VPB 321: Animal Biotechnology (2+1)

WHOLE GENOME SHOTGUN METHOD

• The shotgun sequencing method goes straight to the job of decoding, bypassing the need for a physical map. Therefore, it is much faster. 
• Multiple copies of the genome are randomly shredded into pieces that are 2,000 base pairs (bp) long by squeezing the DNA through a pressurized syringe. This is done a second time to generate pieces that are 10,000 bp long.   
• Each 2,000 and 10,000 bp fragment is inserted into a plasmid, which is a piece of DNA that can replicate in bacteria. The two collections of plasmids containing 2,000 and 10,000 bp chunks of human DNA are known as plasmid libraries.
• Both the 2,000 and the 10,000 bp plasmid libraries are sequenced. 500 bp from each end of each fragment are decoded generating millions of sequences. Sequencing both ends of each insert is critical for the assembling the entire chromosome.
• Computer algorithms assemble the millions of sequenced fragments into a continuous stretch resembling each chromosome.
• The small genomes of several viruses and bacteria and the much larger genomes of three higher organisms have been completely sequenced; they are bakers' or brewers' yeast (Saccharomyces cerevisiae), the roundworm (Caenorhabditis elegans), and the fruit fly (Drosophila melanogaster).
• In October 2001, the draft sequence of the pufferfish Fugu rubripes, the first vertebrate after the human, was completed; and scientists finished the first genetic sequence of a plant, that of the weed Arabidopsis thaliana, in December 2000. Many more genome sequences have been completed since then.