DNA Sequencing

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Fred Sanger

He developed a method for scientists to sequence whole genomes.

The first thing he sequenced was a bacteriophage. He published sequence of the human mitochondrial genome. The genome of the haemophilus influenza bacteria was sequenced. He developed Sanger sequencing. The improved form of sanger sequencing was high throughput sequencing.

DNA that is going to be sequenced is mixed with DNA polymerase, an excess of nucleotides and terminator bases. This acts as a primer.

This mixture is placed in a thermal cycler. It switches rapidly between two cycles: one at 96 degrees – which separates double strands into single, and one at 50 degrees – where the primers anneal to DNA.

At 60 degrees, DNA polymerase begins to build up new DNA strands by adding complementary nucleotides.

Terminators are added to the strand. This stops the synthesis of DNA, so no more bases can be added. This creates fragments of different lengths.

The fragments are separated by capillary sequencing, like gel electrophoresis, which means they separate according to their lengths.

There are fluorescent markers on the terminators. The lasers detect these meaning the last base is recognised and the sequence is ordered.

This order is fed to a computer. It assembles a genome by looking at the overlapping.

High throughput sequencing

Developed in the new millennium

Also called next-generation sequencing

However it has a high error rate and since it generates fragments only, a template must be used.

But it is much quicker

It takes place on a plastic slide instead of a gel or capillaries. This slide is called a flow cell.

PCR is used to replicate millions of DNA fragments. This forms clusters.

Terminator bases are still used to stop sequencing, meaning an image can be taken.

This process is very efficient and fast. It has reduced costs, meaning more genomes can be sequenced.

Used by 100,000 genomes project.

Means 3 billion pairs can be sequenced in days.