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Molecular Pathology

DNA sequencing


Author: Rodney E. Shackelford, D.O., Ph.D., Tulane University (see Reviewers page)
Revised: 24 November 2010, last major update November 2010
Copyright: (c) 2008-2010, PathologyOutlines.com, Inc.


● Nucleic acid sequencing is the determination of the nucleotide base order (adenine, cytosine, guanine, uracil and thymine) in a DNA or RNA molecule


Nucleotide bases and gel for sequencing

References: Wikipedia-nucleotide, Wikipedia-DNA sequencing

Initial sequencing

● The first nucleic acid sequencing began in the mid-1960’s using 2-dimensional chromatography
● The intial protocols were innovative, but inefficient by today's standards
● For example, in 1973 Gilbert and Maxam published the sequence of the 24 bp lac operator using a protocol that required 300-700 grams of bacteria, multiple purification steps, conversion of the selected DNA fragments into RNA and digestion of these sequences with different RNases (Proc Natl Acad Sci USA 1973;70:3581)

Several properties of DNA made the initial attempts at DNA sequencing difficult:
● (1) The chemical properties of different DNA molecules were so similar that separating them appeared difficult
● (2) Compared to amino acids in proteins, DNA was much longer
● (3) No base-specific DNases were known and previous protein sequencing methods had depended upon proteases that cut adjacent to specific amino acids
● Since structurally simpler RNA molecules did not have these drawbacks, they were among the first larger nucleic acids sequenced; the first relatively large nucleic acid sequenced was the Esherichia coli alanine tRNA in 1965 (Science 1965;147:1462, Nucleic Acids Res 2007;35:6227)

Later efforts

● In the 1970’s, Sanger and Maxam-Gilbert developed chain termination and base-specific chemical cleaving methodologies that overcame many of the initial problems, vastly improving sequencing efficiency, especially when applied to DNA
● Since this time, improved sequencing methods, combined with automated analysis and development in bioinformatics, has led to doubling of the known nucleic acid sequences every 16 months for the past 40 years, a logarithmic nine orders of magnitude database increase since 1965

Recent techniques

● Many new “next generation” DNA sequencing technologies and platforms are currently in development and the speed, efficiency, cost-effectiveness and accuracy of DNA sequencing technology has been steadily improving
● Since the early 1990’s, most have employed the Sanger dideoxynucleotide chain-terminating method, although other DNA sequencing technologies improvements are being developed


● The enormous increase in the ability to sequence nucleic acids has allowed the complete sequencing of the human genome and the genomes of over 180 other species, including:
   - Microorganisms: Bacillus anthracis, Caenorhabditis elegans, Helicobacter pylori, Mycobacterium tuberculosis, Saccharomyces cerevisiae, Yersinia pestis
   - Animal kingdom: chicken (Gallus gallus), chimpanzee (Pan troglodytes), dog (Canis familiaris), fruit fly (Drosophila melanogaster), honey bee (Apis mellifera), Japanese puffer fish (Takifugu rubripes), mouse (Mus musculus), rat (Rattus norvegicus ), sea squirt (Ciona intestinalis)
● In addition, the woolly mammoth (Mammuthus primigenius) genome and a “first draft” of the Neanderthal (Homo sapiens neanderthalensis) genome have been published, demonstrating that the DNA sequences of long extinct complex organisms can be sequenced from fossil sources
● DNA sequencing is used in many areas, including forensics, disease diagnosis, personalized medical (pharmacogenomics), tissue identification, transplantation typing, biotechnology, epidemiology, medical research, comparative genomics and evolution, archeology and anthropology
● DNA sequencing has also raised many important bioethical issues related to personal privacy, public health and safety

End of Molecular Pathology > DNA sequencing > History

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