Sanger sequencing, also known as dideoxy chain‑termination sequencing, is the first‑generation DNA sequencing technology and serves as the "gold standard" for gene sequence identification. Its core principle is to employ dideoxynucleotide triphosphates (ddNTPs) to terminate DNA‑chain elongation. Nucleic acid fragments of different lengths are separated by capillary electrophoresis to accurately determine the base sequence. Boasting exceptional single‑base accuracy, stable read lengths, intuitive and reliable results, straightforward operation and low cost, Sanger sequencing remains the preferred technology for gene sequence verification, site‑directed mutation detection and clone identification. Together with next‑generation high‑throughput sequencing (NGS), it constitutes a complementary sequencing system combining low‑throughput and high‑throughput capabilities.
It is applicable to sequencing conventional PCR products and standard plasmids. Unidirectional sequencing suffices for basic verification of short fragments, while bidirectional sequencing allows complementary read‑based correction for long fragments. This approach effectively mitigates signal attenuation toward the ends of unidirectional reads and enhances both the integrity and accuracy of the resulting.
Designed specifically for molecular cloning experiments, it is used to verify plasmid construction, fragment insertion into vectors and gene cloning, to precisely determine whether the cloned sequence matches the target sequence, and to detect problems such as vector‑borne mutations, aberrant fragment insertion, and frameshift mutations.
For precise verification of site‑specific mutations, gene‑editing sites and SNP sites, it can clearly identify homozygous mutations, heterozygous mutations, and base insertions/deletions. It serves as the core verification method for gene function validation and mutant strain screening.
For difficult‑to‑sequence templates featuring high GC content, tandem repeat sequences, hairpin structures, and low‑complexity sequences, an optimized amplification system and sequencing procedure are adopted to overcome interference from template secondary structures and raise the sequencing success rate for complex fragments. Applicable sample types include PCR products, bacterial cell culture sequencing, plasmid sequencing, etc.
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