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6.6: Expressed Sequence Tags

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  • Only a very small percentage (1.2% in humans) of the DNA in vertebrate genomes encodes proteins (the "proteome") because the exons of most genes are separated by much-longer introns between our genes lie vast amounts of DNA much of which appears to regulate the expression of our genes but is not transcribed and translated into a protein product. So even when the complete sequence of a genome is known, it is often difficult to spot particular genes (open reading frames or ORFs).

    One approach to solving the problem is to examine a transcriptome of the organism. Most commonly this is defined as: All the messenger RNA (mRNA) molecules transcribed from the genome. It is "a" transcriptome, not "the" transcriptome, because what genes are transcribed in a cell depends on the kind of cell (e.g., liver cell vs. lymphocyte) and what the cell is doing at that time, e.g.,

    • getting ready to divide by mitosis;
    • responding to the arrival of a hormone or cytokine;
    • getting ready to secrete a protein product.

    Expressed Sequence Tags (ESTs)

    ESTs are short (200–500 nucleotides) DNA sequences that can be used to identify a gene that is being expressed in a cell at a particular time.

    The Procedure:

    • Isolate the messenger RNA (mRNA) from a particular tissue (e.g., liver)
    • Treat it with reverse transcriptase. Reverse transcriptase is a DNA polymerase that uses RNA as its template. Thus it is able to make genetic information flow in the reverse (RNA ->DNA) of its normal direction (DNA -> RNA).
    • This produces complementary DNA (cDNA). Note that cDNA differs from the normal gene in lacking the intron sequences.
    • Sequence 200–500 nucleotides at both the 5′ and 3′ ends of each cDNA.
    • Examine the database of the organism's genome to find a matching sequence.

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