Sanger Sequencing

Definition

Sanger sequencing is a method used to determine the nucleotide sequence of DNA, developed by Frederick Sanger in the 1970s. This technique relies on selective incorporation of chain-terminating dideoxynucleotides during DNA replication, allowing researchers to generate fragments of varying lengths that can be analyzed for their sequences. It's foundational in genomics and has paved the way for advancements in next-generation sequencing technologies.

5 Must Know Facts For Your Next Test

1. Sanger sequencing is often referred to as the chain termination method due to its use of dideoxynucleotides that terminate DNA strand elongation.
2. This technique was instrumental in the Human Genome Project, providing accurate sequence data for the human genome.
3. While Sanger sequencing is highly accurate, it is less efficient for sequencing large genomes compared to next-generation sequencing technologies.
4. The process typically involves amplifying the DNA template through PCR before sequencing, ensuring enough material for analysis.
5. Sanger sequencing is still widely used today for applications such as validating sequences obtained from next-generation methods and for small-scale projects.

Review Questions

• How does Sanger sequencing differ from other DNA sequencing methods in terms of its basic principles and applications?
  • Sanger sequencing differs from other DNA sequencing methods primarily due to its use of dideoxynucleotides that cause chain termination during DNA synthesis. This results in fragments of varying lengths that can be separated by electrophoresis to read the sequence. While it offers high accuracy and is suitable for smaller sequences or validation of NGS results, it is not as efficient or cost-effective for larger genomes compared to next-generation sequencing methods.
• Discuss the role of dideoxynucleotides in Sanger sequencing and how they contribute to determining the DNA sequence.
  • Dideoxynucleotides are essential in Sanger sequencing because they lack a 3' hydroxyl group, which prevents further addition of nucleotides once incorporated into the growing DNA strand. This selective incorporation allows the generation of fragments of different lengths, each terminating at a specific base. By analyzing these fragments through electrophoresis, researchers can determine the exact order of nucleotides in the original DNA template.
• Evaluate the impact of Sanger sequencing on the field of genomics and its transition towards next-generation sequencing technologies.
  • Sanger sequencing has had a profound impact on genomics, providing a reliable method for accurately determining DNA sequences and enabling significant projects like the Human Genome Project. However, as the demand for faster and more cost-effective methods increased, next-generation sequencing technologies emerged, allowing for high-throughput sequencing at unprecedented speeds. Despite this shift, Sanger sequencing remains relevant, particularly in validating sequences and addressing specific research needs where accuracy is paramount.

Related terms

Dideoxynucleotide: A type of nucleotide used in Sanger sequencing that lacks a 3' hydroxyl group, preventing further extension of the DNA strand.

Electrophoresis: A laboratory technique used to separate DNA fragments based on size, crucial for analyzing the products of Sanger sequencing.

Next-Generation Sequencing (NGS): A collection of advanced sequencing technologies that enable rapid and cost-effective sequencing of entire genomes, contrasting with the more time-consuming Sanger method.