DNA SEQUENCING

=DNA Sequencing= The earliest methods of sequencing DNA were essentially a modified form of PCR. Four reaction mixtures are prepared each containing the basic PCR reagents (target sequence, primers, DNA polymerase and free DNA Nucleotides). However, to each reaction mixture a special replication stopping nucleotide is also added. [image:http://i.imgur.com/4N5hc2i.png?1] [image:http://i.imgur.com/j8tG5xk.png?2] For the reaction mixture containing replication stopping cytosine (C): Whenever one of these special cytosine (C) nucleotides is incorporated, replication stops prematurely, yielding an incomplete copy. As the original sequence is being copied, it is purely chance as to whether a normal C will be incorporated, allowing replication to continue, or whether a replication stopping C will be added. This yields a series of different sized copies / fragments each corresponding to the position of a cytosine (C). The fragments in each reaction mixture are labelled using a different coloured fluorescent dye. Gel electrophoresis is then used to separate the fragments according to their size. The size of each fragment corresponds to the position of one of the bases C, G, T or A. For instance we can see here that the size of the blue fragments will correspond to the positions of cytosine (C). [image:http://i.imgur.com/XaQuXMD.png?1] Automated Sequencing: DNA fragments will move down a gel in order of their size, the smallest fragments reaching the end of the gel first. As fragments reach the end of the gel, they pass through a laser beam causing the fragment to fluoresce a particular colour. This colour is detected by the sequencer and recorded in a printout. [image:http://i.imgur.com/NlOCqK6.png] DNA sequencing is based on the use of specialised replication stopping nucleotides. A normal Deoxyribose Nucleic Acid (DNA) nucleotide has an OH group on the 3’ Carbon. This OH group will interact with the phosphate group on the 5’ carbon of another nucleotide, forming a phosphodiester bond. Basically this OH group is essential for linking nucleotides together. [image:http://i.imgur.com/SmEa3ga.png] The special replication stopping nucleotides are known as Di-Deoxyribose Nucleic Acid (dDNA) Nucleotides and are missing the 3’ OH group. This means that no more nucleotides can be added to the 3’ end of the growing nucleotide chain. [image:http://i.imgur.com/UZapuxO.png?1]