Sanger method

Sanger sequencing, also known as the chain-termination method, is a technique for DNA sequencing developed by Frederick Sanger and his colleagues in 1977. This method was a significant breakthrough in the field of genetics, allowing scientists to determine the precise order of nucleotides in a segment of DNA. It has played a crucial role in the advancement of biotechnology and has been instrumental in various scientific discoveries and applications, including the Human Genome Project.

Overview

Sanger sequencing involves the selective incorporation of chain-terminating dideoxynucleotides (ddNTPs) during DNA replication. These ddNTPs are similar to the normal nucleotides that make up DNA, but they lack a hydroxyl group on the 3' carbon, preventing further extension of the DNA chain once incorporated. By using a mixture of normal nucleotides and a small proportion of ddNTPs, it is possible to generate DNA fragments of varying lengths, each ending with a ddNTP. The sequence of the original DNA strand can then be determined by analyzing the lengths of these fragments.

Procedure

The Sanger sequencing process involves several key steps: 1. DNA Fragmentation: The DNA to be sequenced is fragmented into smaller pieces. 2. Primer Annealing: A short DNA primer is attached to the template strand to initiate replication. 3. Extension/Chain Termination: A DNA polymerase enzyme is used to synthesize a new DNA strand by adding nucleotides to the primer. This reaction mixture contains a mixture of normal deoxynucleotides (dNTPs) and a small proportion of ddNTPs. Each ddNTP is labeled with a distinct fluorescent dye, allowing for the identification of the terminating nucleotide of each fragment. 4. Electrophoresis: The resulting DNA fragments are separated by size using gel electrophoresis. The smallest fragments move fastest and furthest on the gel. 5. Data Analysis: A laser and detector system reads the fluorescent signals as the fragments pass by, allowing the sequence of the original DNA strand to be reconstructed based on the order of termination events.

Applications

Sanger sequencing has been utilized in a wide range of genetic research and diagnostic applications. It is particularly useful for sequencing small to medium-sized DNA fragments (up to 900 base pairs). Key applications include: - Genotyping - Mutation detection - Phylogenetics - Functional genomics - Microbial identification

Despite the development of newer, high-throughput sequencing technologies, Sanger sequencing remains a gold standard for the accurate and reliable determination of DNA sequences, especially for clinical and diagnostic purposes.

Limitations

While highly accurate, Sanger sequencing has limitations in terms of throughput and scalability. It is relatively slow and costly for sequencing large genomes compared to next-generation sequencing (NGS) technologies. Additionally, the method requires a significant amount of template DNA and is less effective for sequencing repetitive regions or regions with secondary structures.

Conclusion

Sanger sequencing has been a cornerstone of molecular biology and genetics research for decades. Its development marked a pivotal moment in the history of science, enabling countless discoveries and innovations. Despite the advent of more advanced sequencing technologies, the Sanger method continues to be an essential tool in various research and diagnostic settings.

Sanger method Resources
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