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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. Therefore, isolating mRNA is key to finding expressed genes in the vast expanse of the human genome.

Figure 1. An overview of the process of protein synthesis.
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Protein synthesis is the process whereby DNA codes for the production of amino acids and proteins. The process is divided into two parts: transcription and translation. During transcription, one strand of a DNA double helix is used as a template by mRNA polymerase to synthesize a mRNA. During this step, mRNA passes through various phases, including one called splicing, where the non-coding sequences are eliminated. In the next step, translation, the mRNA guides the synthesis of the protein by adding amino acids,one by one, as dictated by the DNA and represented by the mRNA.
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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.

Figure 2. An overview of how ESTs are generated.
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ESTs are generated by sequencing cDNA, which itself is synthesized from the mRNA molecules in a cell. The mRNA in a cell are copies of the genes that are being expressed. mRNA does not contain sequences from the regions between genes, nor from the non-coding introns that are present within many interesting parts of the genome.
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REFERENCES

http://www.ncbi.nlm.nih.gov/About/primer/est.html

Posted by 西瓜 at July 22, 2003 09:01 PM