Sequencing of the DNA

The sequencing of DNA consists in determining the order of sequence of the Nucléotide S of a fragment of DNA given. Currently, the majority of the sequencings of DNA are carried out by the method of Sanger described below. This technique uses the reaction of polymerization of the DNA using a DNA polymerase and didésoxyribonucléotides (ddNTP).

The Séquence of DNA contains the necessary information with the living beings to survive and reproduce. To determine this sequence is thus useful as well for research aiming at knowing how the organizations as for subjects applied live. In medicine, it can be used to identify, diagnose and potentially find treatments genetic diseases. In Biology the study of the sequences of DNA became an important tool for the classification of the species.

History

The sequencing of the DNA was invented in second half of the years 1970. Two methods were developed independently, one by the team of Walter Gilbert, in the United States, and the other by that of Frederick Sanger, in Great Britain. These two methods are founded on principles diametrically opposite: the approach of Sanger is a method by enzymatic synthesis selective, while that of Maxam and Gilbert is a method by chemical degradation selective. For this discovery, Gilbert and Sanger were rewarded by the Nobel Prize for chemistry in 1980.

Initially, the method of Sanger required to lay out of a DNA simple bit which was used as matrix for the enzymatic synthesis of the complementary bit. For this reason, the first biological organization whose genome was sequence in 1977 is the virus bacteriophage φX174. This virus with the property to have a genome made up of DNA simple bit which is encapsulated in the viral particle.

During 25 last years, the method of Sanger was largely developed thanks to several important technological advances:

the development of adapted vectors of sequencing, as the phage M13 developed by Joachim Messing with the beginning of the year 1980.
the development of the automated chemical synthesis of the Oligonucléotide S which are used as starters in the synthesis.
the introduction of fluorescent tracers to the place of the radioactive markers used initially. This progress with license to leave the sequencing the confined parts, reserved for the use of radioisotopes.
the adaptation of the technique PCR for the sequencing.
the use of automatic sequencers of genes
the use of the capillary electrophoresis for separation and the analysis

The method of Maxam and Gilbert requires toxic chemical reagents and remains limited as for the size of the fragments of DNA which it makes it possible to analyze (<250 nuclétoides). Less easy to robotize, its use became confidential today.

Method of Sanger

The principle of this method consists in initiating the polymerization of the DNA using small a oligonucléotide (starts) complementary to part of the fragment of DNA with séquencer. The elongation of the starter is carried out by the fragment of Klenow (a DNA polymerase I deprived of activity exonucléase 5' →3') and maintaining by DNA polymerases Thermostable S, those which are used for PCR. The four désoxyribonucléotides (dATP, dCTP, dGTP, dTTP) are added, like in weak concentration of the one of the four didésoxynucléotides (ddATP, ddCTP, ddGTP or ddTTP).

These didésoxynucléotides, acts like terminating “poisons” of chain: once incorporated in the new synthesized bit, they prevent the continuation of the elongation. This termination is done specifically on the level of nucleotides corresponding to the didésoxyribonucléotide built-in the reaction. For the complete sequencing of the same fragment of DNA, one repeats this reaction four times in parallel, with the four didésoxyribonucléotides different.

For example, in the reaction where ddGTP was added, the synthesis stops on the level of G. the reactional mixture containing, at the same time dGTP and a little ddGTP, the termination is done in a statistical way according to whether the DNA polymerase uses one or the other of these nucleotides. It results a mixture from it from fragments of DNA of increasing sizes, which finish all on the level of one of G in the sequence. These fragments are then separated by electrophoresis on a polyacrylamide gel, which thus makes it possible to locate the position of G in the sequence.

The detection of the fragments thus synthesized is done by incorporating a tracer in the synthesized DNA. Initially this tracer was radioactive, today, one uses fluorescent tracers, attaches either with the oligonucléotide, or with the didésoxyribonucléotide.

Method of Maxam and Gilbert

This method is based on a chemical degradation of the DNA and uses the reactivities different from the four bases has, T, G and C, to carry out selective cuts. By reconstituting the order of the cuts, one can go back to the sequence of the Nucléotide S of the corresponding DNA. One can break up this chemical sequencing into six successive stages:

Marking : The ends of the two bits of DNA with séquencer are marked by a radioactive tracer (³²P). This reaction is done in general by means of radioactive ATP and of polynucléotide Kinase
Isolement of the fragment of DNA with séquencer. This one is separate by means of a electrophoresis on a gel of Polyacrylamide. The fragment of DNA is cut out gel and is recovered by diffusion
Séparation of bits . The two bits of each fragment of DNA are separated by thermal denaturation, then purified by a new electrophoresis.
specific chemical Modifications . The DNA simple-bit are subjected to specific chemical reactions of the various basic types. Walter Gilbert developed several types of specific reactions, carried out in parallel on a fraction of each bit of marked DNA. For example for G (alkylation by dimethyl sulfates), for G and has (depurination), for C and for C and T (hydrolysis alkaline). These various reactions are carried out under very spared conditions, so that on average each molecule of DNA carries only zero or one modification.
Cut . After these reactions, the DNA is cleaved on the level of the modification by reaction with a base, the Pipéridine.
Analysis . For each fragment, the products of the various reactions are separated by electrophoresis and are analyzed to reconstitute the sequence of the DNA. This analysis is similar to that which one carries out for the method of Sanger.

Sequencing of whole genome

The knowledge of the structure of a genome in its entirety can pass by its sequencing. However, the size of the genomes being several million bases (or mégabases), it is necessary to couple the approaches of molecular biology with that of data processing to be able to treat such a significant number of données.
Two great principles of sequencing of whole genome are used. In both cases, genomic DNA is split up beforehand by enzymatic methods (enzymes of restriction) or physics (Ultrason S).

the first method of sequencing called by hierarchical scheduling consists in classifying the fragments genomic obtained before séquencer
the second method known as total (or whole-genome shotgun), does not make a classification of the fragments genomic obtained but the sequence dand a random order. An analysis Bio-data processing then makes it possible to reorder the fragments genomic by overlapping of the sequences communes.

The principal difference between these two principles is that hierarchical scheduling tries to align a set of clones of big size (~ 100 KB) whereas in the global method the whole Génome is tiny room in fragments of small size which are sequences then aligned.

Hierarchical scheduling

After extraction, the DNA genomic is cut out by sonication in fragments from 50 to 200 KB then clone in a Vecteur adapted like the bacterial artificial chromosomes or VAT. The number of clones must allow a cover from 5 to 10 times the overall length of the studied genome. The overlapping and the scheduling of the clones are carried out either by Hybridation of specific probes, or by analysis of the profiles of restriction, or more frequently by a scheduling after sequencing and hybridization of the ends of the VAT. After having carried out the scheduling of the clones, they are split up and sequences individually, then assembled by alignment bio-data processing.

The advantages of this method are a greater facility of assembly of the fragments thanks to the overlapping of the VAT, the possibility of comparing the fragments with the data banks available, and the possibility of sharing the work of sequencing between several laboratories, each one having charges an area chromosomique.
with it The major disadvantage is the difficulty of cloner fragments containing of the very frequent sequences repeated in certain genomes, like those of the mammals, which makes difficult the analayse final bio-data processing.

Global method

It is about a genomic method of sequencing of DNA initially imagined in the laboratory of Frederick Sanger in Cambridge at the end of the Seventies for séquencer the first genomes of virus.

This method was popularized by Craig Venter for the sequencing of the large genomes, in particular within the company Celera Genomics. The first application was the sequencing of bacterial genomes, then genome of the Drosophile and finally of the genome Humain and Murin.
To carry out a sequencing of complete genome using this technique, two to three banks made up of fragments random of DNA genomic are realized. Between the banks, the fragments diverge as well cuts some as in localization on the Génome. Starting from these banks, many clones are sequences then assembled. The total sequence is obtained by treating the whole of the banks using tools bio-data processing, by aligning the fragments using the overlapping sequences.

The advantages compared to the sequencing by hierarchical scheduling are the speed of the technique and a weaker cost. The disadvantage is that the data processing does not make it possible to align fragments comprising of the repeated sequences of big size which are frequently present in the Génome S of the mammals.

This method is usually indicated under the name of shotgun (sawed-off shotgun), or Whole Genome Shotgun (WGS). This metaphor illustrates the randomness of the initial fragmentation of the DNA genomic: one sprinkles all the genome, a little as disperse leads of this type of firearm.

Other methods

Use of the chips with DNA
….

See too

Sequencer of genes
Sequencing of the genome

References

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