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In 1977, Frederick Sanger developed a procedure for sequencing DNA that was the primary method used for decades until the development of next-generation sequencing technologies in the early 2000s. Today, while next-generation sequencing is used for genome sequencing, Sanger sequencing is still routinely used for generating high-quality sequence data of smaller DNA regions.

Sanger sequencing, also known as the chain termination method, involves mixing the following components in a small tube or well:

  • Single-stranded DNA template
  • DNA polymerase
  • A short DNA primer complementary to 3' end of the DNA fragment to be sequenced
  • dNTPs (a mixture of all 4 deoxyribonucleotides)
  • ddNTPs (a mixture of all 4 dideoxyribonucleotides)

Because each ddNTP lacks the 3'-OH group onto which a subsequent nucleotide would be added (see image below comparing dATP to ddATP), incorporation of a ddNTP results in chain termination, giving the method its name. In recent iterations of the chain termination method, each ddNTP is labeled with a different fluorochrome (fluorescent dye).

Sanger-sequencing. Digital Image. Wikimedia Commons. Wikimedia Foundation, Inc., 19 Dec. 2012. Web. 16 Mar. 2017.

Since the concentration of each ddNTP is low compared to its dNTP counterpart, incorporation of the ddNTPs is random. An array of DNA fragments is generated, representing the incorporation of a ddNTP at every possible position. An example of this is shown below.

Sanger-sequencing. Digital Image. Wikimedia Commons. Wikimedia Foundation, Inc., 19 Dec. 2012. Web. 16 Mar. 2017.

These negatively charged DNA fragments are then subjected to gel electrophoresis, and the smaller the DNA fragment, the faster it will move towards the positive electrode. Thus, by analyzing the order of the fluorescence colors resulting from electrophoresis, one can determine the order of nucleotides in the DNA strand complementary to the original template.

By examining the gel shown below, determine the DNA sequence of the newly synthesized strand.

Auman, Ann. Created for Albert.io. Copyright 2017. All rights reserved.

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