Expressed sequence tags: medium-throughput protocols.
Generating expressed sequence tags is a simple, cheap, and efficient way to sample the genome of a target organism. An expressed sequence tag (EST) is a single-pass sequence derived from a single complementary DNA (cDNA) clone, and the sequence serves to identify the gene from which it derives. We present a set of tested laboratory protocols for setting up and performing an EST analysis of any chosen species. These medium-throughput protocols do not require dedicated genomics equipment, such as robots, and focus on the use of microtiter plates and multichannels. Using these protocols, a single competent research worker should be able to generate 2000 ESTs in 1 mo. In a nonnormalized library, these 2000 ESTs should identify between 1000 and 1500 different genes, and thus possibly between 10 and 20% of the genes of any target parasite.
Whitton
Claire
C
Institute of Cell, Animal, and Population Biology, Ashworth Laboratories, University of Edinburgh, UK.
Daub
Jennifer
J
Thompson
Marian
M
Blaxter
Mark
M
D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary; D020224:Expressed Sequence Tags; D016133:Polymerase Chain Reaction
D018076:DNA, Complementary