DNA sequencing

DNA sequencing encompasses biochemical methods for determining the order of the nucleotide bases, adenine, guanine, cytosine, and thymine, in a DNA oligonucleotide. The sequence of DNA constitutes the heritable genetic information in nuclei, plasmids, mitochondria, and chloroplasts that forms the basis for the developmental programs of all living organisms. Determining the DNA sequence is therefore useful in basic research studying fundamental biological processes, as well as in applied fields such as diagnostic or forensic research. The advent of DNA sequencing has significantly accelerated biological research and discovery. The rapid speed of sequencing attainable with modern DNA sequencing technology has been instrumental in the large-scale sequencing of the human genome, in the Human Genome Project. Related projects, often by scientific collaboration across continents, have generated the complete DNA sequences of many animal, plant, and microbial genome.




The newly synthesized and labeled DNA fragments are heat denatured, and separated by size (with a resolution of just one nucleotide) by gel electrophoresis on a denaturing polyacrylamide-urea gel. Each of the four DNA synthesis reactions is run in one of four individual lanes (lanes A, T, G, C); the DNA bands are then visualized by autoradiography or UV light, and the DNA sequence can be directly read off the X-ray film or gel image. In the image on the right, X-ray film was exposed to the gel, and the dark bands correspond to DNA fragments of different lengths. A dark band in a lane indicates a DNA fragment that is the result of chain termination after incorporation of a dideoxynucleotide (ddATP, ddGTP, ddCTP, or ddTTP). The terminal nucleotide base can be identified according to which dideoxynucleotide was added in the reaction giving that band. The relative positions of the different bands among the four lanes are then used to read (from bottom to top) the DNA sequence as indicated.


There are some technical variations of chain-termination sequencing. In one method, the DNA fragments are tagged with nucleotides containing radioactive phosphorus for radiolabelling. Alternatively, a primer labeled at the 5’ end with a fluorescent dye is used for the tagging. Four separate reactions are still required, but DNA fragments with dye labels can be read using an optical system, facilitating faster and more economical analysis and automation. This approach is known as 'dye-primer sequencing'. The later development by L Hood and coworkers of fluorescently labeled ddNTPs and primers set the stage for automated, high-throughput DNA sequencing.



The different chain-termination methods have greatly simplified the amount of work and planning needed for DNA sequencing. For example, the chain-termination-based "Sequenase" kit from USB Biochemicals contains most of the reagents needed for sequencing, prealiquoted and ready to use. Some sequencing problems can occur with the Sanger Method, such as non-specific binding of the primer to the DNA, affecting accurate read out of the DNA sequence. In addition, secondary structures within the DNA template, or contaminating RNA randomly priming at the DNA template can also affect the fidelity of the obtained sequence. Other contaminants affecting the reaction may consist of extraneous DNA or inhibitors of the DNA polymerase.

Dye-terminator sequencing
An alternative to primer labelling is labelling of the chain terminators, a method commonly called 'dye-terminator sequencing'. The major advantage of this method is that the sequencing can be performed in a single reaction, rather than four reactions as in the labelled-primer method. In dye-terminator sequencing, each of the four dideoxynucleotide chain terminators is labelled with a different fluorescent dye, each fluorescing at a different wavelength. This method is attractive because of its greater expediency and speed and is now the mainstay in automated sequencing with computer-controlled sequence analyzers (see below). Its potential limitations include dye effects due to differences in the incorporation of the dye-labelled chain terminators into the DNA fragment, resulting in unequal peak heights and shapes in the electronic DNA sequence trace chromatogram after capillary electrophoresis (see figure to the right). This problem has largely been overcome with the introduction of new DNA polymerase enzyme systems and dyes that minimize incorporation variability, as well as methods for eliminating "dye blobs", caused by certain chemical characteristics of the dyes that can result in artifacts in DNA sequence traces. The dye-terminator sequencing method, along with automated high-throughput DNA sequence analyzers, is now being used for the vast majority of sequencing projects, as it is both easier to perform and lower in cost than most previous sequencing methods.




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Cycle Sequencing

To sequence a piece of dna you need 1)a Template DNA 2) a short DNA primer that is complementary to the dna you want to sequence, 3)A enzyme called DNA polymerase,(4) Four nucleotides.(A,C,G,T), To this mix ,we also add a second type of nucleotide; one that has a slightly different chemical formula, These dideoxynucleotides(diddtp) can be recognized by a DNA sequencer.

To start the sequencing reaction this mixture is heated to 96C ,so the template DNA's two complementary strand separates,Then the temperature is lowered, so that the short "primer" sequence finds its complementary sequence in the template DNA.Finally the temperature is raised 60c,this allows the enzyme to bind to the DNA and create a new strand of DNA.





The sequence of this new DNA is complementary to the original DNA strand. The enzyme makes no distinction between dNTPs or didNTPs.each time a didNTP is incorporated, in this case didATP,The synthesis stops. Because billion of DNA molecules are present in the test tube, the strand can be terminated at any position. This results in collection of DNA strands of many different lengths.


The sequencing reaction is transferred from the test tube to a lane of a polyacrylamide gel. The gel is placed into a DNA sequencer for electrophoresis and analysis. The fragments migrate according to size and each is detected as it passes a laser beam at the bottom of the gel. Each type of dideoxynucleotide emits colored light of a characteristic wavelength and is recorded as a colored band on a simulated gel image, and finally computer program interprets the raw data and outputs an electropherogram with colored peaks representing each letter in the sequence.the sequence fragments are sorted out according to the size, starting from the shortest to longest one, the stimulated gel image is read from bottom to top, starting with the smallest fragment, Thus we sequences present in template DNA.

















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