cDNAs

Complementary DNA (cDNA) is DNA synthesized from an mRNA template by reverse transcriptase. Because it is derived from processed mRNA, cDNA corresponds to expressed sequences and generally lacks introns, reflecting the exons of expressed genes. cDNA is used for cloning, sequencing, and expression analysis, and serves as a stable DNA representation of an RNA transcript.

Synthesis typically begins with reverse transcription of RNA into first-strand cDNA. Priming strategies include oligo-dT primers targeting the poly-A tail, random hexamers, or gene-specific primers. For many applications, second-strand synthesis produces double-stranded cDNA, enabling cloning or library construction. The cDNA is often ligated into plasmid or phage vectors or prepared as sequencing libraries with adapters.

Applications include construction of cDNA libraries used to capture expressed genes, generation of expressed sequence tags (ESTs), cloning of full-length cDNAs, and sequencing. cDNA is also used in expression profiling, where cDNA probes or arrays measure transcript abundance. Modern RNA-seq workflows frequently convert RNA to cDNA as the basis for high-throughput sequencing.

Limitations and sources of bias include incomplete reverse transcription, preferential representation of abundant transcripts, and loss of very long or GC-rich RNAs. Library preparation steps may introduce artifacts such as chimeric reads. Techniques like normalization, ribosomal RNA depletion, and careful primer design help mitigate biases.

cDNA analysis requires careful quality control, including verification of insert size, sequence accuracy, and absence of genomic DNA contamination. cDNA remains a fundamental tool in molecular biology for studying gene expression and transcript structure.