Sanger sequencing and the human genome project

Deoxy nucleotides (dNTPs) are used in regular chain extension of a DNA chain. 

Dideoxy nucleotides (ddNTPs) are used in Sanger sequencing. They have another missing hydroy group on the 3' carbon, meaning they can join a sequence of DNA put prevent it extending further. 

Sanger sequencing can sequence 800 bp at once. It uses a mix of dNTPs and ddNTPs to extend onto a DNA primer. The mix means that sometimes the chain incorporates a ddNTP which terminates the DNA sequence. This creates different size fragments which seperated using gel electrophoresis. Each ddNTP is done in a seperate reaction and then they are ran in 4 seperate lanes on the gel. This is done so that each lane is a known base and the gel can be read for the sequence. 

In 1977 dye terminator sequencing was invented which was the same principle as Sanger sequencing but used fluorescently labelled ddNTPs which allows the whole process to be completed in one tube. This is because each ddNTP was a different colour. This was the domiant menthod of sequencing for 30 years.  

24 distinct chromosomes containing between them over 3 million base pairs. But over 50% of the human genome is repetitive. 

human genome project initaited in 1990. 

sequencing was a huge challenge as can only read 800 bp at once. 

pieceing the sequence togther was going to be a challenge too. 

• Extract human genomic sample from several annoymous donors 
• Fragment DNA - by sonication, enzymatically or chemically 
• Size selction - 100-200kbp chosen 
• Fragments cloned into bacterial artificial chromosomes (BACs) - BACs can fit inserts of up to 300kbp

Overview of approach:

• Genetic and physical map 
• Fragment and clone DNA into BACs 
• Identify BAC clones containing markers
• BAC end sanger sequencing to identify overlapping clones
• Shotgun sequence BACs and align to form consensus sequence  

IHGSC used genetics and physical maps of human genome in order to sequence in an ordered manner. 

Genetic maps rely on recombination frequency between genetic markers 

Physical maps use molecular biology methods such as restriction digests and FISH 

Establishing a dense set of genetic markers across genome was a major achievement for IHGSC and first step to being able to sequence. the human genome. 

Firstly you need to transform the BACs into individual E.coli cells and grow colonies. Then pick individual colonies and place into tubes, which will then contain millions of copies of the same DNA fragment. This is referred to as the BAC library. 

Then you have to test by PCR if each vector (BAC) has a marker sequence. This is a long process as has to be done for many genetic markers and there are many BACs. If a BAC is negative for marker tested then it is from a differentpart of the genome. Not all BAC will conatin a marker.

Finally use Sanger sequencing to obtain a 800bp read. Then test the BAC library for the end sequence from this read by PCR. The BAC conatining that sequence will likely be the next part of the sequence. This process is called chromosome walking and is used between the genetic markers to work out the sequence. 

Shotgun sequencing is a brute force approach to sequencing large regions of DNA. 

Take a 40-100kb fragment from a BAC clone and break it up into 5-10kb fragments. Clone these into plasmid vectors. Sequence the two ends of the fragment (part connected to the plasmid). If we sequence lots of these "paired ends" we are able to assemble the large fragment as pieces will overlap in random places. This creates a consensus sequence. 

The IHGSC were a not for profit organisation commited to releasing the data about the human genome to the public. 

Celera was a private company who were also trying to sequence the genome using shotgun sequecing. They threatened to patent and commercialise the genome. They had already used shotgun sequecing to sequence the first bacterial genome. 

Competition accelerated the efforts to complete the human genome. Eventually both were published simultaneously in Nature and Science at a joint UK and US conference. 

• fragmentation 
• size selection (2,10 and 50kbp) 
• clone fragments into plasmids to create library
• automated sequencing on ABI 3700 
• assemble reads to create consensus sequence and sequence contigs 
• has five fold coverage so each base is sequenced on average 5 times 

Needed to prove it was feasible for a complex, repeat rich human genome: government funders are risk adverse. Celera didn't have this problem. 

Assembly was easier and could be performed with confidence using the clone by clone apporach. 

Finishing (closing gaps) is easier using clone by clone as gaps can be individually targetted.

Clone by clone is better suited to a diverse international consortium, as BAC contigs could be shared easily between members.

In the end they were wrong. Genomes are now routinely sequenced using massively parallel Next Generation sequencing technologies which employ shotgun sequencing.