Genetics and molecular biology · BEGINNER LESSON
Sanger sequencing
Sanger sequencing is a first-generation DNA sequencing method that determines the order of bases in a targeted DNA fragment by terminating DNA copy growth at different lengths. It is measured by separating and detecting those fragment lengths to infer the sequence.
What it is and what is measured
Sanger sequencing is a method for reading the order of DNA bases in a chosen DNA fragment, not an entire genome at once. A DNA fragment is a piece of DNA, and the readout comes from fragment lengths that were produced when DNA copying stopped at different positions. The method uses capillary electrophoresis, which separates DNA fragments by size so they can be detected and compared. This is why Sanger sequencing is often described as a first-generation sequencing technology. [0,1]
[1] The Evolution of Gene Sequencing Technologies - PMC - NIH [2] Past, present, and future of genomic technologies in cerebellar ataxias
How it works
The core idea is chain termination. DNA is copied from a template, which is the original DNA being read, but some copied strands stop growing early because of a special terminating building block. That creates a set of DNA fragments with different lengths, each ending at a known base position. A capillary electrophoresis instrument then separates those fragments by size, and the pattern of fragment lengths is used to infer the DNA sequence. In practice, the method is used on targeted DNA fragments, often after amplification by specifically engineered primers, which are short DNA pieces that tell the copying reaction where to start. [0,1]
[1] The Evolution of Gene Sequencing Technologies - PMC - NIH [2] Past, present, and future of genomic technologies in cerebellar ataxias
What it can detect and why it mattered
For targeted regions, Sanger sequencing can detect single-nucleotide variants (SNVs), which are one-base changes, and indels, which are small insertions or deletions. It became an important tool because it is accurate and relatively simple to analyze for a few samples or a small number of targets. For that reason, it has been used to confirm key variants and to validate results from newer sequencing methods. [1,2]
[2] Past, present, and future of genomic technologies in cerebellar ataxias [3] www.nist.gov › publications › variant-alleleVariant Allele Characterization in STR Markers Using Next ...
Limitations
Sanger sequencing is slow and labor intensive compared with newer methods. It has limited throughput, meaning it cannot efficiently process large numbers of samples or very large genomic studies. Its useful read length is only for a targeted fragment, so it is not well suited for genome-wide discovery or for very large-scale screening. Sources also note that it has lower sensitivity and discovery power for finding new genes than modern high-throughput sequencing approaches. [0,1,2]
[1] The Evolution of Gene Sequencing Technologies - PMC - NIH [2] Past, present, and future of genomic technologies in cerebellar ataxias [3] www.nist.gov › publications › variant-alleleVariant Allele Characterization in STR Markers Using Next ...
SEE THE IDEA
How Sanger sequencing produces a DNA readout
A chosen piece of DNA is selected for analysis.
Copying stops at different positions, creating fragments of different lengths.
Fragments are separated by size so they can be detected.
The size pattern is used to reconstruct the base order.
Target DNA is copied into length-terminated fragments, then separated and read to infer the sequence.
The method measures fragment lengths to recover the order of bases in a targeted DNA region.
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Sources and further reading
This general lesson was generated from public source material. It explains a concept; it does not interpret a private paper or provide medical advice.