Chain-termination methods

Be a way of DNA sequencing based on the selective uptake of the chain to end the dideoxynucleotide by a DNA polymerase in the DNA replication.Developed of the in vitro by his colleagues and Frederick Sanger in 1977 Sanger sequencing It is the method most widely used of about ’25 bedding. More recently, the Sanger sequence, displaced by the order, “next generation” sequencing method of large-scale in particular, automated analyzes the genome. However, it remains for the small, widely used, especially the method of Sanger reads for the preparation of long DNA sequences of these specific.

Chain termination method developed by colleagues Frederick Sanger in 1977 soon became the method of choice for reliability and its relative ease. I have invented using a smaller amount of radioactivity and toxic less than Gilbert method and Maxam chemicals when chain terminator method. Is a method not its relative ease, be used in the first generation of DNA sequencer for the Sanger method, which is automated. It is a method by Sanger sequencing, won the mid-2000s from the 1980s. During this period, the great strides, for example, automating a common fluorescent labels and capillary electrophoresis, etc., has been made in the art. This leads to cost savings, these developments have allowed the sequence more effective. Ushered in the era of genome, Sanger method is a technology to produce the first human genome in 2001 in the form of mass production. However, after 10 years, one hundred million U.S. dollars down approach fundamentally different on the market, $ 20.01 million price tag of the genome, in 2011.

Chain-termination methods

The chain termination method classic, it requires and, (dNTP of) deoxynucleosidetriphosphates single-stranded DNA template, primer DNA, DNA polymerase, normal modified nucleotides (dideoxyNTPs), he quit the DNA chain elongation it. Chain end these lacks 3′-OH group required for the formation of phosphodiester bonds between nucleotides of the two samples obtained by DNA polymerase to stop the extension of DNA incorporating the ddNTP nucleotides . You can assign a label to detect the fluorescence automated sequencing machine or out radioactive ddNTP. DNA sample is divided into sequencing reactions four separate containing DNA polymerase (a dCTP and dTTP, dATP in the dGTP) deoxynucleotides four all standard. To each reaction, it was added to only one of (the ddTTP ddGTP, or ddCTP,, the ddATP) 4 dideoxynucleotide between nucleotide of the other three were normal. Put it in the order more reasonable, to test the ddNTP all four separate reactions of four, is required for this process. Heat-denatured, DNA fragments after a round of extension of a primer template DNA binding, obtained are separated by size using gel electrophoresis. In many cases, this is polyacrylamide to each of the four reaction run in one (Lane, T, G, C) of lane four separate – is done with the help of urea denaturing gel. DNA bands can be can be visualized by UV light or autoradiography Thereafter, DNA sequence into a gel or images account X-ray film directly.

The right image, X-ray film was exposed to the gel corresponding to DNA fragments of different lengths, a dark band. Dark band of the band shows, the DNA fragment is the result of chain termination after (the ddTTP ddGTP, or ddCTP,, the ddATP) incorporation of dideoxynucleotide. Of the four lanes, the relative position of the different bands were used to read the DNA sequence from the bottom to the top.

DNA fragments are labeled on the label or ddNTP has a fluorescent marker of primer of new DNA chain of the dNTP or radioactive label. Technical variations of chain termination sequence comprises the use of a primer labeled at the 5 ‘end with a fluorescent dye marking or nucleotides containing radioactive phosphorus radiolabeled. The dye primer sequencing, faster, reading will be easier in the optical system for the automated analysis and more economical. Development after a colleague and Leroy Hood of the primers, please set the automated, the stage for high-throughput DNA sequencing with fluorescent-labeled ddNTP. Chain termination method will simplify DNA sequencing considerably. For example, chain termination based kits are commercially available, previously dispensed for use, contains the reagents necessary for sequencing ready. Limits affect the accuracy of the sequence affects the secondary structure of DNA and accurate readings of the DNA sequences, non-specific binding of the primer and the like in the DNA.

Dye-terminator sequencing using four reaction that does not like the tag and label primer method, the permission ddNTP chain terminator sequencing reaction. Dye – The terminator sequence, each of the deoxynucleotide chain terminators 4, each emitting light of different wavelengths is labeled with a fluorescent dye. Current, speed paint terminator sequence and the big speed is the core of the sequence of automated the cause. Hair dye due to the difference in the formation of the dye-labeled chain terminators into DNA fragments obtained in (see figure) DNA sequencing tracks electronic chromatogram of the capillary after electrophoresis and the height unevenness of peak contains its limits are.  Encapsulation of variability, and to minimize the “hairs”, the method for removing stains, this problem is addressed by the modified DNA polymerase enzyme system, and using a dye. Dye has a DNA sequence highly automated analyzers – terminator sequence method is used in most of the sequencing project now.