DNA sequencing refers to the analysis of the base sequence of a specific DNA fragment, the arrangement of adenine (A), thymine (T), cytosine (C) and guanine (G), which can be used to determine the orientation and structure of recombinant DNA and to locate, identify and compare mutations.
Knowledge of DNA sequences has become indispensable in basic biological research and in numerous applications such as diagnostics, biotechnology, forensic biology, and biosystematics. The rapid sequencing speed with modern DNA sequencing technologies has helped to reach the complete DNA sequence, or sequencing of multiple types of genomes and complete DNA sequences of living species, including the human genome and many other animal, plant and microbial species.
Figure 1. The process of Sanger sequencing.
Chemical reagents treat the end DNA fragments, causing specific cleavage of the bases and producing a set of reactive mixtures with various lengths of DNA strands, which are separated by gel electrophoresis.
A DNA polymerase is used to extend the primers bound to the template of the sequence to be determined. This is done until the incorporation of a strand terminating nucleotide. Each sequence assay consists of a set of four separate reactions, each containing all four deoxynucleotide triphosphates (dNTP) mixed with a limited amount of a different dideoxynucleotide triphosphate (ddNTP). The lack of the 3-OH group required for the extension of ddNTP causes the extended oligonucleotide to terminate selectively at G, A, T or C. The termination point is determined by the corresponding double deoxy in the reaction. The relative concentration of each dNTPs and ddNTPs can be adjusted so that the reaction yields a set of chain termination products that are several hundred to several thousand bases long. They have a common starting point but terminate at different nucleotides and can be separated by high-resolution denaturing gel electrophoresis with fragments of different sizes, which can be detected by X-ray film radiolucent autoradiography or non-isotope labeling after gel processing.
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In order to DNA sequencing, we can use the following methods.
Assembly of individual sequence read segments into longer contiguous sequences (neighboring fragments), assembly into correctly sequenced neighboring fragments in the absence of reference sequences (scaffolds).
Accurate confirmation of mutations by orthologous techniques (independent chemical reactions), as well as discovery of new mutations.