Sanger sequencing

Definition

Sanger sequencing is a method for determining the precise order of nucleotides in a DNA molecule, developed by Frederick Sanger in 1977. This technique relies on selective incorporation of chain-terminating dideoxynucleotides during DNA replication, which allows for the generation of fragments that can be analyzed to deduce the sequence. Sanger sequencing has played a foundational role in genome sequencing projects and remains an important technique within DNA sequencing technologies.

5 Must Know Facts For Your Next Test

1. Sanger sequencing was the first widely adopted method for sequencing DNA and is often referred to as the 'chain termination method.'
2. The process involves four separate reactions, each containing a different dideoxynucleotide labeled with a distinct fluorescent dye, which allows for the identification of each base in the sequence.
3. This method has an accuracy rate exceeding 99.9%, making it highly reliable for sequencing short lengths of DNA.
4. Although more advanced techniques like next-generation sequencing have emerged, Sanger sequencing remains essential for validating results and sequencing smaller genomes or specific regions.
5. Sanger sequencing contributed significantly to major projects like the Human Genome Project, which relied on this technique for accurate DNA analysis.

Review Questions

• How does Sanger sequencing utilize dideoxynucleotides to determine the sequence of DNA?
  • Sanger sequencing uses dideoxynucleotides to terminate DNA strand elongation during replication. When these modified nucleotides are incorporated into the growing DNA strand, they prevent further addition of nucleotides due to the lack of a 3' hydroxyl group. This selective incorporation results in a variety of fragment lengths that correspond to the original sequence, allowing researchers to deduce the complete DNA sequence by analyzing these terminated fragments.
• Compare Sanger sequencing with next-generation sequencing (NGS) in terms of their applications and limitations.
  • Sanger sequencing is highly accurate and ideal for smaller-scale projects, such as confirming mutations or analyzing specific genes. However, it is slower and more expensive per base compared to next-generation sequencing (NGS), which can rapidly sequence entire genomes at a lower cost. NGS excels in high-throughput applications, making it suitable for large-scale genomic studies, but it may produce errors in homopolymeric regions. Each method has its strengths and is often used complementarily in research.
• Evaluate the impact of Sanger sequencing on genomic research and its relevance today in light of advancing technologies.
  • Sanger sequencing has fundamentally transformed genomic research since its inception, providing the first reliable means of determining DNA sequences accurately. Despite the rise of next-generation sequencing technologies that allow for massively parallel sequencing, Sanger's reliability makes it indispensable for validating results from NGS or resolving complex genomic regions. Its continued use underscores its importance in contemporary genomics, especially in clinical settings where accuracy is paramount and precise mutations need confirmation.

Related terms

Dideoxynucleotide: A type of nucleotide used in Sanger sequencing that lacks a 3' hydroxyl group, causing DNA strand termination when incorporated into a growing DNA chain.

Polymerase Chain Reaction (PCR): A widely-used technique that amplifies specific segments of DNA, enabling researchers to produce millions of copies of a particular sequence for further analysis, including Sanger sequencing.

Capillary electrophoresis: A method used in Sanger sequencing to separate DNA fragments based on their size, allowing for the visualization and reading of the sequence.