California State University Northridge
Biology 470 - Biotechnology
Lecture 3 - Sanger method
Sanger Method (Dideoxynucleotide chain termination)
Here's an example of how one goes about sequencing by this method.
First, anneal the primer to the DNA template (usually single stranded):
Then split the sample into four aliquots, in tubes labeled "G", "A", "T" and "C" and add the following substrates to the respective tubes:
"G" tube: All four dNTPs, including one that is labeled, plus ddGTP
"A" tube: All four dNTPs, including one that is labeled, plus ddATP
"T" tube: All four dNTPs, including one that is labeled, plus ddTTP
"C" tube: All four dNTPs, including one that is labeled, plus ddCTP
When a polymerase (e.g. Klenow fragment) is added to the tubes, the synthetic reaction proceeds until, by chance, a dideoxynucleotide is incorporated instead of a deoxynucleotide. This is a "chain termination" event, because there is a 3' H instead of a 3' OH group. Since the synthesized DNA contains some radiolabeled (or chemically labeled) substrates, the products can be detected and distinguished from the template.
If, for example, we were to look only at the "G" reaction, there would be a mixture of the following products of synthesis:
5'-GAATGTCCTTTCTCTAAGTCCTAAG 3'-GGAGACTTACAGGAAAGAGATTCAGGATTCAGGAGGCCTACCATGAAGATCAAG-5' 5'-GAATGTCCTTTCTCTAAGTCCTAAGTCCTCCG 3'-GGAGACTTACAGGAAAGAGATTCAGGATTCAGGAGGCCTACCATGAAGATCAAG-5' 5'-GAATGTCCTTTCTCTAAGTCCTAAGTCCTCCGG 3'-GGAGACTTACAGGAAAGAGATTCAGGATTCAGGAGGCCTACCATGAAGATCAAG-5' 5'-GAATGTCCTTTCTCTAAGTCCTAAGTCCTCCGGATG 3'-GGAGACTTACAGGAAAGAGATTCAGGATTCAGGAGGCCTACCATGAAGATCAAG-5' 5'-GAATGTCCTTTCTCTAAGTCCTAAGTCCTCCGGATGG 3'-GGAGACTTACAGGAAAGAGATTCAGGATTCAGGAGGCCTACCATGAAGATCAAG-5' 5'-GAATGTCCTTTCTCTAAGTCCTAAGTCCTCCGGATGGTACTTCTAG 3'-GGAGACTTACAGGAAAGAGATTCAGGATTCAGGAGGCCTACCATGAAGATCAAG-5'
(and so on, if the DNA being sequenced continues to the right)
These products are denatured into single stranded DNA molecules and run on a polyacrylamide/urea gel. The gel is dried onto chromatography paper (to reduce its thickness and keep it from cracking) and exposed to X-ray film. Since the template strand is not radioactively labeled, it does not generate a band on the X-ray film. Only the labeled top strands generate bands, which would look like this:
As you can see from this one reaction (the "G" reaction) the chain termination
events produce individual bands on a gel. The chain terminations closest to the primer
generate the smallest DNA molecules (which migrate further down the gel), and chain
terminations further from the primer generate larger DNA molecules (which are slower
on the gel and therefore remain nearer to the top).
When similar chain termination reactions are run for each nucleotide, the four reactions can be run next to each other, and the sequence of the DNA can be read off of the "ladder" of bands, 5' to 3' sequence being read from bottom to top:
The resolution of the gel electrophoresis is very important in DNA sequencing. Molecules that are 50, 100, or 200 bases in length must be separable from molecules that are 51, 101, or 201 bases in length (respectively). There are several modifications to improve the resolution:
One thing I should confess is that this example has one bit of fiction - you can't
really obtain usable sequence information that close to the end of the primer, unless
your ddNTP/dNTP ratios are quite high. If you increased that ratio, you would make
it more difficult to read sequence 200 to 300 nucleotides further down, because most
of the synthetic products would have terminated before that point.
It is possible to obtain labeled products in one of two ways:
Internal labeling of products:
In this case, one of the dNTP substrates is radiolabeled (or chemically labeled) so that the synthetic products are marked internally, and possibly in many places at once. The multiple sites of incorporation mean that the product will have a higher specific activity.
One disadvantage of this is that nonspecific side reactions (perhaps having nothing
to do with the oligonucleotide) will also be labeled.
End-labeling of products:
An oligonucleotide can be labeled with a P-32 phosphate at its 5' end, for example, by the enzyme polynucleotide kinase and the substrate gamma-P-32 ATP. If this radiolabeled oligonucleotide is annealed to a template and extended with a polymerase, the products will be labeled only at their 5' end.
An advantage of this is that only reactions involving the annealing of the oligonucleotide
will be labeled. A disadvantage is that at most one labeled atom is incorporated
into the synthetic product, so the specific activity is low.
Because of this "spreading out" of the signal, you get bands that are a bit "foggy" looking when you develop the film:
Another disadvantage of P-32 is that radiolabeled substrates not have a long "shelf-life" because the half life of the P-32 nucleus is short.
P-33 phosphate is now frequently used because it solves several of the
problems associated with P-32. The radioactive decay of P-33 is of lower energy,
so a sharper image can be generated on a piece of film. The half-life of P-33 is
about twice as long, so dNTP substrates labeled with it have a longer life as well.
An unfortunate consequence of a longer half-life however, is that the synthetic products
will have a slightly lower specific activity.
S-35 thiophosphate is a second solution to the problems associated with P-32. The radioactive sulfur takes the place of one of the oxygens in the phosphate, which then becomes a "thiophosphate." It has a longer half-life (87 days), meaning that the substrate shelf-life is longer, and a less energetic decay, meaning that the film results are sharper.
Approaches based on machine reading:
How can you avoid having to "read the bases" off of a gel or film yourself? Train a machine to scan the gel with a laser as it is running, and use different fluorescent dyes to indicate the bases. The gel is run using special (and expensive) glass plates that are optically pure, and the laser scans back and forth across the lanes while the DNA fragments migrate past the laser "window."
ABI corporation makes a
machine that we use in Sc4108. They have made a program called EditView, which you can download from
their site, and which shows the types of chromatograms one obtains from the laser
Dye-labeled primers. In this method, the oligonucleotide primers are end-labeled with four different fluorescent dyes, and four separate synthetic reactions are conducted in the presence of dNTPs and ddNTPs:
Upon completion, these four reactions can be combined into one lane on a gel, and run on a machine that can scan the lanes with a laser. The wavelength of fluorescence can be interpreted by the machine as an indication of which reaction (ddG, ddA, ddT, or ddC) the product came from.
The fluorescence output is stored in the form of chromatograms: