DNA sequencing

Sanger Sequencing

A well-established method used to determine the precise nucleotide sequence of a DNA fragment by terminating strand elongation using modified nucleotides.

Sanger Sequencing Advantages

Provides highly accurate, high-quality data through a straightforward method that is ideal for small-scale sequencing projects.

Sanger Sequencing Limitations

Limited to sequencing short DNA fragments, has low sensitivity, and is inefficient for large-scale genome projects.

Sanger Sequencing Applications

Used in medical research/diagnostics, evolutionary biology, agriculture, forensics, biotechnology, virology, and epidemiology.

ddNTPs (Dideoxynucleotide Triphosphates)

Modified nucleotides that lack the 3'-OH group necessary for phosphodiester bond formation, halting DNA elongation when incorporated.

Fluorescent ddNTP Types

The four modified bases (ddATP, ddGTP, ddCTP, ddTTP), each labeled with a distinct color dye to identify the terminal base of a fragment.

Sanger Reaction Preparation

Mixing the target DNA template, primers, DNA polymerase, standard dNTPs, and a low concentration of fluorescent ddNTPs.

Chain Termination Step

During PCR amplification, incorporation of a ddNTP randomly halts strand synthesis, creating fragments of varying lengths that each end at a known base.

Fragment Separation Step

Capillary electrophoresis separates the resulting DNA fragments by size down to single-base resolution.

Sequence Detection Step

A laser reads the distinct fluorescent dye on the terminating ddNTP of each fragment as it passes, reconstructing the exact DNA sequence.