The Transcriptome

• the exons of most genes are separated by much-longer introns
• the genome contains vast amounts of noncoding "junk" DNA

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; that is all the DNA transcripts — mostly messenger RNAs (mRNAs) — being produced.

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)
• 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)

The Procedure:

• Isolate the 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.
• That is the gene that was expressed.