DNA sequencing and analysis

Principles of DNA sequencing (Sanger method)

DNA chopped into fragments with restriction enzymes
Terminator bases (thymine, adenine, cytosine and guanine) that are modified are used
Each base has a fluorescent tag attached - A = green, G = yellow, T = red and C = blue

The DNA for sequencing is mixed with a primer, DNA polymerase, an excess of free nucleotides and the terminator bases
Mixture is put into a thermal cycler (basically PCR - 96C DNA strands separate, 50C primers anneal, 60C the DNA polymerase builds up the complementary DNA strands)
Each time a terminator base is randomly used, the synthesis of DNA is stopped so no more bases are added --> results in many DNA fragments with different lengths
After many cycles, all the possible DNA chains will be produced
The DNA fragments are then separated according to their length by capillary sequencing (gel electrophoresis in capillary tubes)
Lasers detect the different colours of the terminator bases and therefore the order of the sequence
This order of bases is the complementary strand to the original DNA fragments so can be used to work out the original easily
The data from the sequencing is fed into a computer that reassembles the genome by comparing all the fragments and finding areas that overlap
Once the genome is assembled you can identify the genes that code for specific characteristics

Next-generation sequencing

Techniques have become faster and more automated with time
Instead of using gel or capillary tubes, the reaction takes place on a plastic slide now
Millions of fragments are attached to the slide and replicated in situ using PCR to form clusters of identical DNA fragments
All the clusters are sequenced and imaged at the same time so it's known as the massively parallel sequencing or next-generation sequencing
^ constantly being refined and developed
Enables us to use the information from the genome in many new and different ways

21 - Manipulating genomes