What is the Difference Between Sanger Sequencing and Pyrosequencing?

Sanger sequencing and pyrosequencing are both DNA sequencing methods, but they differ in their principles and methods of detection. Here are the main differences between the two:

1. Chain Termination vs. Pyrophosphate Detection: Sanger sequencing, also known as chain termination sequencing or dideoxy sequencing, relies on the synthesis of a new DNA strand complementary to the template strand, using modified, labeled nucleotides that can terminate the synthesis process. In contrast, pyrosequencing detects the release of pyrophosphate and the generation of light upon nucleotide incorporation.
2. Dideoxynucleotides vs. Regular Nucleotides: Sanger sequencing uses modified dideoxynucleotides, which terminate the synthesis process when incorporated into the growing DNA strand. Pyrosequencing, on the other hand, uses regular nucleotides and depends on the detection of pyrophosphate release during nucleotide incorporation.
3. Sensitivity and Mutation Detection: Pyrosequencing has a higher sensitivity for mutation detection, with the ability to clearly identify a mixture containing 5% mutant alleles. Sanger sequencing, while less sensitive, is still used for applications such as sequencing single genes or short fragments.
4. History and Present Use: Sanger sequencing was invented in 1977 and remained the main DNA sequencing method until the 21st century. Pyrosequencing was developed about 20 years after Sanger sequencing and quickly became a standard DNA sequencing method before being replaced by more advanced high-throughput sequencing technologies. Today, both methods are less commonly used in laboratories due to the advent of newer, more efficient, and affordable techniques such as Illumina, PacBio, and Nanopore sequencing.

Comparative Table: Sanger Sequencing vs Pyrosequencing

Sanger sequencing and pyrosequencing are two DNA sequencing methods used in molecular biology. Here is a table comparing the differences between the two techniques:

Sanger sequencing, also known as chain termination sequencing, is a first-generation DNA sequencing method developed by Frederick Sanger and his team. It works by terminating the chain elongation process at specific nucleotide positions, allowing the construction of the nucleotide sequence. However, it is limited by its short read length and lower sensitivity to minor alleles.

Pyrosequencing, on the other hand, is a more recent sequencing method that belongs to the second generation of DNA sequencing techniques. It is based on the "sequencing by synthesis" principle and is faster, less expensive, and more sensitive to minor alleles compared to Sanger sequencing. However, it may be less accurate than Sanger sequencing.