What is clone-by-clone sequencing?
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.
- In clone-by-clone sequencing the genome is broken up into large chunks, 150 kilobases long (150,000 base pairs).
- The location of these chunks on the chromosomes is recorded (mapped) to help with assembling them in order after sequencing.
- The chunks are then inserted into Bacterial Artificial Chromosomes (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.
What are the advantages of clone-by-clone sequencing?
- 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.
What are the disadvantages of clone-by-clone sequencing?
- 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 you cannot sequence using clone-by-clone sequencing methods.
This page was last updated on 2014-11-17