Complementary DNA
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In genetics, complementary DNA (cDNA) is DNA synthesized from a mature mRNA template. cDNA is often used to clone eukaryotic genes in prokaryotes.
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Overview
The central dogma of genetics outlines that in synthesizing proteins, DNA is transcribed into mRNA, which is translated into protein. One difference between eukaryotic and prokaryotic mRNA is that eukaryotic mRNA can contain introns (intervening sequences), which are not coding sequences, per se, and must be spliced out of the mRNA before it is translated into protein. Prokaryotic mRNA has no introns, so it is not subject to splicing.
Often it is desirable to express eukaryotic genes in prokaryotic cells. A simplified method of doing so would include the addition of eukaryotic DNA to a prokaryotic host, which would transcribe the DNA to mRNA and then translate it to protein. However, as eukaryotic DNA has introns, and since prokaryotes lack the machinery to splice them, the splicing of eukaryotic DNA must be done prior to adding the eukaryotic DNA into the host (as well, before placing the eukaryotic DNA into the prokaryote, it must be methylated and a prokaryotic promoter region must be added). This spliced DNA is called complementary DNA.
Synthesis
Though there are several methods for doing so, cDNA is most often synthesized from mature (fully spliced) mRNA using the enzyme reverse transcriptase. This enzyme operates on a single strand of mRNA, generating its complementary DNA based on the pairing of RNA base pairs (A, U, G, C) to their DNA complements (T, A, C, G).
To obtain eukaryotic DNA whose introns have been spliced:
- A eukaryotic cell transcribes the DNA into mRNA.
- The same cell processes the nascent mRNA strand by splicing out introns, and adding a poly-A tail and GTP cap.
- This mature mRNA strand is extracted from the cell.
- A poly-T oligonucleotide is hybridized onto the poly-A tail of the mature mRNA template. (Reverse transcriptase requires this double-stranded segment as a primer to start its operation.)
- Reverse transcriptase is added, along with deoxynucleotide triphosphates (A, T, G, C).
The reverse transcriptase scans the mature mRNA and synthesizes a sequence of DNA that complements the mRNA template. This strand of DNA is complementary DNA.
Applications
Complementary DNA is often used in gene cloning or as gene probes or in the creation of a cDNA library.
External links
- H-Invitational Database (http://www.h-invitational.jp/)de:CDNA