cDNA

Synthesis of complementary DNA (cDNA) from mRNA by reverse transcriptase

mRNA 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 3' end of the ssDNA can fold back on itself to form a single-stranded "hairpin loop" and a short double-stranded region. Once the mRNA is dissolved away with NaOH, DNAPol I is added, which uses the double-stranded region as a primer for synthesis of a new DNA strand. The single-stranded portion of the loop is cut with an S1 nuclease, and the result is a double-stranded cDNA copy of the mRNA. Note that this cDNA will include only the exon portions of the gene, and not the introns, which were spliced out of the mRNA template.