Expressed Sequence Tags: Medium-Throughput Protocols<I>

TLDR

Expressed sequence tags (ESTs) are inexpensive, single‑pass sequences from cDNA clones that enable rapid sampling of a target organism’s genome and identification of the originating gene. The authors provide tested laboratory protocols for setting up and performing EST analysis in any chosen species. The protocols are medium‑throughput, relying on microtiter plates and multichannel pipetting rather than dedicated robotics. A single researcher can generate 2,000 ESTs in one month, yielding 1,000–1,500 distinct genes—about 10–20% of a target parasite’s genome—in a nonnormalized library.

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.