Worksheet IX.1. Synthesis of cDNA.
Synthesis of cDNA
Genes from higher organisms are normally not expressed in E. coli, as these organisms use different signals controlling gene expression. Cloned eukaryotic genes are not expressed in E. coli quite simply because the enzymes of the bacterium do not recognise the regulatory sequences used in eukaryotic organisms. Additionally, no splicing mechanism occurs in prokaryotes.
A cloning method that uses the mRNA of eukaryotic genes would be particularly useful because splicing of introns has already occurred. mRNA itself cannot be used for the actual cloning process, but it can be converted into DNA by complementary DNA (cDNA) synthesis.
The key to this method is the enzyme reverse transcriptase that will synthesise a DNA polynucleotide strand complementary to an existing RNA strand. Once the cDNA strand has been synthesised, the RNA strand of the hybrid molecule can be partially degraded by treating it with a ribonuclease. The remaining RNA fragments then serve as primers for DNA polymerase I, which will synthesise the second cDNA strand. The result is a double stranded DNA fragment consisting only of the coding exons (Figure 1, Brown 1991, slightly modified).
Figure 1. cDNA synthesis comprises three main steps
(a) first strand synthesis
(b) RNA degradation
(c) second strand synthesis
RNase H = special type of ribonuclease (RNase) used in cDNA synthesis
DNA pol I = DNA polymerase I