Basic methods

Early efforts at sequencing the gene arduous, and using a method known as wandering spot analysis, Gilbert and Maxam, time and effort, reported the sequence of 24 base pairs. The explorer Frederick Sanger, due to the fact of the DNA sequence, fortunately, began to change in the middle of 1970, the situation in this area some, when you have developed a technology faster, more efficient as it is connected, the work of Sanger is, to receive the Nobel Prize in Chemistry in 1980 innovative.

Over the last few decades, advances in technology that have been automated, Sanger sequencing process to accelerate dramatically, more sophisticated. Also called dideoxy method or chain termination, Sanger sequence comprises the use of a DNA polymerase enzyme that has been purified to synthesize DNA chains of varying length. An important feature of the reaction process, is that the triphosphate Sanger dideoxynucleotide (a ddNTP) is included. 3 ‘hydroxyl necessary to form a phosphodiester bond next nucleotide DNA chain elongation in chain terminating dideoxynucleotide These are not the (OH) group. Therefore, when the dideoxynucleotide is incorporated into the growing chain, and further, to inhibit chain extension. As a consequence, many of these reactions, it is the number of DNA fragments of different lengths. These fragments were separated by size by using the method to pull the molecules present in the fiber capillary electrophoresis tubes or gels, electric field, such as capillary or hair gel base. This procedure is sensitive enough to distinguish DNA fragments of different sizes by a single nucleotide.

In the method of Sanger, parallel sequencing reactions four, using the same sample in the array. Each reaction includes DNA polymerase, (a dCTP and dTTP, dATP in and dGTP) single-stranded template, specific primers to initiate the reaction, four deoxynucleotide standard. The polymerase I will add a base of the DNA chain complementary to the single-stranded template sample. One of the four dideoxynucleotide was added to each reaction at lower concentrations than the standard deoxynucleotides (the ddTTP ddGTP, or ddCTP,, the ddATP). Dideoxynucleotide is not present at all the 3 ‘OH group is incorporated at the end of a DNA polymerase DNA, to grow them. And is used for the chain to prevent terminated (i.e., T A, G, or C) always in the same nucleotide, a ddNTP four different. I can be obtained along the length of different analysis. Then, in the shot from the sample obtained in the four columns of the gel (by dideoxynucleotide added), the researchers will be able to know what is at the end of each fragment-based look at the fragment of size . This creates a DNA sequence that is easy to read.

In 1986, a company called Applied Biosystems, and will start to produce an automatic machine for DNA sequencing based on the Sanger method. The reaction is performed in one column, so as to be able to read the color, these machines are using the fluorescent dye to label the nucleotides. By running the 24 samples at a time, $ 100,000 machine provides 12,000 “letter” of DNA per day. machine is more expensive than gel and glass plate traditional when the investment is made, but also from the traditional way, on a machine with these cheaper than array data, and faster. In fact, Craig Venter, These are machines used to unify the Human Genome Project to a higher gear.

Basic Sanger method is used for the genome of consistency whole still, go to expensive you are limiting the number of potential genomic considerations of cost and time are met. Over time, the (DNA synthesis technology) and sequencing, alternative strategies separate for visualization of parallel sample of other advanced by a more complex strategy. Result of the machine, you will be able to process 96 samples in normal time today. Further, gels traditional that can be read from a single reaction, to produce 250-500 bases of the DNA sequence of the reaction, the 750-1000 base pair sequence on the basis of the beginning of the ABI machine and Sanger sequencing can, On the other hand, it is the sequence option much cheaper than used to. Further, gel is used sequencing technology is not being designed to further improve the efficiency of sequencing. Flow cytometry, scanning microscopes, mass spectrometry, and these include hybridization strategy.

Today, scientists are using 454 sequencing (Roche-house development then) and method of DNA sequencing called pyrosequencing addition of new single nucleotide more. In pyrosequencing, the number of nucleotides is limited to the cutting out DNA synthesis. Then, unlike the method of Sanger, chain extension may be reconstituted by the addition of nucleotides. Shown as a series of peaks called Pyrograms corresponding to the order of nucleotides in the character the amount of visible light that is added to the growing chain are each nucleotide is small, it is generated by enzymatic action, was added the light, and finally The sequence has revealed the primary DNA. Thus, it is possible by aligning and blinking when it is located at the time the sample nucleotides, researchers stretch of DNA sequence.

In the commercial version, by a low cost compared with the previous method, applying the technique of pyrosequencing the array easy picotiter plate of mass of DNA, 454 sequence, and can be read in 20 million base-running. Method does not rely on DNA cloning template but (ie, skip, heterochromatin-free unclonable segment) it is read in order. However, one major disadvantage of the approach is what you pyrosequencing incomplete expansion through the simple repetition of the same nucleotide or homopolymer. The difficulties that the researchers to be able to distinguish the overlapping region longer than the length of the first, each reading is about 100 base pairs at that time. However, pyrosequencing is improved rapidly, the new machine can generate reads a sequence of 400 base pairs.