Introduction Sanger sequencing, also known as the chain termination method, was developed by Frederick Sanger and his colleagues in 1977. This method revolutionized the field of molecular biology by allowing scientists to determine the precise order of nucleotides in DNA. Despite the advent of next-generation sequencing technologies, Sanger sequencing remains a gold standard for DNA sequencing due to its high accuracy and reliability.
Principle of Sanger Sequencing
The principle of Sanger sequencing is based on the selective incorporation of chain-terminating
dideoxynucleotides (ddNTPs) during DNA synthesis. These ddNTPs lack a 3' hydroxyl group, which prevents the formation of a phosphodiester bond, thereby terminating DNA strand elongation. Components DNA Template: The single-stranded DNA to be sequenced. Primer: A short single-stranded DNA that binds to the template and initiates DNA synthesis. DNA Polymerase: An enzyme that synthesizes new DNA strands by adding nucleotides to the primer. Deoxynucleotide Triphosphates (dNTPs): The building blocks of DNA. Dideoxynucleotide Triphosphates (ddNTPs): Modified nucleotides that terminate DNA synthesis. Steps Involved in Sanger Sequencing 1. DNA Template
Preparation The DNA of interest is extracted and purified from the source. This can be done using various methods such as chemical extraction, column-based extraction, or magnetic bead-based extraction. 2. Chain Termination PCR The DNA template is mixed with a primer, DNA polymerase, dNTPs, and a small amount of ddNTPs. The ddNTPs are labeled with fluorescent dyes, each corresponding to a different nucleotide (A, T, C, G). During the PCR reaction, the DNA polymerase incorporates dNTPs into the growing DNA strand until a ddNTP is incorporated, terminating the strand. 3. Separation of DNA Fragments The resulting DNA fragments of varying lengths are separated by size using capillary electrophoresis. The fragments are passed through a capillary filled with a gel matrix, and an electric field is applied to separate them based on size. As the DNA fragments pass through the capillary, a laser excites the fluorescent dyes attached to the ddNTPs. The emitted fluorescence is detected and recorded, allowing the determination of the DNA sequence. Advantages of Sanger Sequencing High Accuracy: Sanger sequencing has an accuracy of around 99.99%, making it ideal for applications requiring precise DNA sequence information. Long Read Lengths: It can produce read lengths of up to 1000 base pairs, which is useful for sequencing small genes and regions of interest. Validation: Often used to validate sequences obtained from next-generation sequencing technologies. Limitations of Sanger Sequencing Low Throughput: Sanger sequencing is not suitable for large-scale sequencing projects due to its relatively low throughput. Cost: It is more expensive and time-consuming compared to next-generation sequencing methods. Labor-Intensive: Requires multiple steps and careful handling of reagents and samples.
Applications of Sanger Sequencing
Clinical Diagnostics: Used for detecting genetic mutations and diagnosing genetic disorders. Research: Employed in various research applications, including gene cloning, mutation analysis, and comparative genomics. Forensics: Utilized in forensic science for DNA profiling and identification.
Recent Advances While next-generation sequencing technologies have largely replaced Sanger sequencing for large-scale projects, Sanger sequencing remains an essential tool for specific applications. Recent advances include the development of automated Sanger sequencing platforms, which have increased the efficiency and accuracy of the method.
References 1. [Sanger Sequencing: Principle, Steps, Applications, Diagram](https://microbenotes.com/sanger-sequencing/) 3. [Sanger Sequencing: Introduction, Principle, and Protocol](https://www.cd- genomics.com/blog/sanger-sequencing-introduction-principle-and-protocol/) 4. Sanger, F., Nicklen, S., & Coulson, A. R. (1977). DNA sequencing with chain-terminating inhibitors. Proceedings of the National Academy of Sciences, 74(12), 5463-5467. 5. Maxam, A., & Gilbert, W. (1977). A new method for sequencing DNA. Proceedings of the National Academy of Sciences, 74(2), 560-564.
