Sanger Sequencing

I think it's amusing when scientific lingo finds its way into popular speech, because it is often distorted in interesting ways. When political commentators talk about a "litmus test" they're not referring to acids and bases. DNA and the broader concept of genetics have exploded into a cultural phenomena of their own. In most circles you can speak of "genes" without being mistaken for "jeans"; everyone has their stance on the so-called nature versus nurture debate; and now personal genomics companies have sprung up that analyze your genome for disease predictors, ancestral heritage, etc. I have already devoted an entire post to DNA itself. Now I am proud to announce a mini-series of posts devoted to reading and writing DNA, the molecule we all think we understand.

The first practical method for sequencing DNA was pioneered by Frederick Sanger, a prolific scientist who won two Nobel Prizes for Chemistry: one for sequencing proteins, particularly insulin, and a second for DNA sequencing. Don't misinterpret this as a cheat because the two methods are completely unrelated. Sanger's method of DNA sequencing, now called Sanger sequencing or chain termination sequencing, relies on some very clever chemical tricks. To run a Sanger sequencing reaction you need template DNA, a single stranded DNA primer to get the reaction started, DNA polymerase, and nucleotides (G's, C's, A's, and T's). However, some of the nucleotides in the pool are chemically modified so that they terminate a growing DNA strand when incorporated. (They lack the 3' OH group required for the next nucleotide to latch on to).

There are several ways of carrying out a Sanger sequencing reaction, but an older way is as follows. For a given template run four different reactions, all containing a pool of the four normal nucleotides but each also including one modified nucleotide. DNA polymerase incorporates nucleotides indiscriminately, so occasionally a modified nucleotide will be incorporated and the strand is cut short. In each of the four reactions a population of DNA molecules of varying lengths all ending in the same nucleotide accumulate. You run the four reactions side by side on a high resolution gel, and if you were clever and radioactively labeled the modified nucleotides, then you can get an image by laying a piece of film on top. Such an "old school" sequencing gel is shown in the image above. Moving down the gel and noting which of the four bands appear in what order give you the DNA sequence.

To my knowledge nobody uses this method anymore, but it is a nice illustration of the technique, and it is not uncommon to see images such as this one. Nowadays Sanger sequencing is usually done by running a single reaction with all four modified nucleotides. Each nucleotide is fluorescently labeled with a different color, and the terminated DNA fragments are sorted using small capillaries in a machine. They pass by a laser that reads the color of the fluorescing fragments in order, and a computer interprets the flashes of color as a DNA sequence. Today Sanger sequencing is fully automated. My lab uses a DNA sequencing company that handles samples robotically, runs and interprets the sequencing reactions by machine, and posts the results online. And what of Sanger himself? Well, the double Nobel laureate retired in 1985 and, now 92 years old, apparently spends most of his time gardening.

In my next post I will contrast Sanger sequencing with modern methods of high throughput DNA sequencing and highlight the impressive economics of the industry.

Image Credit: John Schmidt (via Wikimedia)

Reference:

DNA Learning Center, Cold Spring Harbor Laboratory. DNA from the Beginning. Frederick Sanger (1918-).