Sanger sequencing, also known as dideoxy sequencing, utilizes radio-labeled dideoxynucleotide triphosphates (ddNTPs) to identify DNA sequences. The ddNTPs are present in much lower concentrations than deoxynucleotides (dNTPs), ensuring that only a small fraction of DNA strands are truncated during synthesis. Each reaction uses a specific ddNTP, allowing the identification of the last nucleotide in the truncated strands. The resulting fragments are separated using gel electrophoresis, where smaller fragments travel further than larger ones. By reading the gel from smallest to largest, the DNA sequence can be determined. This method has evolved to use fluorescent ddNTPs instead of radioactive ones, making it safer and more efficient. In this updated method, a capillary gel column is used, and a photo detector identifies the emitted fluorescence, correlating specific colors to the respective nucleotides (e.g., green for adenine, red for thymine, black for guanine, and blue for cytosine).
Another sequencing technique is pyrosequencing, which detects the release of pyrophosphate during DNA synthesis. In this method, DNA sequences are attached to beads in wells, and enzymes such as sulfurylase and luciferase are used. When a nucleotide triphosphate is added to a well, if it is incorporated into the growing DNA strand, pyrophosphate is released. Sulfurylase converts this pyrophosphate into ATP, which activates luciferase, resulting in light emission. By sequentially adding nucleotides and detecting light, the DNA sequence can be determined efficiently.
Ion Torrent sequencing operates on a similar principle but focuses on measuring pH changes instead of light. When a nucleotide is added and incorporated, protons are released alongside pyrophosphate, causing a change in pH. This method detects these pH changes to determine the DNA sequence, showcasing the versatility of modern sequencing technologies.