Robert Feulgen,who was lived between (1884–1955) was a German physician and chemist who, in 1914, developed a method for staining DNA or otherwise called as the Feulgen stain.Robert J. Feulgen published seventy research papers about the chemistry of physiology, histochemistry etc... and he is best known for his discovery of a method for staining nucleic acid, now termeda Feulgen reaction.
Early Life and his Career
Feulgen was born in Werden, Germany, into a working class family, Feulgen paved his way to the medical school through an outstanding student performance that opened to him the doors of the University of Freiburg at Breisgau in 1905. His medical residency was taken in the City Hospital of Kiel, where he wrote his dissertation on the purine metabolism of patients with chronic gout. After finishing residence, he moved to Berlin and started his carrier as an experimental researcher at the Chemistry Department of the Physiological Institute, then under the direction of Hermann Steudel, a nucleic acid chemist.
Feulgen worked with Steudel from 1912 until 1918, improving Steudel's technique for thymonucleic acid staining via biuret reaction. At the time, what later became known as DNA, was termed thymonucleic acid and thyme tissues were used for the extraction of nucleic acids. Biuret, a compound obtained from urea, gives proteins a blue-violet color (biuret reaction), when copper sulfate and strong alkali are added without heating, and it was used by Steudel to stain DNA. However, the color was lost when water was added, resulting in a colorless solution. Feulgen introduced a new staining technique by adding Congo red combined with malachite green and extracting the salts formed, thus avoiding the biuret reaction and the loss of staining. This new staining technique allowed a more reliable analysis of the resulting product and the calculation of nitrogen and phosphorus ratios present in nucleic acids.
Based upon his observations, Feulgen defended the hypothesis that nucleic acids were formed by four nucleotides, which at the time was thought by many to be too few units to carry the enormous wealth of information needed to transmit genetic information.
In 1914, Feulgen made a major contribution to the tetra nucleotide hypothesis (i.e., four nucleotides forming an oligonucleotide) when he discovered that phenyl hydrazine reacted with apurinic acid, what indicated the presence of aldehyde groups.
Using aldehyde-blocking controls, Feulgen found that when thymonucleic acid (now known as DNA) is mildly hydrolyzed to apurinic acid, the loss of purines exposes aldehyde groups. He termed his discovery the nucleal reaction, since it is a reaction specific for aldehydes based on the formation of a magenta-colored compound, when aldehydes react with fuchsin-sulfuric acid. The positive reaction occurs only in the presence of deoxyribonucleic acid (DNA) whereas yeast nucleic acid (RNA) did not give the same reaction.
In 1919, Feulgen defended his doctorate thesis on the state of the art of nucleic acid research, where he also made a critique of Steudel's staining technique. In the same year, after receiving his Doctor of Philosophy (PhD) degree, he was offered a research position in the Physiological Institute at Giessen, where his career gradually unfolded through successive promotions, until he was named director of the Physiological-Chemical Institute in 1931.
Feulgen further applied the nucleal reaction as a histochemical staining assay that confirmed that such reaction only takes place in the cell nuclei of both plants and animals. He was not able to obtain a positive nucleal reaction in pentose nucleic acid obtained from yeast cells, but he rightly inferred from several studies that the pentose nucleic acid (RNA) of yeast was localized in the cytoplasm. Feulgen exposed during the Congress of Physiology in Tübingen that year those results but his conclusions were met with general skepticism.
In the 1920s there was a common assumption that nucleic acids occurred under the form of thymonucleic acid (DNA) only in animals and as pentose nucleic acid (RNA) only in yeast and plants. Another study by Feulgen, in 1937, provided data contradicting such hypothesis. He isolated rye germ nuclei and succeeded in obtaining positive nucleal reactions, therefore proving that DNA is present in the cells of both plants and animals. He also inferred from these experimental studies with nucleal reaction that thymonucleic acid (DNA) in Eukaryotes was located at the nucleus whereas pentose or yeast nucleic acid (RNA) was located at cytoplasm. Although we now know that RNA may also be present in the nucleus, being synthesized there and then transported to the cytoplasm, Feulgen's conclusion was insightful and substantially correct right regarding the general localization of each type of nucleic acid in the cell.
In 1924, Feulgen and Voit discovered a positive nucleal reaction in the cytoplasm without first performing mild hydrolysis. They concluded that such outcome was due to the presence of aldehyde groups in the cytoplasm. Since a further test with the addition of lipid solvents was negative to nucleal reaction, they inferred that a lipid precursor was responsible by the first results obtained, and termed it plasmalogen. Four years later, they isolated the plasmalogen and in 1939, they were able to identify it as an acetal phosphatide.
Another important Feulgen contribution to the understanding of the chemical e molecular structure of DNA came in 1936 through his studies of the nucleic acids in their undegraded state, which led him to conclude that nucleic acids were in fact molecular polymers formed by
oligonucleotides, each comprised of the bases thymine, cytosine, adenine and guanine. Therefore, he also concluded that the extractive techniques utilized so far resulted in a mixture of depolymerized fragments of such a molecule. He studied the viscosity and the optical activity of the polymer and established then two experimental states of the DNA molecule, which he termed form A (undegraded molecule or polymer) and form B (depolymerized molecule). He also succeeded in converting form A to B by using a commercial pancreatic juice to digest the polymer into fragments. The enzyme present in the pancreatic juice, responsible by the depolymerization, was termed nucleogelase by Feulgen; and from the same
commercial juice DNA depolymerase was isolated some years later.
Although modern molecular geneticists now appreciate the great importance of Feulgen's pioneering studies, at his own time little attention was given to his experimental work. The Feulgen reaction is still in use as an effective staining technique in studies of chromosomes and in genetic cytochemistry assays.