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  • Methods for DNA sequencing

    The human genome consists of three billion pairs of nucleotides. Each nucleotide contains one of four nitrogenous bases – A, C, G or T, form the alphabet by which genetic information is stored in the DNA molecule. The grounds of one chain of DNA bases mate with another chain of strictly defined rules (A is paired with T, G – s), it suffices to determine the sequence of bases in one of them.

    To identify the specific grounds in some regions of the genome is required sensor capable subnanometrovoe notice the difference between A, T, G and C. The only physical method that has such a high resolution – scanning tunneling spectroscopy. However, the sequencing of the length of the billions of links are most often used, not the physical and chemical sposoby.Genom man was decoded using the technique developed in the late 1970’s. American biochemist Frederick Sanger. This procedure is preceded by cutting sequencing study of the DNA molecule into fragments, cloning them in E. coli and multiple duplication to obtain millions of copies of each fragment. As a result of the last round of duplication, conducted in special circumstances, receive a set of copies of fragments of various lengths, each of which ends with a fluorescently labeled nucleotide. Fragments are separated by length using electrophoresis, register the light signal from each of them as it passes through the detector and obtain the nucleotide sequence of the original circuit.

    The advantages of the method of Sanger include its relative simplicity and high precision, but despite subsequent improvements, it remains expensive and time consuming. The challenge the founders of alternative ways of sequencing was to increase the speed of the procedure and its cheaper. For this it was necessary to exclude the stages of separation, time-consuming, miniaturized the entire system, while preserving the opportunity to read the sequence millions of fragments.

    Many research groups have based their developments biosynthesis – a process that uses living organisms when playing its genome and removing it damage. Thus, in the cell prepares to divide, untwist the DNA double helix, its constituent chains apart, and then on each of them is synthesized by a new chain (one – continuously, on the other – is intermittent, with the formation of separate fragments). The process of successive additions to a growing chain of nucleotides is catalyzed by specific enzyme DNA polymerase. Another enzyme, ligase, binds the fragments. The result is two new full length polynucleotide chain complementary to the DNA matrix in which they are synthesized.

    Methods for sequencing using the biosynthesis taken as a basis for the stages that process, which occur on a single chain sekveniruemoy DNA. Record the time of accession to the primer hybridized with the DNA-matrix, the complementary nucleotide (extension chain), or the moment matching ligase primer with oligonucleotide probe containing known nucleotide in a certain position.

    There are different ways of data acquisition processes, but usually use one of two types of signals. If the label is attached to a nucleotide fluorophores, then recorded it emits light of a certain wavelength. Fluorescence detection is used for sequencing as a method of lengthening the chains, and the method of ligation. It is used by many researchers, among them – Metsker, Michael (Michael Metzker) from the University of Baylor, Robi Mitra (Robi Mitra) from the University of Washington, and led my laboratory at Harvard Medical School and the Corporation Agencourt Bioscience.

    Published on October 16, 2012 · Filed under: Bioscience, Diagnostics, DNA;
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