Complementary DNA

Output from a cDNA microarray used in testing

In genetics, complementary DNA (cDNA) is DNA synthesized from a mature mRNA template in a reaction catalyzed by the enzyme reverse transcriptase and the enzyme DNA polymerase.^[1] cDNA is often used to clone eukaryotic genes in prokaryotes. When scientists want to express a specific protein in a cell that does not normally express that protein (i.e., heterologous expression), they will transfer the cDNA that codes for the protein to the recipient cell. cDNA is also produced by retroviruses (such as HIV-1, HIV-2, Simian Immunodeficiency Virus, etc.) which is integrated into its host to create a provirus.

Overview

The central dogma of molecular biology outlines that in synthesizing proteins, DNA is transcribed into mRNA, which is translated into protein. One difference between eukaryotic and prokaryotic genes is that eukaryotic genes can contain introns (intervening sequences), which are not coding sequences, and must be removed from the RNA primary transcript before it becomes mRNA and can be translated into protein. Prokaryotic genes have no introns, so their RNA 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. This DNA, which was made as a complementary copy of the RNA, is called complementary DNA (cDNA). To obtain expression of the protein encoded by the eukaryotic cDNA, prokaryotic regulatory sequences would also be required (e.g. a promoter).

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 and C) to their DNA complements (T, A, C and G respectively).

To obtain eukaryotic cDNA whose introns have been removed:

1. A eukaryotic cell transcribes the DNA (from genes) into RNA (pre-mRNA).
2. The same cell processes the pre-mRNA strands by removing introns, and adding a poly-A tail and 5’ Methyl-Guanine cap.
3. This mixture of mature mRNA strands is extracted from the cell. The Poly-A tail of the post transcription mRNA can be taken advantage of with oligo(dT) beads in an affinity chromatography assay.
4. A poly-T oligonucleotide primer is hybridized onto the poly-A tail of the mature mRNA template, or random hexamer primers can be added which contain every possible 6 base single strand of DNA and can therefore hybridize anywhere on the RNA (Reverse transcriptase requires this double-stranded segment as a primer to start its operation.)
5. Reverse transcriptase is added, along with deoxynucleoside triphosphates (A, T, G, C). This synthesizes one complimentary strand of DNA hybridized to the original mRNA strand.
6. To synthesize an additional DNA strand (to make it dsDNA), you need to digest the RNA of the hybrid strand, using an enzyme like RNase A.
7. After digestion of the RNA, a single stranded DNA is left and because single stranded nucleic acids are hydrophobic, it tends to loop around itself. It is likely that the ssDNA forms a hairpin loop at the 3' end.
8. From the hairpin loop, the reverse transcriptase can then use it as a primer again to continue transcribing a complimentary sequence for the ss cDNA.
9. Now, you should be left with a double stranded cDNA with identical sequence as the mRNA of interest.

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.

Note that the central dogma of molecular biology is not broken in this process.

Applications

Complementary DNA is often used in gene cloning or as gene probes or in the creation of a cDNA library. When scientists transfer a gene from one cell into another cell in order to express the new genetic material as a protein in the recipient cell, the cDNA will be added to the recipient (rather than the entire gene), because the DNA for an entire gene may include DNA that does not code for the protein or that interrupts the coding sequence of the protein (e.g., introns). Partial sequences of cDNAs are often obtained as expressed sequence tags.

Viruses

Some viruses also use cDNA to turn their viral RNA into mRNA (viral RNA → cDNA → mRNA). The mRNA is used to make viral proteins to take over the host cell.

References

External links

• H-Invitational Database
• Functional Annotation of the Mouse database
• cDNA Animation (Flash)