What Are ESTs and How Are They Made?

ESTs are small pieces of DNA sequence (usually 200 to 500 nucleotides long) that are generated by sequencing either one or both ends of an expressed gene. The idea is to sequence bits of DNA that represent genes expressed in certain cells, tissues, or organs from different organisms and use these "tags" to fish a gene out of a portion of chromosomal DNA by matching base pairs. The challenge associated with identifying genes from genomic sequences varies among organisms and is dependent upon genome size as well as the presence or absence of introns, the intervening DNA sequences interrupting the protein coding sequence of a gene.

Separating the Wheat from the Chaff: Using mRNA to Generate cDNA

Gene identification is very difficult in humans, because most of our genome is composed of introns interspersed with a relative few DNA coding sequences, or genes. These genes are expressed as proteins, a complex process comprised of two main two steps. Each gene (DNA) must be converted, or transcribed, into messenger RNA (mRNA), RNA that serves as a template for protein synthesis. The resulting mRNA then guides the synthesis of a protein through a process called translation. Interestingly, mRNAs in a cell do not contain sequences from the regions between genes, nor from the non-coding introns that are present within many genes. The problem, however, is that mRNA is very unstable outside of a cell; therefore, scientists use special enzymes to convert it to complementary DNA (cDNA). cDNA is a much more stable compound and, importantly, because it was generated from a mRNA in which the introns had been removed, cDNA represents only expressed DNA sequence.

From cDNAs to ESTs

Once cDNA representing an expressed gene has been isolated, scientists can then sequence a few hundred nucleotides from either end of the molecule to create two different kinds of ESTs. Sequencing only the beginning portion of the cDNA produces what is called a 5' EST. A 5' EST is obtained from the portion of a transcript that usually codes for a protein. These regions tend to be conserved across species and do not change much within a gene family. Sequencing the ending portion of the cDNA molecule produces what is called a 3' EST. Because these ESTs are generated from the 3' end of a transcript, they are likely to fall within non-coding, or untranslated regions (UTRs), and therefore tend to exhibit less cross-species conservation than do coding sequences.

ESTs as Genome Landmarks

Just as a person driving a car may need a map to find a destination, scientists searching for genes also need genome maps to help them to navigate through the billions of nucleotides that make up the human genome. For a map to make navigational sense, it must include reliable landmarks or "markers". Currently, the most powerful mapping technique, and one that has been used to generate many genome maps, relies on Sequence Tagged Site (STS) mapping. A STS is a short DNA sequence that is easily recognizable and occurs only once in a genome (or chromosome). The 3' ESTs serve as a common source of STSs because of their likelihood of being unique to a particular species and provide the additional feature of pointing directly to an expressed gene.

ESTs as Gene Discovery Resources

Because ESTs represent a copy of just the interesting part of a genome, that which is expressed, they have proven themselves again and again as powerful tools in the hunt for genes involved in hereditary diseases. ESTs also have a number of practical advantages in that their sequences can be generated rapidly and inexpensively, only one sequencing experiment is needed per each cDNA generated, and they do not have to be checked for sequencing errors because mistakes do not prevent identification of the gene from which the EST was derived.