Sanger Sequencing

Definition

Sanger sequencing is a method for determining the nucleotide sequence of DNA, developed by Frederick Sanger in the 1970s. This technique involves selectively incorporating chain-terminating dideoxynucleotides during DNA replication, which allows for the identification of the sequence based on the lengths of the resulting fragments. As a foundational method in genomics, Sanger sequencing played a crucial role in the Human Genome Project and is still widely used today for sequencing small regions of DNA and validating results from next-generation sequencing technologies.

5 Must Know Facts For Your Next Test

1. Sanger sequencing is often referred to as chain termination sequencing due to its use of dideoxynucleotides that terminate DNA strand elongation.
2. The method requires a single-stranded DNA template, DNA polymerase, primers, and the four standard deoxynucleotides along with the four dideoxynucleotides.
3. The resulting DNA fragments are separated by size using capillary electrophoresis, allowing for the determination of the sequence based on the order of fragment lengths.
4. Sanger sequencing can read up to about 1000 base pairs accurately, making it ideal for sequencing smaller sections of DNA or verifying NGS results.
5. It was the primary method used in the Human Genome Project, which was completed in 2003 and provided a reference sequence for human DNA.

Review Questions

• How does Sanger sequencing differ from other sequencing methods, particularly regarding its mechanisms and applications?
  • Sanger sequencing differs primarily in its use of dideoxynucleotides that terminate DNA synthesis, resulting in fragments that can be analyzed for their lengths to determine the sequence. Unlike next-generation sequencing methods, which can simultaneously analyze millions of fragments, Sanger sequencing typically analyzes one fragment at a time and is best suited for smaller sequences. Its high accuracy makes it valuable for validating results obtained from other techniques.
• Evaluate the advantages and disadvantages of using Sanger sequencing compared to Next-Generation Sequencing (NGS) in genomic research.
  • Sanger sequencing offers high accuracy and reliability, making it a preferred method for confirming specific sequences. However, it is slower and more expensive per base compared to NGS, which can sequence millions of fragments simultaneously at a lower cost. The choice between these methods depends on the specific needs of a project; Sanger is ideal for targeted sequences while NGS excels in large-scale genomic studies.
• Design an experiment utilizing Sanger sequencing to confirm the presence of a specific mutation in a gene known to be associated with a genetic disorder.
  • To confirm a specific mutation using Sanger sequencing, I would first extract DNA from patient samples suspected of carrying the mutation. Then, I would design primers flanking the region of interest where the mutation is located. Using PCR, I would amplify this segment of DNA and then prepare it for Sanger sequencing by adding dideoxynucleotides. After running capillary electrophoresis, I would analyze the sequence data to identify whether the expected mutation is present by comparing it with a reference sequence.

Related terms

Dideoxynucleotide (ddNTP): A modified nucleotide that lacks a hydroxyl group at the 3' position, preventing further elongation of a DNA strand during replication.

Polymerase Chain Reaction (PCR): A technique used to amplify specific segments of DNA, making it easier to sequence or analyze them.

Next-Generation Sequencing (NGS): A modern sequencing technology that allows for the rapid sequencing of large amounts of DNA, offering higher throughput and lower costs compared to Sanger sequencing.