Only genes that are transcribed into messenger RNA (mRNA) are active, or expressed. Scientists can, therefore, extract the mRNA from cells to study gene expression in different cells and tissues. The scientist converts mRNA into complementary DNA (cDNA) via reverse transcription. Because mRNA does not contain introns (non-coding regions) and other regulatory sequences, cDNA—unlike genomic DNA—also allows researchers to directly determine the amino acid sequence of the peptide encoded by the gene.
cDNA can be generated by several methods, but a common way is to first extract total RNA from cells, and then isolate the mRNA from the more predominant types—transfer RNA (tRNA) and ribosomal (rRNA). Mature eukaryotic mRNA has a poly(A) tail—a string of adenine nucleotides—added to its 3’ end, while other types of RNA do not. Therefore, a string of thymine nucleotides (oligo-dTs) can be attached to a substrate such as a column or magnetic beads, to specifically base-pair with the poly(A) tails of mRNA. While mRNA with a poly(A) tail is captured, the other types of RNA are washed away.
Next, reverse transcriptase—a DNA polymerase enzyme from retroviruses—is used to generate cDNA from the mRNA. Since, like most DNA polymerases, reverse transcriptase can add nucleotides only to the 3’ end of a chain, a poly(T) primer is added to bind to the poly(A) tail to provide a starting point for cDNA synthesis. The cDNA strand ends in a hairpin loop. The RNA is then degraded—commonly with alkali treatment or RNase enzymes—leaving the single-stranded cDNA intact.
A second DNA strand complementary to the cDNA is then synthesized by DNA polymerase—often using the hairpin loop of the first cDNA strand or a nicked piece of the mRNA as a primer.
The resulting double-stranded cDNA can be inserted into bacterial or viral vectors and cloned using standard molecular biology techniques. A cDNA library—representing all the mRNAs in the cells or tissue of interest—can also be constructed for additional research.
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