logo
AAT Bioquest

What are the steps of Sanger sequencing?

Posted March 24, 2022


Answer

Sanger sequencing is a process used to determine the precise order of the nucleotides in a given DNA fragment. It is also known as the chain termination method. Sanger sequencing consists of 6 steps:

  • The dsDNA (double-stranded DNA) is denatured into two ssDNA (single-stranded DNA) using heat.
  • The temperature is lowered and a DNA primer that is complementary to the template DNA is attached to one end. This marks where the sequencing will begin. 
  • The fragments are added to 4 polymerase solutions. Each solution contains the 4 types of bases (dNTPs) but only one type of termination nucleotide (ddNTP). 
  • The DNA synthesis reaction gets initiated and the complementary chain grows until a termination nucleotide is randomly introduced.
  • The resulting DNA fragments are denatured to obtain a series of ssDNA of various lengths.
  • Gel electrophoresis is used to separate the denatured fragments and determine the sequence.
Additional resources

Sanger Gap Sequencing for Genetic Alphabet Expansion of DNA

MagaDye™ 4 Color Sanger Sequencing Terminator Kit