosities and decode the mystery around the molecular sexing of birds.

The essential is invisible to the eyes

When Saint-Exupery wrote that sentence for the “Little Prince” it seemed he was predicting the description of the DNA made by Watson and Crik 10 years later. When we are unable to distinguish males and females based on their plumage or size (among others) we have to rely on the genetic material to determine the sex of the bird. DNA, the mysterious code that encloses our evolutionary history and determines who we are, is the ace up our sleeve that will let us get to know the sex of our birds. Our mission in the laboratory is making visible “the essential” and concluding if that DNA belongs to a male or a female. How do we do it? First, we need to find a region of the DNA that let us distinguish males from females (a genetic marker) and after that we have to be able to visualize that marker in order to know the sex of the bird.

A factory of DNA in a drop of water

The genetic marker used for the molecular sexing test is called CHD (a simpler way to name the gen of the Chromodomain-helicase DNA-binding protein) and, as would be expected, it is located in the sexual chromosomes Z and W. Its function is similar in both chromosomes, but the DNA segment found in each chromosome has a different size: this is the difference that we have to spot in order to identify males and females at the molecular level.  Females have a “big” and a “small” fragment which belong to the chromosomes Z and Y –respectively-. In the case of males, they only have two copies of the “big” fragment located in the chromosome Z.

Having into account that each cell contains two meters of DNA, that the fragment of CHD is not bigger of a micrometer (one thousandth of a millimeter) and that each sample analysed contains millions of cells, how do we get to distinguish between “big” and “small” fragments of the copies of CDH? Looking for a needle in a haystack is a daunting task, but having millions of needles in the same haystack might facilitate meeting that challenge. Something similar is done in the laboratory of molecular biology. With a volume inferior to a drop of water we create millions of identical copies of the fragments of CHD that are located in the DNA extracted from the samples received. The American biochemist Kary B. Mullis worked on that and his discovery (named polymerase chain reaction or PCR) was rewarded with the Nobel Prize in Chemistry in 1993.  PCRs are based in cycles of temperature changes (heating and cooling cycles during programmed times) and are performed in devices called thermo cyclers.

Those “thermomixes” of DNA generate millions of copies of the fragment of the CHD marker. Once the PCR is finished, we should be able to determine if in that “drop of water” there are “big” or “small” fragments of the CHD marker and, this way we are able to determine the sex of every species.  We place the resulting products of the PCR in a solid medium derived from the agar (the same substance used for making jellies). After that, we apply an electric field to the DNA molecules. This technique is known as electrophoresis and thanks to it we can separate the “big” DNA molecules and the “small” ones according to its size (which is directly related to the electric charge of the molecules). As a result, males only show a single band of DNA in the agarose gel (big” fragments of CHD, all of the same size) and females present two bands (one “big” and one “small”).

Thus, every time you receive the result of a DNA test, think that the work carried out by two Nobel Prizes and a team of professionals and experts in molecular biology concludes by putting the DNA of your bird in a tray of jelly.