Expressed sequence tags: medium-throughput protocols

Claire Whitton, Jennifer Daub, Marian Thomson, Mark Blaxter

Research output: Contribution to journalArticlepeer-review

Abstract

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.
Original languageEnglish
Pages (from-to)75-92
Number of pages18
JournalMethods in Molecular Biology
Volume270
DOIs
Publication statusPublished - 2004

Keywords

  • Base Sequence
  • Cloning, Molecular
  • DNA, Complementary
  • Expressed Sequence Tags
  • Polymerase Chain Reaction

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