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Sequencing
The theory behind sequencing.
The old way to do it!
New tools, new techniques.
The theory behind sequencing
 ^Same Base |
Here is a simple observation. Figure 1 shows a collection of fragments of DNA, copies of the same sequence, starting with the same nucleotides, and having various sizes. They have been sorted by size. Imagine now that you could
identify the last nucleotide. If you read the last nucleotide of each fragment, starting with the smallest one, then, you are actually reading the sequence of the gene.
It is easy to separate DNA according to size.
We have shown that elsewhere. The main theoretical
problem is to obtain these special fragments in the first place. They must all start at the same point, and we must be able to identify their last nucleotide |
Maxam and Gilbert Method
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We start with a DNA fragment whose 5' phosphates are labeled with the radioactive isotope 32P. Both extremities are labeled. The next step is to get rid of one of the ends, because it could confuse the results (figure 2). We use a restriction enzyme to cut the fragment end, and get rid of it, via a gel filtration column if it is small, or via a gel purification. Gel purification is actually quite awkward, as the fragment will be radioactive. |
The mixture is aliquoted into four tubes,
in which different reactions will be performed. DNA will be cleaved by
enzymes that will cut at specific sites. Traditionally, we use enzymes
that will cut
A=Adenine, T=Thymine, C=Cytosine, G=Guanine. After the gel has run, (and sometimes we perform a double
run Try and read this sequence before checking the result! Note: this method is rarely used: it requires 32P, involves lots of steps and takes almost a week to do all the reactions for just one sample. |
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Sanger's method
Sanger had a more constructive approach. He decided not to cut, but
polymerize DNA from one known primer, and make sure it stops polymerizing
at a precise nucleotide. The nucleotides that make up DNA are deoxyribonucleotides
: they have an OH removed from the normal ribose sequence, but still
have two left, the 5' and the 3' which are essential to the building of
DNA. As we have seen elsewhere, DNA is replicated normally from 5' to 3',
and nucleotides are added to the 3' OH, if there is an OH end there. If
it is missing, no nucleotide can be added, and the polymerization stops.
So if we add some dideoxy-nucleotides , which lack the 3' OH, to the reaction mixture and if one of them is incorporated
in the sequence during the polymerization, the polymerization will stop there. This dideoxynucleotide
can be incorporated at any time, and the chance it is incorporated at the
beginning or at the end of the sequence is equal. It means, that after
a while, the tube where the reaction takes place should contain fragments
of all sizes.
To perform Sanger's sequencing method, one prepares 4 aliquots of our template DNA, in four tubes, and incubates them in a similar medium. The only thing that will change is the dideoxynucleotide used. Each tube will contain one type of dideoxynucleotide (A, T, C, G) (and all the deoxynucleotides), and a radioactive (35S) dATP. Therefore
- in tube A, at the end of the reaction we will obtain a selection of fragments of DNA, of different sizes, all finishing with A.
- in tube C, at the end of the reaction we will obtain a selection of fragments of DNA, of different sizes, all finishing with C.
- in tube T, at the end of the reaction we will obtain a selection of fragments of DNA, of different sizes, all finishing with T.
- in tube G, at the end of the reaction we will obtain a selection of fragments of DNA, of different sizes, all finishing with G.
The following is easy: load all four tubes in four different tracks of a gel, run the gel, and read the sequence. All the fragments will be labelled, and an autoradiography of the gel will reveal the position of the bands. Figure 4 represent the same sequence as read with the Maxam Gilbert method, but this time the Sanger's method has been used. 
The old way to do it!
A method used in laboratories until quite recently:
- Prepare plasmid DNA : that could take one to three days depending on
the sample. You need about 200 ng of DNA to
be able to sequence it successfully.
- We use a kit containing an enzyme designed for sequencing.
- We still have to prepare a gel, which is a delicate and time consuming
method. Two heavy plates are specially coated, and assembled together,
and a polyacrylamide gel is poured in
between. Polyacrylamide gels are used for sequencing because they can separate fragments of DNA that differ only by one base pair, while agarose gels do not have this resolution power. The gap between the plates is only a fraction of a millimeter.
- While the gel is polymerizing (this could take from 30 minutes to one hour, and it's best to do it the day before you run the gel), prepare your sequencing mixtures (sequencing buffer with nucleotides,radioactive dATP, and sequencing primers), and four small tubes per template fragment you want to sequence. .
- The sequencing is done in two steps, which are performed successively, and need to be well timed, as the polymerase enzyme works fast.
- Once the polymerase is loaded into the tube containing the DNA, there is a three minutes break before you have to aliquot this mixture in the four labeled ATGC tubes which each contain the respective dideoxynucleotide. Once the aliquot is added to the dideoxynucleotide, the reaction will begin stopping at random.
- After a few minutes, you can put the tube on ice, add a blue dye, and load each aliquot onto four adjacent wells of your gel. The run will take 4 to 6 hours, and then you will have to "fix" it, and put it in a cassette for exposure. After a few days you will be able to read your sequence on the autoradiograph.
New tools, new techniques
New tools have appeared on the market such as PCR,
(which we discussed earlier), fluorescent marking, laser technology and
computers. All together, they help the researcher by lightening their load
and reducing the time between the preparation of a sample and getting results (its
sequence).
You start with your sample from wich you extracted few molecules
of DNA. This takes about half a days work. Two nested
PCR reactions will give you a concentrated sample of the DNA you want
to sequence. You perform the first PCR in the afternoon, and the second
PCR the following morning.
Incubate a small amount of this DNA, with a powerful Taq polymerase,
and the four different dideoxynucleotides fluorescently marked (each dideoxynucleotide is marked with a different fluorescent dye to distinguish them from each other). You can perform
the reaction in a single tube. Put your tube in the PCR machine with the appropriate
programme, and wait about 3 hours. At the end of the 20 cycles, you need to
rinse your DNA and give it to the technician in charge of running the "Sequencer
Machine". The "sequencer" includes a gel and a laser which records the peaks of fluorescence that pass through it which corresponds to the dideoxynucleotides' dies.
It is attached to a computers which record the sequence read. The computer will give you back a graphic file representing
your sequence and a text file of the DNA's sequence. You have
to make changes where the computer couldn't decide which base occupies
a particular space in the sequence. The total time is still two or three
days, but your actual time is reduced if you use a technician to man the "Sequencer".
This time can be used to perform other experiments, or write and read papers.
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