Online Education Kit: Sequencing Reactions: Transcript

National Human Genome Research Institute

National Institutes of Health
U.S. Department of Health and Human Services


Sequencing Reactions: Transcript

A DNA sequencing reaction includes four main ingredients, "Template" DNA copied by the E. coli; free bases, the building blocks of DNA that come in 4 types; short pieces of DNA called "primers"; and DNA polymerase, the enzyme that copies DNA.

The chemical reaction that makes DNA in a test tube is similar to what happens in a living cell: both rely on DNA polymerase and, in both cases, DNA strands have a head end, which is called the 5' end, and a tail end, which is called the 3' end. A DNA strand can grow only from its 3' end.

Making DNA in cells and sequencing DNA in test tubes both depend on complementary base pairing. The building blocks on opposite strands of DNA pair specifically - a C always pairs with a G, and an A always pairs with a T.

The primer sequence binds to its complementary sequence on the template DNA.

Free bases that match the template sequence can attach to the new strand's growing (3') end.

Among the free bases in the solution are a few that have a fluorescent dye attached to them. When a dye-bearing base attaches to the growing strand, it stops the new DNA strand from growing any further. A different colored dye is attached to each of the four kinds of bases.


Last Reviewed: March 29, 2012