cDNA cloning
Also known as: complementary dna cloning
cDNA cloning is the technique of copying mature mRNA into complementary DNA with reverse transcriptase and inserting that DNA into a vector for propagation. Because cDNA is built from spliced mRNA, it contains only exons and no introns.
The procedure starts with mRNA isolated from a chosen tissue. Eukaryotic mRNA carries a poly-A tail, so an oligo-dT column or bead selectively captures it, and the same oligo-dT can serve as the primer. Reverse transcriptase, an enzyme from retroviruses, then synthesizes a DNA strand complementary to the mRNA template. The RNA strand is degraded, DNA polymerase builds the second DNA strand, and the resulting double-stranded cDNA is ligated into a plasmid or phage vector and transformed into bacteria.
The defining feature is that cDNA is intron-free. Introns were already removed when the pre-mRNA was spliced, so the cloned sequence corresponds to the coding portion of the gene. That matters practically, because bacteria have no spliceosome and cannot process a eukaryotic gene cloned straight from genomic DNA. Cloning human insulin or growth hormone for bacterial production therefore requires cDNA rather than the genomic sequence.
A collection of clones made this way is a cDNA library, and it differs fundamentally from a genomic library. A genomic library represents the entire genome of an organism and is essentially the same regardless of source tissue. A cDNA library represents only the genes being transcribed in that tissue at that moment, so a liver cDNA library and a neuron cDNA library have different contents. Comparing libraries is one way to study differential gene expression.
cDNA underlies many downstream techniques — RT-PCR and quantitative PCR, microarray and RNA-seq workflows, hybridization probes, and recombinant protein production. The MCAT covers cDNA cloning within recombinant DNA and biotechnology in the biological and biochemical foundations section, where the most commonly tested points are the role of reverse transcriptase, the absence of introns, and the tissue-specific nature of a cDNA library.
Key takeaways
- cDNA cloning uses reverse transcriptase to copy mature mRNA into DNA, which is then inserted into a vector.
- Because it derives from spliced mRNA, cDNA contains exons only and lacks introns and regulatory regions.
- cDNA is required to express eukaryotic genes in bacteria, which cannot splice out introns.
- A cDNA library reflects the genes expressed in one tissue at one time; a genomic library reflects the whole genome.
- cDNA supports RT-PCR, expression analysis, hybridization probes, and recombinant protein production.