ddNTPbased

ddNTP-based methods refer to techniques that utilize dideoxynucleoside triphosphates (ddNTPs) for DNA sequencing and analysis. ddNTPs are modified nucleotides lacking a 3'-hydroxyl group, which prevents the addition of further nucleotides during DNA synthesis. This property makes ddNTPs essential in terminating DNA chain elongation selectively, enabling precise determination of DNA sequences.

The most prominent application of ddNTP-based technology is in Sanger sequencing, developed by Frederick Sanger in the 1970s. In this method, DNA polymerase incorporates standard deoxynucleoside triphosphates (dNTPs) along with fluorescently labeled ddNTPs. When a ddNTP is incorporated, synthesis terminates at that point, producing DNA fragments of varying lengths. These fragments are then separated by electrophoresis, and the terminal ddNTP’s label indicates the corresponding nucleotide in the sequence.

ddNTP-based sequencing remains valuable for its accuracy and simplicity, particularly for small-scale projects and confirmation of sequences obtained through high-throughput methods. Modern variations incorporate fluorescent labeling and automated detection, increasing throughput and ease of use.

Beyond sequencing, ddNTPs are used in various molecular biology applications, such as mutation detection, genotyping, and clone verification. The key advantage of ddNTP-based techniques is their ability to produce high-fidelity, single-nucleotide resolution data.

While newer sequencing technologies have largely supplanted ddNTP-based methods for large-scale genome projects, these techniques continue to be relevant in research and diagnostic laboratories for targeted and confirmatory analysis.