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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 3' end of the ssDNA can fold back on itself to form a single-stranded "hairpin loop" and produce a short dsDNA region. The mRNA is removed by treatment with NaOH. DNAPol I is then 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 nuclease. 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.


Figure © 2002 by Griffiths et al.; all text material © 2012 by Steven M. Carr