DNA Sequencing

Summary

DNA (deoxyribonucleic acid) sequencing is a technique used to determine the order of the nitrogenous bases (adenine, guanine, cytosine, and thymine) that make up a gene, DNA molecule, or entire genome. Genome sequencing has been completed for many organisms, including animals, plants, and humans. Applications of this technology can advance the understanding, diagnosing, and treatment of disease; enable personalized healthcare; and produce innovative techniques that can be used in forensics, agriculture, and archaeology.

Definition and Basic Principles

DNA sequencing is a laboratory technique that allows scientists to determine the structure of DNA at its highest level of resolution. DNA is a double-stranded helix made of building blocks called deoxyribonucleotides. Deoxyribonucleotides are nucleotides that contain the deoxyribose sugar as well as a phosphate group and a nitrogenous base. The information in DNA is stored as a code made up of the nitrogenous bases adenine (A), guanine (G), cytosine (C), and thymine (T).

DNA sequencing has a number of applications that may revolutionize medicine, agriculture, anthropology, and archaeology. The prospective uses of these applications include personalized medicine, the decoding of genes, and the identification of mutations causing genetic diseases. Sequencing, coupled with genetic engineering and agriculture, has produced plants with improved nutritional quality, greater resistance against insects, and better ability to withstand poor soil and drought. Sequencing of entire genomes has been completed for several organisms, including extinct species. This has provided extensive insight into human migration and the evolution of all living organisms.

Despite the prospective and generated benefits of DNA sequencing, the use of this technique has raised ethical, legal, and social questions. Concerns exist regarding genetic determinism and discrimination, the manipulation of an individual's attributes, the loss of genetic privacy, and the modification of food. Programs have been created in response to these concerns.

Background and History

In 1953, James D. Watson and Francis Crick proposed the double-helical structure of DNA. This discovery has since yielded revolutionary insights into the genetic code and protein synthesis. However, because of certain properties of DNA, it took about fifteen years before the first sequencing experiments were completed.

The first nucleic acid to be sequenced was yeast alanine transfer RNA (tRNA) because of its size and availability for purification. Following this accomplishment, scientists began to purify genomes of bacteriophages and pursue sequencing. However, whole genome sequencing did not become an actuality until after the discovery of restriction enzymes by Hamilton Smith, Werner Arber, and Daniel Nathans in 1970 and the development of more modern methods of DNA sequencing by Frederick Sanger and Alan R. Coulson in 1975. In 1977, the first genome, belonging to the bacteriophage phiX174, was sequenced.

The increasing amount of information created a need for computer programs capable of compilation and analysis of DNA, followed by databases with rapid searching programs (such as Genbank, created in 1982). These developments, as well as several advances in laboratory techniques (such as automated sequencing), led to the completion in 1998 of the first genome for an animal, a nematode called Caenorhabditis elegans, and ultimately the complete mapping of the human genome in 2003.

How It Works

Plus and Minus Method. Before the development of direct DNA sequencing, DNA had to be converted into RNA, sequenced, and then decoded. In 1975, Sanger and Coulson introduced plus and minus sequencing, the first direct DNA sequencing method. Plus and minus sequencing begins with several asynchronous cycles of DNA synthesis with radioactively labeled deoxynucleoside triphosphates (dNTPs). The asynchronous cycles lead to an array of DNA fragments varying in nucleotide length (1, 2, 3, … 100). Products are separated into eight containers, and dNTPs are added. However, each container receives either one of the dNTPs (the plus reactions) or three of the four dNTPs (minus reactions). This allows for the termination of synthesis in a sequence-specific manner. The products are separated via electrophoresis on a polyacrylamide gel. Subsequently, the gel is exposed to X-ray film that results in a series of bands corresponding to the radiolabeled DNA fragments, allowing the sequence to be constructed. Although this method revolutionized how sequencing was completed, it was inefficient, and therefore, other techniques were developed.

Maxam and Gilbert Method. In 1977, Allan Maxam and Walter Gilbert developed a sequencing method that replaced the plus and minus method. This method was similar, as it required a radioactive label, gel electrophoresis for fragment separation, and the use of X-ray autoradiography for product visualization and inference of the sequence. However, the method differed by allowing the direct analysis of purified double-stranded DNA and using another way of creating products ending in a specific nucleotide.

In the Maxam and Gilbert method, the double-stranded DNA is radioactively labeled, cut with restriction enzymes, and denatured. Subsequently, the DNA is treated chemically in four separate reactions, which cut DNA at different nucleotides. The first reaction, called the A+G reaction, cuts the nucleotides adenine (A) and guanine (G). The second reaction, called the G reaction, cuts at G. The third and fourth reactions are similar but involve cytosine (C) and thymine (T) in the C+T and the C reaction. The products of these four reactions are run through gel electrophoresis in four adjacent wells and analyzed for the sequence. The G reactions determine the placement of G, the A+G reactions determine the location of A, and so forth. This method is rarely used in the twenty-first century.

Sanger Sequencing Method. Named after Sanger, this method was developed in December 1977 by Sanger and Gilbert. Because of its efficiency and limited use of chemicals and radioactivity, it became the most widely used technique. This method takes advantage of dideoxynucleoside triphosphates (ddNTPs), analogs of the dNTPs. The ddNTPs are nucleotides lacking the 3'-hydroxyl function on their deoxyribose sugar required to form phosphodiester bonds between two nucleotides of a developing DNA strand. Therefore, the ddNTPs are used during the synthesis of DNA strands to terminate DNA extension, resulting in products of different lengths.

Originally, this method required four separate reactions. Each aliquot contained a template DNA primed with an oligonucleotide, DNA polymerase to extend the sequence, and dNTPs. Each reaction received one of the chain-terminating ddNTPs labeled radioactively for detection. Later, sequencing could be completed in one reaction by substituting the radioactive label with four unique fluorescent dyes corresponding to each ddNTP. Further progress was made in 1983 when Kary Mullis introduced polymerase chain reactions (PCR), which allowed target sequences of DNA to be amplified in a fraction of the time. After PCR, the strands are separated and analyzed with automated sequencers, resulting in a chromatogram with a series of four-colored peaks representing each of the DNA bases. Computers are used to assemble sequences and analyze them for a variety of characteristics.

Applications and Products

Genetic Diagnostics. The increased knowledge of genes and the organization of the genome has had a significant impact on medicine. Any disorder is caused by a combination of the environment and genetics. However, the role of genetics may be large (as in Huntington's disease) or small (as in diabetes). Sequencing has helped elucidate the genetic variation and mutations responsible for predisposing a person to disease, modifying the course of a disease, or causing the disease itself. Understanding the molecular mechanisms of disease allows for the development of tests, diagnoses, treatments, and even cures and preventative options.

Personalized Medicine. Medicine is moving in the direction of using specific treatments based on patients' individual attributes, a development termed personalized medicine. Although personalized treatments are being developed in many medical fields, the most striking examples are in oncology. For example, physicians are measuring the levels of human epidermal growth factor receptor 2 (HER2) in patients with breast cancer, and if the test is positive, the person is treated with trastuzumab.

Disease Control. DNA sequencing can also play a key role in helping to prevent or at least slow the spread of some infectious diseases. For example, during the global COVID-19 pandemic in the early 2020s, scientists turned to DNA sequencing as a method for studying the coronavirus responsible for the disease. The data yielded through this research helped public health officials develop responses to the crisis and adapt those responses as needed when new virus variants emerged.

Agriculture. Sequencing the genomes of plants and animals has made it possible to create transgenic organisms, which incorporate desired characteristics from other organisms. For example, genes from the bacterium Bacillus thuringiensis have been successfully transferred to crops such as rice, cotton, corn, and potatoes, thereby producing plants that are protected from insects. The alteration of genomes has also produced plants that resist drought and disease. Golden Rice, a genetically modified strain of rice, contains high levels of beta-carotene, which is converted to vitamin A in the human body. This rice has the potential to fight vitamin A deficiency in less-developed countries. Interestingly, bananas have also been modified to produce human vaccines against diseases such as hepatitis B.

Comparative/Evolution. The ability to sequence DNA has led to the study of the evolution of all forms of life, including humans. Since sequencing began, genomes have been mapped for innumerable organisms, including chimpanzees, mice, fish, fruit flies, plants, yeasts, bacteria, and viruses. The Human Genome Project, completed in 2003, identified 20,000 to 25,000 genes and about 3 billion bases in humans. DNA sequencing has also been completed on ancient DNA from clinical, museum, archaeological, and paleontological specimens. These data have greatly increased knowledge of genetic variation, thus increasing understanding of human differences, similarities, evolution, and origins.

Microbial Genomics. In 1994, the US Department of Energy (DOE) launched the Microbial Genome Project. Scientists realized that these organisms, with their ability to withstand extremes of temperature, radiation, acidity, and pressure, provided an excellent resource for the development of applications of renewable energy production, environmental cleanup of toxic waste, management of environmental carbon dioxide, and industrial processing of antibiotics, insecticides, and enzymes. Although this project ended in 2005, the DOE continued its research in the Genomic Science Project and the Joint Genome Institute's Community Sequencing Program.

Biological Weapons. Although highly controversial, genetic sequencing has led to the development of materials for biowarfare. One example is invisible anthrax, which was developed by introducing a gene that altered the immunological properties of the microorganism Bacillus anthracis. Access to the DNA of virulent agents and strains is regulated and restricted, thus preventing the introduction of genes to create novel properties. However, with the advancement of microbiology, it is becoming increasingly possible to synthesize agents artificially. In 2002, the poliovirus was synthesized using only the information of the genetic sequence.

Careers and Course Work

Students interested in pursuing careers involving genomic research must take a cross-disciplinary approach. Students should develop a solid background in science, including biology, chemistry, physics, and mathematics, at the undergraduate level. Pursuing higher degrees in the basic sciences or combining studies of journalism, law, computer science, anthropology, archaeology, bioethics, medicine, and pharmaceuticals can result in further specialization. Although higher education, including a master's degree, a medical degree, or a doctorate, is required for many advanced research positions in academic institutes or industries, opportunities are available for individuals without advanced degrees. Careers may take several paths, including medicine, public health, pharmaceuticals, agriculture, computer science, engineering, business, law, history, archaeology, and anthropology. Individuals interested in medicine can pursue careers as genetic counselors, medical geneticists, or genetic nurses. Pharmacy students with backgrounds in genetics can pursue innovative research and development of personalized medicine and pharmacogenomics. Scientists interested in agriculture may be involved in the genetic modification of food. Other possibilities include programming and maintaining DNA databases, marketing and promoting new technologies, paternity testing, and forensic science. Many of the positions in genetics are likely to be in research into evolution, diagnostic testing, and development of the proteomes and HapMaps.

Social Context and Future Prospects

DNA sequencing has come a long way since its inception in 1975. Enormous accomplishments have created new careers, research, developments, and innovative applications. Armed with the blueprint of life, scientists have begun working to unlock some of biology's most intricate and complex processes, including determining how a human develops from a single cell, how genes regulate the functions of organs and tissues, and what is involved in the preposition of disease.

Completing the Human Genome Project and initiating projects such as the International HapMap Project have demonstrated the commitment of government and society to understanding the nature and role of genetics. However, DNA sequencing and genetic engineering developments have also raised profound ethical and social concerns. The heightened ability to determine an individual's genetic profile has raised concerns about confidentiality and privacy, possible stigmatization, negative consequences in the areas of employment and insurance, and the psychological effects of knowing one's predisposition to diseases and conditions. The commercialization of DNA products is another area of concern. In response, many US governmental agencies, such as the National Institutes of Health, have created bioethics programs. The Ethical, Legal, and Social Implications (ELSI) Research Program, part of the National Human Genome Research Institute (NHGRI), supports research on the ethical, legal, and social implications of genetics research. Other programs have included a bioethics component.

The NHGRI Genome Technology Program and its Advanced DNA Sequencing Technology awards supported extensive DNA sequencing automation and technology advancements in the early twenty-first century. These allowed scientists to quickly and routinely sequence individual genes relatively cheaply. By the mid-2020s, the cost of sequencing an entire genome was around UDS$2,000.

Bibliography

Brown, Stuart M. Next-Generation DNA Sequencing Informatics. 2nd ed., Cold Spring Harbor Laboratory Press, 2015.

Brown, T. A. DNA Sequencing. IRL P at Oxford UP, 1995.

Brown, T. A. Gene Cloning and DNA Analysis: An Introduction. 8th ed., Wiley, 2021.

Dash, Hirak Ranjan, Kelly M. Elkins, and Noora Rashid Al-Snan. Next Generation Sequencing (NGS) Technology in DNA Analysis. Academic Press, 2024.

"DNA Sequencing Fact Sheet." National Human Genome Research Institute, US National Institutes of Health, 27 June 2023, www.genome.gov/about-genomics/fact-sheets/DNA-Sequencing-Fact-Sheet. Accessed 20 June 2024.

Hudson, Andre and Crista Wadsworth. "Genomic Sequencing: Here's How Researchers Identify Omicron and Other COVID-19 Variants." The Conversation, 20 Dec. 2021, theconversation.com/genomic-sequencing-heres-how-researchers-identify-omicron-and-other-covid-19-variants-172935. Accessed 11 Jan. 2022.

Janitz, Michal. Next-Generation Genome Sequencing: Towards Personalized Medicine. Wiley-VCH, 2008.

Jones, Martin. “Archaeology and the Genetic Revolution.” A Companion to Archaeology. Edited by John Bintliff, Timothy Earle, and Christopher S. Peebles, Blackwell, 2004.

Lynch, April, and Vickie Venne. The Genome Book: A Must-Have Guide to Your DNA for Maximum Health. Sunrise River, 2009.