Tuesday, December 16, 2008

The Sanger Method of DNA Sequencing

 
In 1976 Frederick Sanger developed a method for sequencing DNA enzymatically using the Klenow fragment of E. coli DNA polymerase I. Sanger was awarded his second Nobel Prize for this achievement (he received his first Nobel Prize for developing a method for sequencing proteins). The advantage of using the Klenow fragment for this type of reaction is that the enzyme lacks the 5′ → 3′ exonuclease activity, which could degrade newly synthesized DNA. However, one of the disadvantages is that the Klenow fragment is not very processive and is easily inhibited by the presence of secondary structure in the single-stranded DNA template. This limitation can be overcome by adding SSB or analogous proteins, or more commonly, by using DNA polymerases from bacteria that grow at high temperatures. Such polymerases are active at 60° to 70°C, a temperature at which secondary structure in single-stranded DNA is unstable.

The Sanger sequencing method uses 2′,3′-dideoxynucleoside triphosphates (ddNTPs), which differ from the deoxyribonucleotide substrates of DNA synthesis by lacking a 3′-hydroxyl group (see below). The dideoxyribonucleotides, which can serve as substrates for DNA polymerase, are added to the 3′ end of the growing chain. Because these nucleotides lack a 3′-hydroxyl group, subsequent nucleotide additions cannot take place and incorporation of a dideoxynucleotide terminates the growth of the DNA chain. When a small amount of a particular dideoxyribonucleotide is included in a DNA synthesis reaction, it is occasionally incorporated in place of the corresponding dNTP, immediately terminating replication. The length of the resulting fragment of DNA identifies the position of the nucleotide that should have been incorporated.

Chemical structure of a 2′,3′-dideoxynucleoside triphosphate.
B represents any base.
DNA sequencing using ddNTP molecules involves several steps (as shown below). The DNA is prepared as single-stranded molecules and mixed with a short oligonucleotide complementary to the 3′ end of the DNA to be sequenced. This oligonucleotide acts as a primer for DNA synthesis catalyzed by DNA polymerase. The oligonucleotide-primed material is split into four separate reaction tubes. Each tube receives a small amount of an α[32P]-;abelled dNTP whose radioactivity allows the newly synthesized DNA to be visualized by autoradiography.

Next, each tube receives an excess of the four nonradioactive dNTP molecules and a small amount of one of the four ddNTPs. For example, the A reaction tube receives an excess of nonradioactive dTTP, dGTP, dCTP, and dATP mixed with a small amount of ddATP. DNA polymerase is then added to the reaction mixture. As the polymerase replicates the DNA, it occasionally incorporates a ddATP residue instead of a dATP residue, and synthesis of the growing DNA chain is terminated. Random incorporation of ddATP results in the production of newly synthesized DNA fragments of different lengths, each ending with A (i.e., ddA). The length of each fragment corresponds to the distance from the 5′-end of the primer to one of the adenine residues in the sequence.

Adding a different dideoxyribonucleotide to each reaction tube produces a different set of fragments: ddTTP produces fragments that terminate with T, ddGTP with G, and ddCTP with C. The newly synthesized chains from each sequencing reaction are separated from the template DNA.

Finally, the mixtures from each sequencing reaction are subjected to electrophoresis in adjacent lanes on a sequencing gel, where the fragments are resolved by size. The sequence of the DNA molecule can then be read from an autoradiograph of the gel.


This technique has also been modified to allow automation for high throughput applications like genomic sequencing. Instead of using radioactivity automated sequencing relies on fluorescently labeled dideoxynucleotides (four colors, one for each base) to detect the different chain lengths. In this system the gel is “read” by a fluorimeter and the data are stored in a computer file. Additionally, the sequencing machine can also provide a graphic chromatogram that shows the location and size of each fluorescent peak on the gel as they passed the detector.


The bottom figure is from Wikipedia. The other figures and the text are from Horton, H.R., Moran, L.A., Scrimgeour, K.G., Perry, M.D., and Rawn, J.D. (2006) Principles of Biochemistry 4th edition, Pearson Prentice Hall, Upper Saddle River, New Jersey, USA.
© Laurence A. Moran, Pearson Prentice Hall

5 comments :

  1. Thanks for this post. I'm sure that I must have slept through this portion of biochem. I suppose I must have crammed it in for the exam but it promptly left immediately after (as crammed info is wont to do). Anyway, how sequencing works (or at least some sequencing) has been one of those little nagging questions that was on my I-should-look-that-up-at-some-point list. Now I can scratch it off.

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  2. Larry, are there other online tutorials/videos that you know of, or recommend, that go into detail on how researchers analyze DNA?

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  3. Thanks. This was very helpful.

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  4. very thanks for your post.i read so many times about this from other site.i cant understand.finally i got it from your blog.it is easy to understand the way of presentation with diagram.plz,upload other topics also.

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  5. respected sir thanx for the post...
    there`s a request to u can u please post something regarding denaturing polyacrylamide gel electrophoesis and silve staining

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