454 sequencing (pyrosequencing)

Definition

454 sequencing, also known as pyrosequencing, is a method of DNA sequencing that relies on the detection of pyrophosphate release during nucleotide incorporation. It allows for the rapid sequencing of short to medium-length DNA strands.

5 Must Know Facts For Your Next Test

1. 454 sequencing uses a 'sequencing by synthesis' approach where nucleotides are added one at a time and detected by light emission.
2. The technology involves four key enzymes: DNA polymerase, ATP sulfurylase, luciferase, and apyrase.
3. It was one of the first next-generation sequencing (NGS) technologies to be commercialized.
4. Pyrosequencing can accurately sequence homopolymeric regions up to around 8-10 bases long but struggles with longer homopolymers.
5. This method generates reads that are typically shorter than those produced by newer NGS methods like Illumina sequencing.

Review Questions

• What is the role of pyrophosphate in 454 sequencing?
• Name the four key enzymes involved in the pyrosequencing process.
• What type of DNA regions pose a challenge for accurate reading in pyrosequencing?

Related terms

Next-Generation Sequencing (NGS): A category of high-throughput DNA sequencing technologies that have revolutionized genomic research.

Illumina Sequencing: A widely used NGS technology that utilizes reversible dye terminators for detecting nucleotide incorporation.

Sequencing by Synthesis: A method where nucleotides are incorporated into a growing DNA strand and detected in real-time.