Sequencing technologies are unable to sequence the entire human genome at once. Thus, the genome must be broken into smaller chunks of DNA, sequenced and then put back together in the correct order using bioinformatics approaches. There are several methods of DNA sequencing, including clone-by-clone and whole-genome shotgun methods.
Clone sequencing method
This method requires the genome to have smaller sections copied and inserted into bacteria. The bacteria then can be grown to produce identical copies, or “clones,” containing approximately 150,000 base pairs of the genome that are desired to be sequenced. Then, the inserted DNA in each clone is further broken down into smaller, overlapping 500 base pair chunks. These smaller inserts are sequenced. After sequencing is performed, the overlapping portions are used to reassemble the clone. This approach was used to sequence the first human genome using Sanger sequencing. This approach is time-consuming and costly, but it is reliable.
- During clone-by-clone sequencing, a map of each chromosome of the genome is made before the DNA is split up into fragments ready for sequencing.
- The chunks are inserted into the Bacterial Artificial Clones (BACs) and put inside bacterial cells to grow.
- The chunks of DNA are copied each time the bacteria divide to produce lots of identical copies.
- The DNA in the individual bacterial clones is then broken down into even smaller, overlapping fragments. Each fragment is 500 base pairs long so that they are a more manageable size for sequencing.
- These fragments are put into a vector that has a known DNA sequence.
- The DNA fragments are then sequenced, starting with the known sequence of the vector and extending out into the unknown sequence of the DNA.
- Following sequencing, the small fragments of DNA are pieced together by identifying areas of overlap to reform the large chunks that were originally inserted into the BACs.
- This ‘assembly’ is carried out by computers which spot areas of overlap and piece the DNA sequence together.
- Then, by following the map constructed at the beginning, the large chunks can be assembled back into the chromosomes as part of the complete genome sequence.
The clone-by-clone approach was used during the 1980s and 1990s to sequence the genomes of the nematode worm, C. elegans, and the yeast, S. cerevisiae.
Clone-by-clone sequencing was the preferred method during the Human Genome Project, which was completed in 2001.
Every fragment of DNA is taken from a known region of the genome, so it is relatively easy to determine where there are any gaps in the sequence. Assembly is more reliable because a genome map is followed so the scientists know where the larger fragments are in relation to each other. As each fragment is distinct many people can work on the genome at one time.
- Making clones and generating genome maps takes a long time.
- Clone-by-clone sequencing is generally more expensive than other sequencing methods.
- Some parts of the chromosomes, such as the centromeres, are difficult to clone. This is because they contain long repetitive sections which makes them difficult to cut and clone into BACs. As a result we cannot sequence using clone-by-clone sequencing methods.