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What is DNA Sequencing? It Is Easier Than It Seems

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Posted August 1, 2018

Since the discovery of DNA in 1869, genetics as a science has been dramatically improving year after year. Though, DNA sequencing became possible only in a century.

DNA double helix. Image source: Max Pixel, CC0 Public Domain

Presently, when genomics as a branch of genetics is being popularized as one of the most promising vectors of human progress, we hear about DNA sequencing almost every day, though many of us have only a basic understanding of what it really is. In short, this is a process of determining the exact sequence of molecules within a single DNA molecule. The necessity of such procedure was stressed by Francis Crick, who believed that DNA sequencing has a potential to give the humanity a better understanding of biochemistry in general, and how living beings can be improved.

Possibly, the knowledge gained from experiments with human genome will help us find the answers to a question that has been haunting humanity for ages. The issues include, among others, the longevity of life, presently incurable diseases, and the expansion of human mental and physical capabilities.

Brief Outline of DNA Sequencing History

After Crick and Watson discovered the DNA structure, they put their effort into creating a theoretical basis that would allow scientists to understand the replication and transcription of DNA better. In spite of their, undoubtedly, tremendous efforts in that direction, it had been impossible to define the precise sequence of nucleotides (organic molecules) within a single DNA fragment for nearly two decades. The main difficulty lied in the fact that nucleotides are very similar to each other in their composition that consequently complicated the process of their analysis as well as that one of DNA.

DNA model built by Crick and Watson in 1953, on display in the National Science Museum of London. Image credit: Alkivar via Wikimedia, Public Domain

The breakthrough happened when the methods of analytical chemistry combined with the methods of selective processing of RNase while considering the features of nucleotide base of RNA. As a result, in 1965, Robert Holly and his research group “read” the sequence of tRNA. They used the sequencing method of Frederick Sanger, which is based on the detection of partial fission fragments marked with a radioactive isotope. Using the same method, in 1972, in the laboratory of Walter Fiers were able to dismantle the first complete sequence of the coding protein of the MS2. Four years later, it was possible to carry out the sequencing of its full genome.

The event mentioned above signified the beginning of the real history of DNA sequencing, which was also contributed to by the developed method of rectification of the genome of Bacteriophages (a kind of a virus that replicates with other bacteria when infecting an organism). Ray Wu and Dale Kaiser used the observation of complementarity of nucleotide bases. They took the DNA polymerase, marking the ends with radioactive nucleotides. Then they picked up the complementary nucleotides for the sequence. All this happens shortly before this method is finalized, and researchers will be able to determine the order of nucleotides on any part of the chain. However, the definition of the grounds was still limited to short DNA segments, and the research report included a large number of analytical chemistry methods and fractionation procedures.

The next step was to replace the 2d fractionation with electrophoresis in the polyacrylamide gel. This method was used in 1970 in the “Plus/Minus” method of Alan Coulson and Frederick Sanger, as well as in 1975 in the method of chemical cleavage of Allan Maxam and Walter Gilbert. It should be noted that, despite the use of polyacrylamide gel in both cases, the methods are different.

The Maxam-Gilbert sequencing DNA method involves splitting the polynucleotide chain into fragments using chemical fission. Splitting is carried out on certain bases of nucleotides; therefore after “a break” in a gel and determination of the length of fragments, it is possible to determine the position of the tagged nucleotides. Moreover, eventually, collect the DNA sequence.

However, the first complete sequencing of the DNA of bacteriophage φx174 was carried out by Sanger. Sanger’s method played a significant role in the further development of DNA sequencing; though, both methods are the ancestors of present-day methods utilized in the field of Genomics.

Even though Sanger’s method is quite expensive, it has managed to become the widely-acceptable standard of reliability. Consequently, the technique was further refined to correspond to modern challenges with greater ease. First, the radioactive label was replaced by fluorescent, and, secondly, the methodology of capillary electrophoresis was introduced. All this allowed automating the process by introducing technical devices for sequencing DNA.

However, the first-generation of DNA sequencing machines were not allowed to analyze long fragments of the genome, so a “shotgun method” was developed. The approach implied cloning of DNA fragments, which were then assembled into silicone and then – into one long continuous sequence. Finally, Leroy Hood invented ABI Prism sequencer that allowed for the sequencing of more complex genomes.

The development of DNA sequencing resulted in the launch of the human genome project. Two teams of scientists had been working on the project for over a decade, and their results are worth more than 3 billion US dollars. The event signified the beginning of the new era, the era of modern genomics.

Even though it is already possible to acquire a full copy of a person’s genome, it is still being decoded. The results promise to be revolutionary, as personal medicine will be possible meaning that every individual will have a chance to get medication or treatment that is specially designed for their genome; thus having the most effective impact on their organism, but most importantly, the deciphered genome will let us reach the new stage of evolution a lot quicker!

The article was prepared by Genomics Science Group

References: Heather, J. M. and Chain, B. (2016). The sequence of sequencers: The history of sequencing DNA. Genomics. DOI: 10.1016/j.ygeno.2015.11.003

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