Sequenziamento di Sanger

Dideoxynucleotides (ddNTPs) are modified bases lacking the hydroxyl group found in regular deoxynucleotides, which prevents them from forming phosphodiester bonds with adjacent nucleotides. This property causes DNA chain termination when incorporated during synthesis. Each ddNTP is labeled with a different fluorescent label to enable detection and identification of terminated DNA fragments.

After DNA amplification produces fragments of varying lengths, each terminating with a labeled ddNTP, the fragments are run on a capillary gel to separate them by size. Automated software then analyzes the emission spectra from each fragment to decipher the genetic sequence. This separation and analysis process reveals the order of nucleotides in the original DNA template.

The first step is to denature the template DNA into single-stranded DNA. This separation of the double helix is essential before primers can bind to the region of interest and DNA polymerase can begin adding nucleotides during the chain termination sequencing reaction.

Sanger sequencing can only sequence approximately 300-1000 base pairs of DNA in a single run, making it unsuitable for large-scale projects. Additionally, sequence quality is poor at the primer binding site, affecting the first 15 to 40 nucleotides. Despite these constraints, its simplicity and reliability make it ideal for small-scale targeted sequencing applications.

DNA polymerase adds nucleotides to the growing DNA strand during synthesis. Occasionally, it incorporates a dideoxynucleotide instead of a regular deoxynucleotide. Since ddNTPs lack the hydroxyl group needed for phosphodiester bond formation, incorporation of a ddNTP terminates DNA amplification, creating fragments of defined lengths for sequencing analysis.

Sanger sequencing remains widely used because of its simplicity, reliability, and cost-effectiveness for targeted applications. The conventional method was adapted into a semi-automated approach, making it more accurate and faster. Today, it is frequently used for small-scale targeted sequencing projects where high throughput is not required.

Each dideoxynucleotide is labeled with a different fluorescent label, enabling automated detection and identification of terminated DNA fragments. When fragments are separated by capillary gel electrophoresis, the fluorescent labels emit spectra that are analyzed by software to determine which ddNTP terminated each fragment, revealing the nucleotide sequence.