Sanger sequencing
Sanger sequencing is a widely used method for determining the DNA sequence of an organism, which involves identifying the specific arrangement of chemical bases that encode hereditary information. Developed by Nobel Prize-winning scientist Frederick Sanger in 1977, this method marked a significant advancement over earlier sequencing techniques. The process begins by unwinding the DNA double helix using heat, followed by the addition of free nucleotides and a DNA polymerase enzyme to synthesize new DNA strands. Special chain-terminating nucleotides are incorporated, resulting in DNA fragments of varying lengths.
These fragments are then sorted using gel electrophoresis, where they are separated based on size. Following this, scientists examine the sorted strands under X-ray or UV light to determine their nucleotide composition. In more modern adaptations, fluorophores are used to enhance visualization, allowing for faster analysis with the help of computer technology. While newer sequencing methods have emerged, Sanger sequencing remains a popular choice due to its reliability and speed, enabling comprehensive DNA analysis in a matter of days instead of years. This technique continues to play a crucial role in genetic research, including studies related to cancer and various genetic disorders.
On this Page
Sanger sequencing
Sanger sequencing is a common method for sequencing the DNA of an organism. Sequencing DNA refers to determining the specific patterns of chemicals that make up the hereditary information found in all living organisms. When it was developed and introduced, the Sanger method was a significant improvement over the original method for sequencing DNA. Over time, it has been improved by using fluorophores and computer assistance. Modern scientists working with the Sanger method can sequence the DNA of an organism over several days instead of the years required in the past.
The Sanger method begins by using heat to unbind the DNA from its double helix pattern. Scientists then introduce new chemicals and catalysts to the mix, encouraging the DNA to bind with them and creating strands of DNA of many different lengths. These strands are sorted using gel electrophoresis. Once the strands have been sorted by length, they are carefully examined by scientists to determine the pattern of the nucleotides. This pattern may be observed by viewing the strands under a UV light, the use of X-rays, or the use of specialized lasers.
Background
Deoxyribonucleic acid, also called DNA, is the hereditary material found in most living things. Almost every cell contains DNA, most of which is in the cell’s nucleus. Some DNA may be found in the mitochondria, which are cell organelles that generate chemical energy for the cell. DNA is made of four chemical components: adenine, guanine, cytosine, and thymine. These bases are arranged in a specific sequence that acts as a set of information for the body. The bases are arranged in the characteristic double-helix pattern associated with DNA.
During the process of cell division, new copies of a body’s DNA are constructed. This allows the body to supply new cells to reference the DNA, which lets them follow the blueprints laid out by its chemical components. Most members of the same species have extremely similar DNA. For example, two humans share roughly 99 percent of genetic data. All the hereditary, biological differences between two people can be found in the remaining 1 percent.
Because DNA is important to understanding the body, many researchers worked to develop DNA sequencing. DNA sequencing is the process by which scientists determine the precise sequence of the base chemicals. Because of the immense amount of information found in an organism’s complete genome, most sequencing projects focus on small segments of the genome. As new methods for DNA sequencing have been developed, it has become more practical for scientists to quickly break down an organism’s genome into a format that can be studied.
Because scientists can study DNA sequences much more easily, researchers may be able to develop new treatments based on the human genome, due to increased understanding of the link between genetics and cancer. For example, the Cancer Genome Atlas Project uses DNA sequencing to study the genetic markers that may lead to the development of cancer and the role of genes in the regulation of cancer. Other medical researchers are using DNA sequencing to study the role of genes in heart diseases, diabetes, and many developmental disorders.
Overview
Sanger sequencing is the second method of DNA sequencing to be developed. It was created by the Nobel Prize-winning scientist Frederick Sanger and was first used by the scientific community in 1977. Since its creation, faster and cheaper methods for DNA sequencing have been developed by scientists. However, Sanger sequencing is still commonly used.
Sanger sequencing is a complex process that must be carried out properly to yield valuable data. It begins with scientists isolating the section of DNA that they hope to sequence. The DNA is heated, causing the double helix shape to unwind into two single strands. Once the DNA unwinds, scientists lower the temperature and add chemicals called free nucleotides. These nucleotides contain one of the four chemical bases that make up DNA. Scientists also add a DNA polymerase, an enzyme that speeds up chemical reactions.
Scientists again heat the DNA. When it reaches the correct temperature, they add chain-terminating nucleotides to the mixture. These nucleotides are similar to the nucleotides that make up traditional DNA. However, they have been carefully modified so that once bound, the chemical reaction stops. These elements are randomly distributed throughout the DNA and incorporated into the process as new strands of DNA are created within the mixture.
Whenever a chain-terminating nucleotide is used in a chemical reaction, the strand of DNA that was forming is stopped. Scientists repeat this process, continuing to add chain-terminating nucleotides until all strands of DNA have incorporated chain-terminating nucleotides. This results in the creation of many strands of different lengths. Scientists use a process called gel electrophoresis to sort these segments. This process involves adding pieces of DNA to a gel base, then exposing that base to controlled amounts of electricity. Smaller DNA segments move the farthest, while larger DNA segments may not be able to move through the gel at all.
Once the segments of DNA have been sorted, scientists examine the strands under an X-ray or UV light. This allows scientists to see which nucleotides make up different parts of the DNA segments. In some instances of Sanger sequencing, scientists add fluorophores to each type of nucleotide. Fluorophores give off a specific type of light. Scientists working on DNA sequencing can then read the results by the colors they give off when a laser is shined through the segments. This process is much faster than reading the results by X-ray or UV light.
When coupled with computers, the Sanger method has become a fast and reliable method for sequencing DNA. In the past, it took scientists years to sequence the genome of a single organism. However, scientists using the Sanger method with the assistance of computers can sequence the entirety of a DNA sample in just days.
Bibliography
“DNA Sequencing.” Khan Academy, 2021, www.khanacademy.org/science/high-school-biology/hs-molecular-genetics/hs-biotechnology/a/dna-sequencing. Accessed 4 June 2021.
“DNA Sequencing Fact Sheet.” National Human Genome Research Institute, 2021, www.genome.gov/about-genomics/fact-sheets/DNA-Sequencing-Fact-Sheet. Accessed 4 June 2021.
“Frederick Sanger.” Nobel Prize, 2021, www.nobelprize.org/prizes/chemistry/1958/sanger/biographical/. Accessed 4 June 2021.
“Genome Sequencing.” Genome News Network, www.genomenewsnetwork.org/resources/whats‗a‗genome/Chp2‗1.shtml. Accessed 4 June 2021.
“Sanger Sequencing.” Let’s Talk Science, 17 Aug. 2021, letstalkscience.ca/educational-resources/backgrounders/sanger-sequencing. Accessed 4 June 2021.
Smith, Yolanda. “DNA Sequencing.” Medical Life Sciences, www.news-medical.net/life-sciences/DNA-Sequencing.aspx. Accessed 4 Jun. 2021.
“What Is DNA?” MedlinePlus, 2021, medlineplus.gov/genetics/understanding/basics/dna/. Accessed 4 June 2021.
“What Is Sanger Sequencing?” Sigma Aldrich, 2021, www.sigmaaldrich.com/technical-documents/articles/biology/sanger-sequencing.html. Accessed 4 June 2021.