Is this right for sequencing of the genome?
• Genomes are mapped to find out which part of the Genome they come from. Information that is already known is used such as the location of microsatellites.
• Samples of the Genome are mechanically broken down into sections of around 100,000 base pairs long.
• These sections are then placed onto Bacterial Artificial Chromosomes (BAC’s) and then transferred to E.coli.
• As the cells grow these sections are copied many times, and this is referred to as a clone library.
• To sequence the BAC’s, cells containing the BAC are taken and cultured. DNA is extracted from the cells and cut into smaller fragments. Different restriction enzymes are used on a number of sections to give different fragment types.
• The sample of DNA is then placed into a reaction mixture with primers, free nucleotides and taq polymerase. However, also in the reaction mixture are nucleotides referred to as ‘terminator nucleotides’ and they are fluorescent in colour. They bind to DNA in the same way as free nucleotides but they stop the fragment from replicating any further. Each of the four nucleotides A, T, G, and C is a different fluorescent colour.
• As the template is copied many times, each time terminator nucleotides bind at random in different positions, and stop it replicating any further. This means that each fragment is a different length. The shortest is one nucleotide in length, the second shortest two nucleotides in length and so on up to the longest fragment which will be the one being sequenced. All the fragments are known as a set of Nested fragments.
• The fragments are then placed in an electrophoresis tank. The shorter the fragments the faster it will travel through the gell. As the fragments reach the end of the gel, a laser reads the colour of the end nucleotide (terminator nucleotide).
• Is then shown as a graph showing direct readings of the results.
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