Strings and Genomes - Infobiotics: Information in Biotic Systems - page 47

in each vessel, say in A, we get sequences of different lengths which correspond
to the positions where a ball of color A is located in the original sequence. In this
manner, by measuring the lengths of the sequences obtained in the four vessels,
we can discover the positions of the original sequence where A are located, those
where T are located, those where C are located, and those where G are located. In
conclusion, the structure of the original sequence can be completely deduced. This
process was realized by Sanger, who won his second Nobel Prize for it, by using
in each vessel a small part of nucleotides without the Oxydryl, in such a way that
when polymerase enzyme uses one of them, then the extension process stops. It is
important that the amount of these nucleotides is not so big and not so small. In fact
in the first case only short fragments are obtained, while in the second case only
long fragments are obtained. However, in almost all sequencing methods the role of
polymerase extension is essential. In a new class of methods each step is constituted
by four sub-steps, where polymerase can use only one of the four nucleotides, and
its use is made evident by coupling to it a phenomenon which produces some effect.
In this way according to which sub-step provides the effect, the kind of nucleotides
added by polymerase can be deduced; therefore all the extended sequence can be
deduced.

2.3.2 Plasmide Cloning Algorithm

DNA cloning of double strands is a process that produces many identical copies of
a given double DNA molecule. Plasmid cloning is performed by means of bacteria.
In fact, in many bacteria there are some double circular forms of DNA, called plas-
mids . Therefore, the main idea of plasmid cloning procedure consists in inserting
a target DNA molecule inside a bacterium plasmid and then letting the bacterium
proliferate in many cells which, being descendant of the same cell, have the same
DNA and consequently the same plasmid including the target DNA molecule. In
this way, we can get many copies of the initially inserted DNA, just by recovering
all these plasmids and by extracting from them the copies of the target molecule.
The following is the detailed (abstract) procedure.

1. Choose a plasmid, called also vector which includes a gene encoding the re-
sistance to an antibiotic A and a second gene encoding the resistance to an
antibiotic B.
2. Cut this circular vector by means of a suitable restriction enzyme occurring once
in the plasmid and in the middle of the sequence of gene A (see Fig. 2.23). As
indicated in the figure, the cut of the restriction enzyme realizes two specific
single strand flanking terminals.
3. Extend the target molecule in a such way that it begins and ends with sticky
ends that can hybridize with the sticky ends provided by the restriction enzyme
(see Fig. 2.24).