Complementary DNA

Almost every cell in the body has the same DNA.

However, different cell types, such as neurons and muscle cells, express different sets of genes. These genes are transcribed into different messenger RNAs, or mRNAs, in each cell.

In the laboratory, mRNA can be used as a template to synthesize complementary DNA, or cDNA. Since cDNA is synthesized from cellular mRNA, it represents the genes expressed by the cell.

One common method begins with extracting RNA from cells.

Following cell lysis and removal of cellular debris, the mRNA is isolated from other types of RNA, such as ribosomal RNA and transfer RNA. This is commonly done by adding the sample to a column containing beads with short stretches of thymine nucleotides attached.

These thymine stretches bind to the poly-A tail, a chain of adenine nucleotides found at the 3-prime end of eukaryotic mRNA, through complementary base pairing.

Other types of RNA lack poly-A tails, so they do not bind to the column and are washed away.

An elution buffer with a low salt concentration is then added. This disrupts the interaction between the poly-A tail and the thymine stretches, releasing the bound mRNA from the beads.

After the mRNA is isolated, an oligo-dT primer binds to the poly-A tail and provides a starting point for reverse transcriptase enzymes to synthesize a single-stranded cDNA copy of the mRNA.

RNase enzymes are then added to degrade the RNA strand of the RNA-cDNA hybrid.

Next, DNA polymerase enzymes synthesize a complementary DNA strand, producing double-stranded cDNA.

This cDNA can then be inserted into plasmids or viral vectors for molecular biology research.