Microbiology
A DNA molecule's nucleotides (adenine, thymine, cytosine, and guanine) must
be numbered to be sequenced. It aids scientists in figuring out the genetic code and the
roles genes play in the body.
Frederick Sanger created the Sanger DNA sequencing method in the late 1970s.
It is also known as the chain termination method. To extend DNA chains, it requires
using nucleotides with a 3'-OH group removed, called dideoxynucleotides (ddNTPs).
To accomplish this, the standard DNA nucleotides (dNTPs) and four different
fluorescently-tagged ddNTPs are used in four individual sequencing procedures. At
random intervals during DNA chain elongation, a ddNTP is integrated by DNA
polymerase, resulting in chain termination. Gel electrophoresis separates the fragments,
and the DNA sequence is deduced by examining the fluorescence bands from the
various fragment sizes (Garner, 2021).
Newer sequencing methods, such as next-generation sequencing (NGS) or
high-throughput sequencing, have profoundly impacted the discipline. In contrast to
Sanger sequencing, NGS permits the massively parallel sequencing of many DNA
fragments at once. The process entails simultaneously sequencing billions of DNA
fragments of varying lengths. Sequencing-by-synthesis with reversible terminators,
DNA fragmentation, amplification, and adapter ligation are the cornerstones of next-
generation sequencing (NGS) techniques like Illumina sequencing. Reconstructing the
original DNA sequence requires massively parallel data generation and computational
analysis (Ding et al., 2021).
Sanger sequencing offers a distinct advantage over the other approach because
of its high accuracy and low error rates. Short-read sequencing is still commonly used
for specialized purposes like sequencing individual genes or verifying NGS results
since it is well-suited to these tasks and can handle shorter DNA fragments. However,
compared to Sanger sequencing, NGS has several benefits. It allows enormous
genomes to be sequenced, unusual variations to be found, and complicated DNA mixes
to be analyzed. Newer sequencing technologies (NGS) are more efficient and
productive in terms of both time and money. However, it needs a large amount of
computing analysis to comprehend the data, and it is especially error-prone in repeating
sections (Bowater & Gates, 2015).
To recap, DNA sequencing is the method used to establish the nucleotide
sequence of a DNA molecule. Next-generation sequencing enables massively parallel
sequencing of numerous DNA fragments simultaneously, while the traditional Sanger
approach relies on chain termination. Each approach has benefits and drawbacks, and
selecting one will rely on factors including research objectives, sample size, and
available funds (Witte & Herde, 2019).
References
Bowater, R. P., & Gates, A. J. (2015). Nucleotides: Structure and Properties. eLS, 1–9.
[Link]
Ding, T., Song, G., Liu, X., Xu, M., & Li, Y. (2021). Nucleotides as optimal candidates for
essential nutrients in living organisms: A review. Journal of Functional Foods, 82,
104498. [Link]
Garner, A. L. (2021). Nucleosides, Nucleotides and Nucleic Acids as Therapeutics: A Virtual
Special Issue. [Link] [Link]
Witte, C., & Herde, M. (2019). Nucleotide Metabolism in Plants. Plant Physiology, 182(1), 63–
78. [Link]
