Hello everyone, this is the supplementary video for this week. Here I will introduce to you the classical Sanger sequencing and how it is carried out in lab. Sanger sequencing is the classical first-generation DNA sequencing method. It is easy to carry out and have a long read length, but its throughput is relatively low. The principles of Sanger sequencing is really simple. As displayed, in a Sanger sequencing reaction system are the target DNA segment, the deoxy-ribonucleoside triphosphates (dNTPs), the dideoxy-ribonucleoside triphosphates (ddNTPs), the sequencing primers, and the DNA polymerases. The core of the sequencing reaction is the ddNTPs. Such a nucleotide cannot form with a dNTP the phosphodiester bond due to a lack of 3’-hydroxyl. These ddNTPs can thus be used to abort the extension of DNA sequences. With such settings the DNA sequence [synthesis] will abort at A, G, C, or T, and form different lengths of DNA fragments in different reactions. These fragments can be separated and displayed later by gel electrophoresis. This is an example of Sanger sequencing result. Each band represents a DNA fragment resulting from the abortion of DNA synthesis by introducing a ddNTP. The DNA sequence can be read (in the bottom-to-top order) from the relative positions between bands in the four lanes. The order of bases in the target fragment can thus be obtained. Now let’s have a look at how the Sanger sequencing is carried out. We illustrate the Sanger sequencing with the 3730xl sequencing system from AB and the BigDye Terminator v3.1 kit. This is the BigDye Terminator v3.1 kit, the red solution. It is in fact a Sanger sequencing reaction system. In the solution are the deoxy-ribonucleoside triphosphates (dNTPs), the dideoxy-ribonucleoside triphosphates (ddNTPs), the sequencing primers, the DNA polymerases, and the buffer for the reaction. We dilute this BigDye as needed, add the templates, and put the reaction system into the PCR machine to start the reaction. Here we run with a 96-well plate. After the PCR ends, we still need to purify the sample to remove the remaining polymerases, dNTPs, and ddNTPs. This video shows our student taking out the sample after the reaction has done. This is the 3730xl sequencer. Briefly, what 3730xl sequencer actually does is more like an electrophoresis machine. It examines the fluorescence signals emitted from the DNA fragments synthesized with different lengths during the PCR. The corresponding signal files are automatically outputted into the connected computer. The 3730xl sequencer is extremely automatic. We need do nothing more than putting into the sample. It can accept multiple 96-well plates in one run. We can see that the machine will process these plates one by one. We can see from the supporting software in the connected computer the event list of the sequencer to tell whether the sequencer is running well or not. The 3730xl will output files will a suffix of “.ab1” automatically. This is a binary file with the signal data of the four lanes corresponding to A, C, G, and T, respectively. Using some softwares, such as chromas, we can visualize the signal data in axes as a series of peaks. Of course, if needed we can also use some packages of R or python to extract the data manually for further analyses. We will not go into the details here. That’s all for the Sanger sequencing. Thank you!
