The Gilbert chemical cleavage method and the Sanger dideoxy chain-termination method were groundbreaking techniques for determining DNA sequences with high accuracy. They propelled gene structure analysis, making it possible to identify disease-related genes and study evolution at unprecedented speed. Combined with PCR and fluorescent labels, they later led to automated sequencers. These methods are the roots of today’s next-generation sequencing (NGS). Sequence information is now applied to medicine, agriculture, and ecosystem conservation.

In the Sanger method, DNA polymerase incorporates dideoxynucleotides (ddNTPs) randomly, terminating the growing strand at specific bases. After size separation, radioactive or fluorescent labels reveal bands, and the sequence is read from the electrophoretic pattern. The Gilbert method uses piperidine-induced chemical cleavage to break single-stranded DNA at defined bases, followed by electrophoresis to deduce the sequence. Sanger’s approach became dominant because of milder reaction conditions, compatibility with longer templates, and easy transition from radioactivity to fluorescence; it was adopted for the Human Genome Project. The underlying principles evolved into capillary electrophoresis, cycle sequencing, and NGS’s chemistry-based signal detection.

In Sanger sequencing, optimization of the ddNTP/dNTP ratio and removal of the 3ʹ→5ʹ proofreading activity via the Klenow fragment enabled automation with fluorescent-labeled primers. Transition to capillary electrophoresis extended read lengths to 800–1000 bp, and cycle sequencing with 30-mer primers provided high S/N even from trace templates. The Gilbert or Maxam-Gilbert method combines chemical backbone modification with depurination/depyrimidination to yield base-specific cleavage patterns; despite toxicity and complexity, it is still used for specialized tasks such as methyl-cytosine detection. Both techniques laid the groundwork for quality-score concepts, bioinformatics pipelines, and NGS error-model construction.