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.