The shotgun method (also known as shotgun cloning) is a method in cloning genomic DNA . It involves taking the DNA to be cloned and cutting it either using a restriction enzyme or randomly using a physical method to smash the DNA into small pieces. These fragments are then taken together and cloned into a vector. The original DNA can be either genomic DNA (whole genome shotgun cloning) or a clone such as a YAC (yeast artificial chromosome ) that contains a large piece of genomic DNA needing to be split into fragments.
If the DNA needs to be in a certain cloning vector, but the vector can only carry small amounts of DNA, then the shotgun method can be used. More commonly, the method is used to generate small fragments of DNA for sequencing. A DNA sequence can be generated at about 600 bases at a time, so if a DNA fragment of about 1100kb is cloned, then it can be sequenced in two steps, with 600 bases from each end, and a hundred base overlap. The sequencing can always be primed with a known sequence from the vector and so any prior knowledge of the sequence that has been cloned is not necessary. This approach of shotgun cloning followed by DNA sequencing from both ends of the vector is called shotgun sequencing.
Shotgun sequencing was initially used to sequence small genomes such as that of the cauliflower mosaic virus (CMV), which is 8kb long. More recently, it has been applied to more complex genomes. Usually this involves creating a physical map and a contig (line of overlapping clones) of clones containing a large amount of DNA in a vector such as a YAC, which are then shotgun cloned into smaller vectors and sequenced. However, a whole genome shotgun approach has been used to sequence the mouse, fly and human genomes by the private company Celera. This involves shotgun cloning the whole genome and sequencing the clones without creating a physical map. It is faster and cheaper than creating a physical gene map and sequencing clones one by one, but the reliability of reassembling all the sequences of the small fragments into one genomic sequence has been doubted. For example, a part of the fly genome was sequenced by the one-by-one approach and the whole genome shotgun method. The two sequences were compared, and showed differences. 60% of the genes were identical, 31% showed minor differences and 9% showed major differences. The whole genome shotgun method generated the sequence much more quickly, but the one-by-one approach is probably more accurate because the genes were studied in more detail.
See also Cloning, application of cloning to biological problems; Yeast artificial chromosome (YAC); Yeast genetics
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