MadSci Network: Molecular Biology

Re: When using the Sanger Method, how is the first base identified?

Area: Molecular Biology
Posted By: Michael Onken, WashU
Date: Wed Mar 19 10:04:25 1997
Message ID: 857160449.Mb

When DNA sequencing - literally reading the genetic code of a strand of DNA - was first invented, two different laboratories developed techniques with very different approaches to sequencing. The Maxam-Gilbert Method1 involved the sequential removal of bases from one end of the DNA molecule, while the Sanger Method2 (now refered to as Dideoxy Sequencing) involved the sequential addition of bases to a primer annealed to a complementary strand of DNA, called the template. Though more reliable, Maxam-Gilbert sequencing uses toxic chemicals and large amounts of radioactivity, and so it is not very popular in most laboratories. Most of us use the Sanger Method for the majority of DNA sequencing.

The Sanger Method uses the natural process of DNA Replication to drive its reaction. In this reaction, DNA polymerase (nowadays, we use a recombinant, truncated form with higher activity) is mixed with a single-stranded DNA template, and allowed to form a complementary strand of DNA from free deoxynucleotides (dNTP's: the building blocks of DNA) in the solution. By adding dideoxynucleotides (ddNTP's: deoxynucleotides missing the 3' hydroxyl group, which is essential for DNA polymerization) to the mix, DNA polymerase will stop and fall off of the template each time it incorporates a ddNTP into the sequence. By doing the same reaction in different tubes with a different ddNTP (ddGTP, ddATP, ddTTP, ddCTP) in each, you get four sets of products corresponding to each base. The end result, after running the products on a denaturing polyacrylamide gel, is a ladder of DNA bands that exactly follows the sequence of terminations by incorporation of each ddNTP, which follows the sequence of the template DNA.

Since the Sanger Method relies on the addition of nucleotides to the primer, it is very rare for an experimenter to read closer than about 5 bases from the end of the primer. For this reason, the ends of the sequence are not usually used to determine the location of the sequence on the template; but rather landmarks (like enzyme sites, and previously determined sequences) within the sequence are used to localize the new sequence to its place along the template.

1. Maxam, A.M., and Gilbert, W. (1977), "A new method of sequencing DNA." Proc. Natl. Acad. Sci. USA 74: 560-564.

2. Sanger, F., Nicklen, S., and Coulson, A.R. (1977), "DNA sequencing with chain-terminating inhibitors." Proc. Natl. Acad. Sci. USA 74: 5463-5467.

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