Thursday 21 November 2013

Frederick Sanger The Father Of DNA Sequencing

 
Frederick Sanger, an outstanding English Biochemist has laid the foundation for the field of Genomics. The field of science which has opened a Pandora box and we continue to explore for decades to come. His technique of protein sequencing has provided the scientists with tools to decode the sequence of any protein in the body and led to the development of new drugs like the Human Growth Hormone. Fred Sanger a two times Nobel Prize winner and Pioneer of DNA sequencing died at the age of 95 in Britain on November 19th. As of 2013, he is the only person to have received the Nobel Prize two times in Chemistry. Other scientists who have received two Nobel Prizes are John Berdeen for Physics (1956 and 1972), Marie Curie (for Physics in 1903 and Chemistry in 1911), Linus Pauling (for Chemistry 1954 and Peace 1962).

Sanger was born on August 13th 1918 in a family of Quakers in Gloucestershire, England. His father was a medical practitioner. Both his father and older brother Theodore have been a great influence on young Sanger for his interest towards biology, science and the scientific method. At school and all through his initial days of college he was above average but not outstanding. He wanted to pursue a career in Medicine but then decided against it as he would see his father visit patient after patient every day. Instead he thought he would be suited for career where he could concentrate all his activities and interests on a single goal. During that time Biochemistry was recently introduced in the Cambridge University and he got interested in it when he first heard about it through Ernst Baldwin. It was the time of Second World War but he could obtain an exemption from the military service as a conscientious objector.

Sanger began his PhD under A. Neuberger 1940 on lysine metabolism and a more practical problem concerning the nitrogen of Potatoes. He was awarded the Doctorate Degree in 1943. He started working under A.C. Chibnall who succeeded F.C. Hopkins, Professor in the Department of Biochemistry, Cambridge University and started working on proteins, especially Bovine Insulin. Till 1943 he has to support himself on his own. From the year 1944 till 1951 he received Beit Memorial Fellowship for Medical Research. After that he became staff member of Medical Research Council. This was most exciting period for working on Protein Chemistry as several fractionation and Chromatography techniques were developed. These techniques helped in determining the fundamental components of living matter. What was known before he sequenced the composition of the Insulin protein is that proteins are made up of amino acids.

Sanger’s first triumph came when he sequenced Insulin protein and revealed that it is made of two polypeptide chains A and B linked by three disulphide bonds. Till then proteins were considered as amorphous substances. He used a chemical fluorodinitrobenzene (FDNB) also called Sanger’s Reagent to determine the composition of the polypeptide chains. Using Sanger’s technique scientists have sequenced several proteins. This approach of Protein sequencing has earned him Nobel Prize for Chemistry in 1958. This discovery has been crucial for the sequence hypothesis proposed by Crick later, which states that sequences of bases (A, G, T, C) in the genetic material determine the sequence of amino acids of a particular protein. The Nobel prize has helped him to work with more enthusiasm and interest as finding funding, facilities and collaborators became little easy for him.

In 1962, Sanger has moved to Laboratory of Molecular Biology in Cambridge where he contemplated the possibility of sequencing RNA using specific nucleases (enzyme which chew nucleic acids). He developed several methods for sequencing small sequences of RNA. By 1965, Robert Holley of Cornell University for the first time sequenced the 77 ribonucleotides of Alanine tRNA in Saccharomyces cerevisiae.

 Sanger then took up the task of sequencing the DNA. Scientists knew that like proteins, DNA is made up of long chain of nitrogenous bases (Adenine, Guanine, Thymine and Cytosine or simply ATGC). The order of these bases has to be determined to know the sequence of the DNA template. In 1975, with Alan Coulson he published the sequence of DNA using DNA Polymerase using the radiolabelled nucleotides. This technique was called “Plus and minus” technique. This was an improvement over earlier methods as it could sequence 80 nucleotides at a single stretch but was very laborious.  Later he came up with a more fool proof, relatively rapid and stringent method which became popular as Dideoxy chain termination method or Sanger method for sequencing DNA molecules. In 1977, he decoded the sequence of ΦΧ bacteriophage174 (5375 nucleotides), human mitochondrial DNA (16,338 nucleotides) and of bacteriophage λ (48,500 nucleotides). This Sanger method of sequencing has increased the efficiency of sequencing DNA by over thousand times. This discovery has earned him the second Nobel Prize in Chemistry in 1980, which he shared with Walter Gilbert and Paul Berg.

Fred Sanger with his colleagues has pioneered the use of thin gels to provide for resolution of DNA bases and 30 years back he has developed the first shot gun genome sequence of the bacterial virus lambda. Even now, scientists around the World use this technique to attack the genome 3,000,000,000 base pairs long. He retired in 1983. He was awarded Order of Merit by the British Government but declined the honour of Knighthood as he didn’t want be addressed as “Sir”. He worked in laboratory throughout his entire career and preferred to do the work on his own instead of assigning it to juniors which is a common precedent in most of the labs. He has little aptitude towards administration or teaching.

Sequencing is the only technique which enables us to decode the genetic information stored in DNA. Since Sanger method is amenable for computer automation, it has allowed scientists to decipher more complicated genomes. The contributions made by Sanger are of par excellence- as the sequencing techniques developed by him have enabled the scientists to determine the building blocks of proteins and DNA. His research has allowed us to understand the language of life. In lieu of his contributions towards the science of genomics, Wellcome Trust has named their institute after him in the year 1993 that specialises in understanding Genomics. His contributions have opened up the field of Molecular Biology, Genetics and Genomics. The modern day biological science is dominated by Genomics which has revolutionised the field of biomedical sciences with its transformative improvements in healthcare. “He is the one the most important scientists of 20th century. He twice changed the direction of scientific world” recalled, J. Craig Venter, the scientist who first sequenced the human genome.

In a review published in Annual Reviews of Biochemistry in 1988 he commented, “Of the three activities involved in scientific research, thinking, talking and doing I much prefer the last and am probably best at it. I am right at thinking, but not much good at talking”.
 
It is my personal tribute to the great Biochemist, whose sequencing technique has formed the backbone of my thesis work and without which several of my observations would have been confounded. The thousands of the gels which I ran day in and day out remind of the veracity of the Nobel Prize winning discovery.
 
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