Its alteration by mutation causes the appearance of diseases or impairments such as cancer and muscular dystrophy . To decipher the genomes of individuals could target these mutations for patients to understand the changes causing the disease, can make predictions for predisposition to certain diseases, making early detection and propose targeted treatment. This is the key to both preventive medicine and completely personalized.
However , these promises can be held only if the decryption – the sequencing of DNA – is fast and inexpensive. Knowing that it took 15 years of efforts, between 1989 and 2004 , and over a billion dollars to achieve completely decipher the human genome under the Human Genome Project ( HUGO ) , must be put in place techniques reducing the costs and time by a factor of 100,000. That’s what the program ” $ 1,000 Genome ” in place in the United States since 2004 in which nanotechnology plays a central role [1,2] . $ 9.5 million of funding has been invested by the National Human Genome Research Institute ( NHGRI ) in this program for Fiscal Year 2010.
DNA and current methods of sequencing
Four building blocks (adenine , thymine, cytosine and guanine – A, T , C and G ) which follow two complementary strands coiled double helix DNA can be compared to a long string of characters . In the case of man, should be 1000 books 700 pages to write all 3 billion letters of the genome . DNA sequencing is complete reading of the 1000 book , letter by letter. The most effective method seems to start by the first page of the first book and perform the decryption letter after letter. But the double helix of DNA has a diameter of 4 nm and the nitrogen bases that constitute the concatenation are spaced 0.34 nm . No instrument can now go to “read ” the molecule at this scale . Researchers have developed the techniques with macroscopic methods available. The analogy with the books can get an idea of the monumental effort it represents .
You have before you 1000 pounds of 700 pages each. On each page , you know there are characters write in invisible ink . You can make a character appear randomly on both the page and whether it is an A, T, C or G. You can also make a layer of the page that allows you to copy the position of letter that you showed .
The Sanger method involves first to tear a page in a book . You get to see a letter on the page and you base the page . The letter goes. Can you get a second letter and get a new layer. The problem is that you can not choose the letter that you appear. We must therefore make thousands of layers to be on seeing all the letters appear on the page. The next step is to order all the layers obtained in order to reconstruct the full sequence of characters on the page . It ‘ll just repeat this work for each of the 700,000 pages to read . The last step is to put these pages in order . This is only possible using supercomputers, capable of finding the overlap between the different pages in order to reconstruct the succession .
This method , long expensive, was the one used in the HUGO project . Copies deciphered sections are made with enzymes whose reliability is not perfect and produce a number of errors. The multiplication of portions to decipher also causes the proliferation of reading errors . It is thus necessary to repeat the operation several times to achieve decoding error rates of 1 per 10,000 bases, set in the context of HUGO .
The pyrosequencing represents the second generation of methods and is now the most common technique employed . It is a sequencing by synthesis. An enzyme creates the complementary strand of the strand to sequence by adding nucleotides one after the other . At each addition, a chemical reaction causes the appearance of a light signal that identifies the nitrogenous base that has been integrated . This method has the advantage of not requiring the reproduction of multi -stranded DNA sequencing . It reduces the costs and delays of a factor 3 compared to the Sanger method .
These last three years, a third generation methods, ” Single Molecule Sequencing Real Time ” made its appearance . She is already using nanotechnology in progress to optimize the pyrosequencing methods . It is again to build the complementary strand of DNA using an enzyme. This time , the fluorophores are directly attached to nucleotides . A nanophotonic structure , called “zero -mode wave guide ” , composed of holes 70 nm in diameter and 100 nm long in a layer of aluminum deposited on a glass ensures effective detection of light signals and allows parallel sequencing many strands . The Companies Pacific Biosciences has received a grant of $ 6.6 million under the program ” $ 1000 genome ” the NHGRI to commercialize the method developed at Cornell University [ 3] .
The methods do not allow second- generation sequencing as pieces composed of a few tens of nitrogenous bases . The third generation improves the performance for the sequencing of strands up to 1000 bases at a speed of 10 bases per second. However , these techniques are limited by the relatively slow speed with which enzymes generate the complementary strand. To overcome these techniques , researchers are working on methods not requiring enzymes and based on “solid state nanopores ” .
The nanopores , key enablers of future sequencing
The development of nanotechnology now allow us to consider the drastic simplification of the sequencing methods . The control of matter at the nanometer scale allows one to create means of direct reading of the sequence of nitrogenous bases of DNA. These techniques are based on the translocation of a strand of DNA through a nanopore .
These nanopores are holes with a diameter of few nanometers , which are made in different materials depending on the technique used. The material separating two liquid media . The strand of DNA , negatively charged , is immersed in an environment negatively charged and is attracted by this solution of the other side of the nanopore , positively charged. This is where the DNA strand through the nanopore that scrolls the reading of nitrogenous bases that is can take place.
The use of nanopores allows playback of long chains of DNA directly without having to duplicate them in quantities as important as what was required by the old methods. The recombination sequences decoded is also simplified because they are much less numerous.
The group of Prof. Amit Meller of Boston University College of Engineering has developed a method for optical detection [4 ]. The technique used is an indirect method of reading DNA . The rope is first converted , each base is translated as a single assembly of two oligonucleotides (series of some bases ) , a method called circular DNA conversion . This single strand of DNA “lying ” is then probed using fluorophore labeled oligonucleotides to obtain double stranded helix ( Figure 2 ).
Passing through the nanopore , drilled in a silicon nitride layer 50 nm thick, the hybridized portion of the strand is detached causing the excitation of fluorophores and the emission of a characteristic light signal . The registration of the succession of these signals by a CCD camera to trace the sequence of oligonucleotides and therefore nitrogenous bases of DNA strand initial [5,6].
The frequency of reading is between 50 and 250 bases per second. By improving the technology through the use of four fluorophores instead of 2 now, researchers hope to reach frequencies of 500 bases per second. Restricting the technique is linked to the procurement capacity of the CCD camera becomes the limiting factor . By improving the camera, it will be possible to accelerate the process since the speed of the blade through the nanopore is controlled precisely.
A quick calculation to realize that even at a rate of 500 continuous bases read per second , it takes 70 days in order to decipher the entire genome of an individual. The main advantage of optical detection lies in the fact that the parallel between nanopores presents no difficulty. Thus , instead of using a single pore , the researchers plan to build hundreds of nanopore arrays facing an acquisition matrix CCD ( Figure 3 ) . Each pixel of the CCD acquires the light signals emitted by a nanopore.
This net benefit outweighs the main drawback of the method is the conversion step of the DNA strand in a string of oligonucleotides and hybridization . The commercialization of this method is considered by the creation of a start – up, NobleGen Biosciences , Inc. .
Other methods are developed based on the detection of variation of electric current at the nanopore during the passage of different nitrogen bases during translocation . These techniques require immobilization of the DNA strand at each nitrogenous base to allow the acquisition of the measure .
To check this , the IBM researchers have developed a transistor DNA . It is a multilayer structure of nanometric thickness is dug in which a nanopore . The succession of conductive and insulating layers of the structure to control the passage of DNA strands in the nanopore to carry out the measures necessary for the identification of nitrogenous bases. To conduct this work, the IBM researchers are associate researchers at Roche to combine expertise in microelectronics , information technology and computational biology skills in the first sequencing of the second. Roche is in fact the current leader of the sequencing methods with automatic equipment developed by his firm 454 Life Sciences was founded in June 2000  .
The main advantage of the electrical detection comes from the fact that the strands of DNA can be analyzed directly . However , the nitrogen bases are separated by only 0.34 nm , this involves securing an extreme precision in the manufacture of multilayer transistor DNA . For now , theoretical models and numerical simulations indicate that this is possible . The practical realization of the structure remains to be demonstrated. A conversion step , identical to that used by the team of Prof. Miller might be necessary.
This need for precision could be obtained using graphene . A team from the University of Pennsylvania has developed a structure comprising a nanopore in a graphene layer (Figure 1) [ 8]. The layer is monatomic, its thickness is less than the distance between two nitrogen bases . However, to improve the robustness of the nanopore and improve the signal to noise ratio of the measurement of electric current , the graphene layer is coated with titanium oxide . If the detection step is improved by the layer of graphene , the team must work on controlling the scrolling of the strand of DNA in the nanopore.
A team of researchers at Arizona State University have used carbon nanotubes as nanopores  . Variations of currents in the nanotubes recorded during the passage of DNA strands that would suggest possible to control and perform a reading of nitrogenous bases using nanotubes. However, modeling is needed to understand the mechanisms of electronic interaction between the nanotubes and the DNA strands .
The main difficulty of using nanopore technology is to control the speed of DNA migration . The methods developed using all cylindrical nanopores . A team from Sandia National Laboratories in New Mexico is developing a nanopore sawtooth slowing the translocation of DNA by a factor of 5 (Figure 4 ) [ 10 ].
Another advance has been published by researchers at the University of Washington [ 11 ]. Jens Gundlach ‘s team used a nanopore of organic origin . This is a barrel -shaped protein , porin , which ensures the exchange of certain molecules in the membranes of bacteria. It took a change of these bacteria , M. smegmatis to produce a suitable nanopore . The transition of DNA in the nanopore is a variation of ion current to the translocation of the strand . Each nitrogenous base induces a different variation of this current which identifies them .
But it takes a millionth of a second nitrogenous base that passes through the nanopore. Too fast for a reading of the strand. To control the speed of translocation , the researchers used a method of extending the genome. They have added a section of DNA double strand between each of the nitrogenous bases of single-stranded sequence . The double strands are too large to pass through the pore . They then blocked the translocation till they split . Researchers have created a barrier that stops the translocation for a few milliseconds, the time to capture the signal of the nitrogen base to identify.
In 10 years, the evolution of techniques of DNA sequencing has been spectacular . The first generation method is exceeded, the second is heavily used , the third is nearing commercialization , and in their laboratories , researchers are preparing already the fourth generation that looks revolutionary.
Nanotechnology will soon allow ” read “the DNA in the same way that a laser beam reads the estate of a CD . However, beyond the technical difficulty of developing an effective reader , the sequencing of DNA leads to other problems . How , for example , store and manage the huge amount of data represented by the DNA sequencing of millions of people ? How to protect data ?If knowledge of the individual genome will provide individualized medical responses , it may also be the source of discrimination, particularly on the part of health insurance policies . To solve these ethical problems, Congress passed, almost unanimously , May 21, 2008 , the Genetic Information Nondiscrimination Act (GINA) . This law prohibits discrimination on the genome. Will there be enough to ensure a beneficial use of sequencing the human genome?