Automated DNA sequencing

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Automated DNA sequencing

This is based on interrupted PCR and Electrophoresis

The reaction mixture contains DNA polymerase, many copies of the single-stranded template DNA fragment, free DNA nucleotides and primers.

Within the squencing mixture, some of the free nucleotides carry a fluorescent markers.

These nucleotides are modified and, if the yare added to the growing chain, the DNA polymerase is 'thrown off' and the strand cannot have any further nucleotides added.

Each nucleotide type has a different coloured fluorescent marker

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The primer joins at the 3', allowing DNA polymerase to attach

DNA polymerase adds free nucleotides according to base-pairing rles, so the strand grows-this is essentially the same as natural DNA replication and PCR.

If a modified nucleotide is added, the polymerase enzyme and thrown off and the reaction stops on that template strand. 

As the reaction proceeds, many molecules of DNA are made. The fragments generated vary in size. In some of them, the template strand has only one additional nucleotide added before the polymerase is thrown off, in others, the template strand is completed. in each case, the final added nucleotide is tagged with a specific colour

As these strands run through the machine (the same way DNA strands move in electrophoresis) a laser reads the colour sequence, from the strand with only a single nucleotide added, to the one with 2 nucleotides added, then three, then four and so on. The sequence of colours, and so the sequence of the bases, can then be displayed 

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The Human Genome Project

Set it's aim in 1990 to determine the whole human genome sequence.

DNA from a few individuals, taken from a pool of 100 volunteer, was used.

Laboratories around the world contributed to the project.

In 2004, the completed human genome sequence was published.

Sequencing, as described above, requires sections of DNA to be sequenced between 6 and 10 times in order to be confident that the base sequence information is accurate. 

Each contribute laboratory worked on different parts of the genome, sharing their sequence information with others to build the whole sequence.

Probably the most fascinating fact is that at the outset, scientists expected to find around 100,000 coding genes on the genome.

By 2004 the estimate was that humans only have around 25,000-not many more than a worm.

It seems now that human complexity has far more to do with regulation of gene expression than with the number of genes.

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