Synthesis of complementary DNA (cDNA) from mRNA by reverse transcription
mRNA (with a poly-A tail) is isolated from an organism of interest. A short TTTT primer is synthesized at the 3' end of the message, and extended by the enzyme reverse transcriptase, which "reverses" the central dogma by synthesizing a single-stranded DNA molecule from the mRNA template. The DNA strand is thus "complementary" to the mRNA. The bulk of the mRNA template is removed by treatment with ribonuclease. DNAPol I then uses the remaining RNA-primed regions to complete synthesis of the second strand, followed by excision of the RNA primers and ligation of the DNA ends, just as in in vivo replication. The result is a double-stranded cDNA that includes only the exon portions of the gene, and not the introns, which were spliced out of the hnRNA template to make mRNA. Sequencing of the cDNA then gives the sequence of expressed gene regions.