Part 5 - DNA Sequencing

From the last step, you have 12 tubes that contain the final PCR product, a mix of DNA pieces of variable length. All DNA pieces in each tube start with the same primer but end with a different nucleotide tagged with a fluorescent marker (different color for each nucleotide A, T, G, C). What remains to be done is to separate the individual DNA pieces and identify the end nucleotide.

This is done by using an automatic sequencer that performs gel electrophoresis on the DNA in each tube. Gel electrophoresis is a method to separate molecules based on differences in size. The sequencer used in this lab has a thin capillary tube attached at one end to a syringe mechanism that contains a buffer solution. The tube is filled with the buffer solution and the other end inserted into one of the tubes containing the DNA pieces. Then, an electric current is applied so that the end of the tube in contact with the DNA has a negative charge and the syringe end a positive charge. Since DNA molecules are negatively charged, they start to move through the tube toward the positively charged syringe end, with the smaller pieces moving faster than the larger ones. Near the syringe end, the capillary tube passes through a laser beam that excites the fluorescent markers, and optical detectors detect the color of the fluorescence.

We can assume that a complete set of DNA pieces, all differing in size by exactly one nucleotide, were generated in the previous step. The smallest piece of DNA that has a fluorescent tag attached to it is the primer. This DNA fragment will travel faster than the other ones. By reading the color (in our example, red), we determine that the first nucleotide beyond the primer sequence is thymidine (T). The next smallest piece of DNA will fluoresce with the color (green) representing the next nucleotide in the sequence (A) and so on (see the animation). By reading the sequence of nucleotides based on their fluorescence as the DNA pieces pass through the laser beam, the sequence of the DNA can be reconstructed.

The sequencer automatically flushes out the buffer from the tube, moves the tray, and runs the electrophoresis again, repeating the program until all 12 tubes have been examined. The resulting sets of sequences are collated by a computer program to build the complete sequence of the 16S rRNA gene.