ADN Analyze

ADN request is too character character bazo nucleotide (As, Ts, Cs and Gs) in a ADN fractional segment. Failed to resolve the gen of the global character is of the physical materials with ADN large amount is a complex complex. This requires an cut the gen ADN system to more than nhỏ hơn, after which extracting and sort the small blocks to a string to a long string or all a whole resolution. For the previous process of the world of the world of the world will be lost many many time and job. However, do this do on the new method is already found in the two cross, over the gen to the gen of the gen to the rapid and very fine, cost for the global suite Rẻ hơn.

Methods of DNA sequencing

Sanger sequensing
DNA sequencing by Sanger was developed by British chemist Fred Sanger and colleagues in 1977. This method is often used to sequentially divide short sections of DNA below 900 base pairs.

Ingredients for Sanger Sequence

Sanger DNA sequencing performs the same steps as PCR techniques. The composition of the reaction also includes substances necessary for DNA replication, or for polymerase chain reaction (PCR), copying of DNA in vitro. Include:

An enzyme of DNA polymerase
A primer, is a short piece of single-stranded DNA combined with a DNA template and acts as a "starter" for the polymerase
4 DNA nucleotides (dATP, dTTP, dCTP, dGTP)

DNA sequence
In addition, the Sanger method requires the addition of a special ingredient:

Dideoxy nucleotides of all four types of nitrogenous bases (ddATP, ddTTP, ddCTP, ddGTP).

The dideoxy nucleotide is similar to the nucloeotide or deoxy common. However, they lacked the hydroxyl group at the 3 'end of the sugar molecule. The 3'-OH group acts as a hook, allowing a new nucleotide to be added to the chain. When dideoxy nucleotides are added to the chain, there is no 3'-OH group, so the nucleotide is not added, the synthesis will stop. Thus, when performing a PCR reaction with the eight nucletide components above, DNA strands of 1 length are formed. These DNA molecules are then denatured and run electrophoresis, to determine the sequence.

Sanger ordering method

The DNA fragment needs to determine the order in which the line is inserted into the splitting vector. Simultaneously, four DNA synthesis reactions were performed in four different test tubes. Each reaction was a mixture of DNA sequencing DNA, DNA primer, DNA polymerase, 1% ddNTP marked radioisotope for each reaction, and 4 dNTP types. Synthesized DNA sequences of varying lengths due to the ddNTP molecule are randomly assigned to stop the reaction and to be detected by electrophoresis.

The products of the four test tubes were also dissociated on the same gel that made the radiation image possible to observe the DNA strips on the gel. Based on the position of the lines visible on the gel, the nucleotide sequence of the gel was determined in a 5 -3 direction from bottom to top.

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Progress and limitations

The Sanger sequence can be made with relatively long pieces of DNA (about 900 base pairs). This method is often used to arrange individual DNA, such as bacterial plasmids or DNA copied in PCR.

However, Sanger's DNA sequencing is costly and ineffective for larger-scale projects, such as genome sequencing or metagenome sequencing.

New generation DNA sequencing: Pyrosequensing 454
Porysequending 454 by 2 scientists Nyren and Ronaghi of the Stockholm Institute of Technology invented and developed by the 454 Life Science Company, a highly sequential two-step DNA sequencing system with a capacity of Much more than the Sanger sequencing system.

Principles

Techniques based on the "synthetic sequence" principle include: initiating a sequenced DNA strand and complementary filament sequences by enzyme reactions. This is a system that can amplify a large number of DNA fragments in a picotter well. The principle of "sequencing by synthesis" is also based on the recognition of the pyrophosphate (PPi) released during nucleotide embedding, which produces a light signal, more efficient than dideoxynucleotide sequencing. .

Method of operation

454 pyrosequensing is a method of generating high-homogeneous DNA fragments. First, the DNA was cleaved of the first segments by and attached to the oligonucleotide adapter at both ends. These sections are then attached to a glass bead and amplified by PCR in oil-water droplets, producing multiple copies of DNA on each glass bead. Glass beads are then kept in picotter wells on a fibrous substrate and sequenced.

The sequencing process takes place in 5 steps. First, the yarn, a single-stranded PCR product, was crossed with a sequencing primer and then incubated with DNA polymerase enzymes, ATP sulfurylase, luciferase and apyrase as well as 5 'phosphosulfate adenosine substrates (APS) and luciferin. When the first dNTP is added, it is attached to the DNA strand by DNA polymerase and attached to a PPi. PPi is converted to ATP by the enzyme ATP sulfyrelase with the involvement of APS, after which a reaction catalyzed by luciferase converts luciferin to oxylciferin - producing visible light with a brightness corresponding to ATP create. This light is detected by a camera and analyzed by a software program.

Apyrase degrades unmatched nucleotides and ATP. After degradation, the reaction begins again with the other nucleotide attached. After a continuous process, complementary DNA strands were synthesized and nucleotide sequencing was determined from the residue