cDNA

Complementary DNA (cDNA) is DNA synthesized from an RNA template by the enzyme reverse transcriptase. It is typically produced from mature messenger RNA (mRNA) and represents the expressed portion of the genome at the time of RNA extraction. Unlike genomic DNA, cDNA generally lacks introns and many regulatory sequences present in the genome.

Synthesis of cDNA involves reverse transcription of RNA into a single-stranded cDNA, usually using primers such as oligo(dT) that target the polyadenylated tail of mRNA or using random hexamers for broader coverage. After first-strand synthesis, a second-strand DNA synthesis step creates double-stranded cDNA that can be cloned, sequenced, or amplified by PCR for downstream applications.

Applications of cDNA include the construction of cDNA libraries for sequencing, cloning of coding sequences into expression vectors, and measurement of gene expression by reverse transcription PCR (RT-PCR) or quantitative RT-PCR (RT-qPCR). cDNA is also used in microarrays and as input material for RNA sequencing (RNA-seq) library preparation, where the abundance of transcripts is inferred from the sequence data.

Limitations and caveats exist: reverse transcription can introduce biases and errors, and synthesis may be incomplete, leading to underrepresentation of certain transcripts or biased 5' ends. Representation depends on the quality and quantity of starting RNA and on the expression level of genes. Because cDNA lacks introns, analyses that rely on genomic structure may require careful interpretation.