Sanger sequencing is a “chain termination method” for determining DNA’s nucleotide sequence. Frederick Sanger, a two-time Nobel laureate, and his associates discovered the technique in 1977; thus, the term “Sanger Sequence.”
How is Sanger sequencing implemented?
The foundation of Sanger sequencing, also known as the enzymatic chain termination method, combines normal deoxynucleotides (dNTPs) with fluorophore-labeled dideoxynucleotides (ddNTPs). The addition of ddNTPs by the DNA polymerase during chain extension stops the DNA strand elongation because ddNTPs lack the hydroxyl group required for nucleotide binding.
By repeating the primer annealing and DNA extension cycles, fragments containing fluorophore-labeled nucleotides at each position can be produced, which allows for the identification of each nucleotide in the DNA template. Below is a step-by-step process of Sanger sequencing:
- A polymerase chain reaction (PCR) uses the patient’s DNA as a template. The PCR reaction uses a combination of chain termination bases (ddNTPs) and regular bases (dNTPs). Adding a random chain-terminating base stops a developing DNA chain in its tracks. That results in DNA fragments of various lengths. A chain-terminating base marks the end of each fragment.
- The next step involves employing capillary electrophoresis to separate the DNA fragments based on size.
- A distinct fluorescent marker exists for each of the four chain termination bases (A, T, C, and G). A laser excites these fluorescently labeled bases at the end of each fragment.
- In the sequence, shorter bits appear first, then progressively longer fragments.
- By recording the fluorescence of the base that ends each segment of DNA, a chromatograph is produced that indicates which base is present at each location along the fragment.
- Any variations are found by comparing the chromatogram to a reference file.
Selecting between NGS and Sanger Sequencing
The Sanger sequencing method is still the “gold standard” for fundamental and clinical research applications, with an accuracy rate of over 99%. Most clinical laboratories use Sanger sequencing to confirm gene variants (such as insertions, deletions, and single-nucleotide variants) that NGS initially discovered. More research, however, is coming out that calls into question the needless use of an expensive and time-consuming validation procedure and supports the accuracy of NGS techniques for variant detection.
Sequencing one DNA fragment at a time is possible with the Sanger method. Thus, sample volume remains the fundamental differentiator when deciding between Sanger and next-generation DNA sequencing methods, which are quite precise. Next-generation techniques would reduce costs and turnaround times associated with discovery applications that need sequencing of large numbers of genes (e.g., over 100 gene targets) or high sensitivity and consequently increased reading depth, as required for complicated samples (e.g., tumor tissue).
However, in molecular biology labs, Sanger sequencing is still the method for confirming the sequences of genes and plasmids. This is partially because Sanger sequencing does not require complex data analysis and can read up to 500–700 bps per reaction.
As a result, it is comparatively simpler and quicker, particularly for gene sequences that contain repeats, which continues to be a significant obstacle for NGS systems that need to connect short sequence reads.
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Conclusion
Sanger sequencing is still the most accurate type of DNA sequencing used in clinical laboratories.
