GENETIC ENGINEERING.

1. DNA SEQUENCING.
DNA sequencing is the process of determining the sequence of nucleotides (As, Ts, Cs, and Gs) in a
piece of DNA. There are two types
 In Sanger sequencing, the target DNA is copied many times, making fragments of different lengths.
Fluorescent “chain terminator” nucleotides mark the ends of the fragments and allow the sequence to be
determined.
 Next-generation sequencing techniques are new, large-scale approaches that increase the speed and
reduce the cost of DNA sequencing
Sanger sequencing: The chain termination method
Regions of DNA up to about 900 base pairs in length are routinely sequenced using a method
called Sanger sequencing or the chain termination method. Sanger sequencing was developed by the
British biochemist Fred Sanger and his colleagues in 1977.
In the Human Genome Project, Sanger sequencing was used to determine the sequences of many
relatively small fragments of human DNA. (These fragments weren't necessarily 900900900 bp or less,
but researchers were able to "walk" along each fragment using multiple rounds of Sanger sequencing.)
The fragments were aligned based on overlapping portions to assemble the sequences of larger regions
of DNA and, eventually, entire chromosomes.
Although genomes are now typically sequenced using other methods that are faster and less expensive,
Sanger sequencing is still in wide use for the sequencing of individual pieces of DNA, such as fragments
used in DNA cloning or generated through polymerase chain reaction (PCR).
Ingredients for Sanger sequencing
Sanger sequencing involves making many copies of a target DNA region. Its ingredients are similar to
those needed for DNA replication in an organism, or for polymerase chain reaction (PCR), which copies
DNA in vitro. They include:
 A DNA polymerase enzyme
 A primer, which is a short piece of single-stranded DNA that binds to the template DNA and acts as a
"starter" for the polymerase
 The four DNA nucleotides (dATP, dTTP, dCTP, dGTP)
 The template DNA to be sequenced
However, a Sanger sequencing reaction also contains a unique ingredient:
 Dideoxy, or chain-terminating, versions of all four nucleotides (ddATP, ddTTP, ddCTP, ddGTP), each
labeled with a different color of dye

_Image credit: "Whole-genome sequencing: Figure 1," by OpenStax College, Biology (CC BY 4.0)._
Dideoxy nucleotides are similar to regular, or deoxy, nucleotides, but with one key difference: they lack
a hydroxyl group on the 3’ carbon of the sugar ring. In a regular nucleotide, the 3’ hydroxyl group acts
as a “hook," allowing a new nucleotide to be added to an existing chain.
Once a dideoxy nucleotide has been added to the chain, there is no hydroxyl available and no further
nucleotides can be added. The chain ends with the dideoxy nucleotide, which is marked with a particular
color of dye depending on the base (A, T, C or G) that it carries.
[Where is the dye attached?]
Method of Sanger sequencing
The DNA sample to be sequenced is combined in a tube with primer, DNA polymerase, and DNA
nucleotides (dATP, dTTP, dGTP, and dCTP). The four dye-labeled, chain-terminating dideoxy
nucleotides are added as well, but in much smaller amounts than the ordinary nucleotides.
The mixture is first heated to denature the template DNA (separate the strands), then cooled so that the
primer can bind to the single-stranded template. Once the primer has bound, the temperature is raised
again, allowing DNA polymerase to synthesize new DNA starting from the primer. DNA polymerase
will continue adding nucleotides to the chain until it happens to add a dideoxy nucleotide instead of a
normal one. At that point, no further nucleotides can be added, so the strand will end with the dideoxy
nucleotide.
This process is repeated in a number of cycles. By the time the cycling is complete, it’s virtually
guaranteed that a dideoxy nucleotide will have been incorporated at every single position of the target
DNA in at least one reaction. That is, the tube will contain fragments of different lengths, ending at each
of the nucleotide positions in the original DNA (see figure below). The ends of the fragments will be
labeled with dyes that indicate their final nucleotide.
[Will all the fragments be labeled?]

Image modified from "Sanger sequencing," by Estevezj (CC BY-SA 3.0). The modified image is licensed
under a (CC BY-SA 3.0) license.

After the reaction is done, the fragments are run through a long, thin tube containing a gel matrix in a
process called capillary gel electrophoresis. Short fragments move quickly through the pores of the gel,
 while long fragments move more slowly. As each fragment crosses the “finish line” at the end of the
tube, it’s illuminated by a laser, allowing the attached dye to be detected.
The smallest fragment (ending just one nucleotide after the primer) crosses the finish line first, followed
by the next-smallest fragment (ending two nucleotides after the primer), and so forth. Thus, from the
colors of dyes registered one after another on the detector, the sequence of the original piece of DNA
can be built up one nucleotide at a time. The data recorded by the detector consist of a series of peaks in
fluorescence intensity, as shown in the chromatogram above. The DNA sequence is read from the
peaks in the chromatogram.

2. GEL ELECTROPHORESIS.
A technique used to separate DNA fragments and other macromolecules by size and charge.
As the name suggests, gel electrophoresis involves a gel: a slab of Jello-like material. Gels for DNA
separation are often made out of a polysaccharide called agarose, which comes as dry, powdered flakes.
When the agarose is heated in a buffer (water with some salts in it) and allowed to cool, it will form a
solid, slightly squishy gel. At the molecular level, the gel is a matrix of agarose molecules that are held
together by hydrogen bonds and form tiny pores.

3. PCR.
Cette technique décrite en 1985 (K. MULLIS et collaborateurs) permet d’amplifier des séquences
d’ADN de manière spécifique et d’augmenter de manière considérable la quantité d’ADN dont on
dispose initialement. Elle nécessite de connaître la séquence des régions qui délimitent l’ADN à
amplifier. Ces séquences serviront à synthétiser des amorces oligonucléotidiques complémentaires (de
longueur de 20 à 30 nucléotides en général). Ces oligonucléotides serviront à délimiter la portion
d’ADN à amplifier. L’ADN polymérase les utilisera comme amorces
4. Cloning.