Referred to as Sanger sequencing, this is basically dideoxy sequencing, where you use radio-labeled dideoxynucleotide triphosphates. Radio-labeled means they have some sort of radioactive tag on them so that you can identify them. As we discussed, you use a much smaller concentration of the dideoxynucleotides compared to the deoxynucleotides, so that only a small amount of the strands are truncated or shortened during synthesis. Of course, you only use one type of dideoxy which determines which base is present when you find a shortened strand because if there's only one type of dideoxynucleotide that you used in the synthesis, anytime you find a shortened strand, you know that the last nucleotide on that strand is going to be whichever one you were using in that particular reaction. You then use gel electrophoresis to separate the fragments of different lengths. You can see that right here. This is our gel, and you can see our fragments from our reaction with G are found here, and our fragments from reaction C are along this path, A along this path, and T along this path. If you were to try to read this code, you'd see that the actual code here is something like 'ATGCTTCG'. I'm going to stop right there. This 'G' is corresponding to this mark right here. So all I'm doing is reading down the gel more or less to determine the code. Of course, I know this to be the order because the smaller strands will travel further in the gel, and this arrow going up this way shows us the direction of travel. The strands down on this end right here are going to be the smallest, and the largest will be at the opposite end. So, you just read from smallest to largest, and that's your sequence. Eventually, these radio-labeled dideoxynucleotide triphosphates were replaced with fluorescent ones. The reason is that it's easier in the lab to scan for the light emitted from fluorescent molecules as opposed to working with radioactive substances. Also, you usually have to use some pretty nasty chemicals when working with these radioactive substances. So, it's more efficient, and you don't have to use these nasty chemicals. That's why there was this transition to using fluorescent ones. But basically, the exact same concept except instead of using gel electrophoresis, you use a capillary gel column. It's like electrophoresis gel except you're running it in a column and you're eluting out the various strands. As the strands come out, with the smaller ones coming out first and the bigger ones later, you have a photodetector that scans and determines what frequency or wavelength of fluorescence each strand is emitting. Based on the color that they're emitting, it's going to determine which labeled dideoxynucleotide triphosphate you have present. So, much in the same way we went through the gel, if we were using a spectrophotometer, we'd read as the strands come out: green, red, black, blue, and we'd know that green means A, red means T, black means G, and blue means C. Next, let me explain more about pyrosequencing and ion torrent sequencing.
Review 1: Nucleic Acids, Lipids, & Membranes
DNA Sequencing 2