Synthesis of complementary DNA (cDNA) from mRNA by reverse transcription
3' 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 extends the double-stranded region as a primer
for synthesis of a new DNA strand.
The single-stranded portion of the loop is cut with a 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.