Dye-terminator method of DNA sequencing. The single reaction mixture contains all four unlabeled dNTPs, all four fluorescently labeled ddNTPs (shown here as blue, red, yellow, and green colors), DNA polymerase, template (the DNA to be sequenced; it can be double- or single-stranded), and specific primer (oligonucleotide). The newly synthesized strands all start with the same primer (and therefore have identical 5'-ends) and terminate with a specific ddNTP. The DNA is resolved on a denaturing sequencing gel. Only "colored" DNA is detected. The "color" (fluorescence) of each band identifies which ddNTP was incorporated to terminate further DNA synthesis. The sequence is read 5'-to-'3' from the bottom-to-top of the gel. (See a real sequencing gel below - Click here)
A real sequencing gel (shown below). It is easy to read by eye the sequence of each of the 28 samples in this particular gel: green bands represent A, blue bands represent C, yellow bands represent G and red bands T. If you begin at the bottom and read up, you can follow the actual sequence of the original DNA fragment we started with when we performed the sequencing reaction. Once the gel has finished running, the computer performs this same operation: reading from bottom to top, it reads the signal along the vertical path for each sample and creates from this information a data file with the nucleic acid sequence.
