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Shotgun Cloning

dna sequencing vector genome

Shotgun cloning (also known as the shotgun method) is a method to duplicate genomic DNA. The DNA to be cloned is cut using a restriction enzyme or by 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. DNA sequence can be generated at about 600 bases at a time. The sequencing can always be primed with known sequence from the vector and the 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). More recently, it has been applied to more complex genomes, including the human genome. 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 clone into smaller vectors and sequenced. 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.

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