Sanger Sequencing

Chain-Terminator Sequencing

Sanger sequencing, also known as dideoxy sequencing, was invented by Frederick Sanger in 1977. This chain-termination method, though no longer used today, set up the foundation for all the future sequencing technologies. The key principle of the Sanger method was the use of the dideoxynucleotide triphosphates (ddNTPs) as DNA chain terminators. These dideoxynucleotides lack an 3'-OH group required to form another phosphodiester bond, thus terminating DNA strand extension. (13)

 The Process

1.  The classical chain-terminator method requires a single-stranded DNA template, a DNA primer, a DNA polymerase, and all four fluorescently labeled nucleotides (dATP, dGTP, dCTP, and dTTP). 


3. The polymerase begins to add on deoxynucleotides, however whenever one of the dideoxynucleotides is added, it terminates DNA strand elongation. The end result is a sample with varying length of DNA, all of them ending with the known ddNTP. 

4 Figures above (13) 

2. This mixture is then separated into four different tubes, and added to each tube is one of the four types of the ddNTPs (ddATP, ddGTP, ddCTP, and ddTTP). 



4. The newly synthesized and labeled DNA fragments are heat denatured, and run on a gel electrophoresis with a resolution of just one nucleotide. This separates the fragments by size, the smaller ones traveling the furthest. Each of the four reactions are run in one of four individual lanes, and the DNA bands are then visualized by autoradiography or UV light. Then the DNA sequence can be directly read on the X-ray film or gel, by reading up the lanes. 




PCR, or Polymerase Chain Reaction, is a commonly used method to amplify DNA. PCR was developed by Kary B. Mullis in 1983. (9) The method involves using specified primers to isolate the desired region of the DNA. Once the two primers bond to the correct starting and ending sites on the denatured DNA, the Taq Polymerase begins to add nucleotides to the end of each primer. After repeating the process several times, the amount of the desired fragment of DNA is increased exponentially.  (11)

    Ingredients for a PCR (11)
  • DNA- contains the segment of chosen target DNA
  • Primers- usually around 20-30 nucleotides long, and complementary in sequence to the ends of the target DNA
  • Taq Polymerase- derived from hot-springs bacteria and can tolerate the heat needed to denature DNA in the PCR reaction
  • Free nucleotides- used by the Polymerase to amplify the target DNA

Figure (10) 

Dye-Terminator Sequencing

Dye terminator sequencing is a variation of Sanger sequencing, which includes the labeling of each of the dideoxynucleotide with a different fluorescent dye, permitting sequencing in a single reaction rather than four. (12) 

 In dye-terminator sequencing, each of the four ddNTPs are labelled with fluorescent dyes, each of which emit light at different wavelengths. The method is a lot more efficient than the original Sanger sequencing. In the figure at the right, the dye-terminator method is compared to the original method.

Figure (5) 

Though the dye-terminator method was slightly more efficient, these methods were still only the starting blocks of the technology. Nowadays the technologies are so advanced, they can sequence whole genomes in a matter of days. Though Sanger methods are no longer used today, they still hold the fundamental concepts to sequencing technologies. Click here to learn about the next generation sequencing technologies.


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