|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. Two teams came up with two different approaches: one team, Maxam and Gilbert's , chose a 'somewhat digestive approach', while the other one, Sanger's, found something more 'constructive'. Both methods have led to numerous applications still in use today.
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) one can read the bands from the bottom, and interpret the results. Figure 3 show what the autoradiography looks like. A band in 1, and not in 2 is an A, a band found in track 1 and in track 2 is a G, and so on.
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.
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
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.
A method used in laboratories until quite recently:
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.