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Complementary DNA (cDNA) is DNA created from single-stranded messenger RNA (mRNA). cDNA contains only the protein coding region of a gene and the flanking untranslated regions (not made into protein). Cloning cDNA begins with the isolation of mRNA from the organism of interest. The mRNA is then bound by a short sequence of DNA (primer) that recognizes the conserved poly-A tail end of mRNA. Reverse transcriptase copies the mRNA into single-stranded cDNA and the mRNA is degraded using RNAse. DNA polymerase then synthesizes the double-stranded cDNA. The cDNA is inserted into a plasmid, which is a DNA molecule for transport of DNA into a cell, and added to bacterial cells. The bacterial cells replicate the plasmid, creating several copies, which are then purified from the cells. Uses for cDNA include cloning genes with large non-coding (intronic) regions that are otherwise too long for polymerase chain reaction (PCR), verifying the correct start and stop sites for the coding region of a gene, and expressing eukaryotic genes in bacteria. (Credit: Brooke Anderson-White)
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