Complementary DNA

Complementary DNA (cDNA) lacks introns and regulatory sequences present in genomic DNA, allowing researchers to directly determine the amino acid sequence of encoded peptides. Since cDNA is synthesized from mRNA, it represents only the genes actively expressed in specific cells or tissues, making it ideal for studying gene expression patterns across different cell types.

Mature eukaryotic mRNA has a poly-A tail—a string of adenine nucleotides—added to its 3' end, while transfer RNA and ribosomal RNA do not. Researchers attach thymine nucleotides (oligo-dTs) to a column or magnetic beads, which specifically base-pair with the poly-A tail. This allows mRNA to be captured while other RNA types are washed away.

Reverse transcriptase is a DNA polymerase enzyme from retroviruses that synthesizes a single-stranded cDNA strand from mRNA. Since reverse transcriptase can only add nucleotides to the 3' end of a chain, a poly-T primer binds to the poly-A tail to provide a starting point for cDNA synthesis.

After reverse transcriptase creates single-stranded cDNA, the original mRNA template must be removed to allow DNA polymerase to synthesize the complementary second strand. RNA is degraded using alkali treatment or RNase enzymes, leaving the single-stranded cDNA intact for the next synthesis step.

DNA polymerase synthesizes a second DNA strand complementary to the first cDNA strand, often using the hairpin loop at the end of the first strand or a nicked piece of the mRNA as a primer. This produces double-stranded cDNA, which can be inserted into bacterial or viral vectors for molecular biology research.

A cDNA library is a collection representing all the mRNAs expressed in specific cells or tissues of interest. Researchers construct cDNA libraries to comprehensively study gene expression patterns, identify active genes in particular cell types, and preserve genetic information for additional research and analysis.

Although neurons and muscle cells contain the same DNA, they express different genes because only certain genes are transcribed into mRNA in each cell type. By extracting and analyzing mRNA from these different cell types, researchers can use cDNA synthesis to study which genes are active and understand cell-type-specific gene expression patterns.