Complementary DNA
From Wikipedia, the free encyclopedia
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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[edit] 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 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. This spliced DNA is called complementary DNA. To obtain expression of the protein encoded by the eukaryotic cDNA prokaryotic regulatory sequences would also be required (e.g. a promoter).
[edit] 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 cDNA whose introns have been spliced:
- A eukaryotic cell transcribes the DNA (from genes) into RNA (pre-mRNA).
- The same cell processes the pre-mRNA strands by splicing out introns, and adding a poly-A tail and 5’ Methyl-Guanine cap.
- This mixture of mature mRNA strands are extracted from the cell.
- A poly-T oligonucleotide primer 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.
Note that the central dogma of molecular biology is not broken in this process.
Also, cDNA has no introns.
[edit] Applications
Complementary DNA is often used in gene cloning or as gene probes or in the creation of a cDNA library. Partial sequences of cDNAs are often obtained as expressed sequence tags.
[edit] External links
- Flash animation at McGraw-Hill genetics-cDNA
- H-Invitational Database
- Functional Annotation of the Mouse database
| Major Families of Biochemicals | ||
| Peptides | Amino acids | Nucleic acids | Carbohydrates | Lipids | Terpenes | Carotenoids | Tetrapyrroles | Enzyme cofactors | Steroids | Flavonoids | Alkaloids | Polyketides | Glycosides | ||
| Analogues of nucleic acids: | Types of Nucleic Acids | Analogues of nucleic acids: |
| Nucleobases: | Adenine | Thymine | Uracil | Guanine | Cytosine | Purine | Pyrimidine | |
|---|---|---|
| Nucleosides: | Adenosine | Uridine | Guanosine | Cytidine | Deoxyadenosine | Thymidine | Deoxyguanosine | Deoxycytidine | |
| Nucleotides: | AMP | UMP | GMP | CMP | ADP | UDP | GDP | CDP | ATP | UTP | GTP | CTP | cAMP | cADPR | cGMP | |
| Deoxynucleotides: | dAMP | TMP | dGMP | dCMP | dADP | TDP | dGDP | dCDP | dATP | TTP | dGTP | dCTP | |
| Ribonucleic acids: | RNA | mRNA | piRNA | tRNA | rRNA | ncRNA | sgRNA | shRNA | siRNA | snRNA | miRNA | snoRNA | LNA | |
| Deoxyribonucleic acids: | DNA | mtDNA | cDNA | plasmid | Cosmid | BAC | YAC | HAC | |
| Analogues of nucleic acids: | GNA | PNA | TNA| LNA | morpholino | |
