cDNA
The cDNA ( English complementary DNA , German complementary DNA ) is a DNA that is synthesized from RNA (such as mRNA and ncRNA ) using the enzyme reverse transcriptase . The cDNA is used in molecular biology , transcriptome analysis and in medical diagnostics .
synthesis
With the help of the enzyme reverse transcriptase , the complementary cDNA can be produced from RNA in the course of an RT-PCR . This specific, RNA-dependent DNA polymerase , like other DNA polymerases, requires a primer (a short, complementary DNA segment) for synthesis, which binds to the RNA. Usually an oligo-dT nucleotide (15-25 deoxythymidines ) is used as a primer , which is complementary to the poly-A tail of the eukaryotic mRNA, or random hexamer oligonucleotides (consisting of six randomly assembled nucleotides ). However, specific primers can also be used in order to isolate a transcript in a targeted manner. The product is now a cDNA strand that has hybridized with the original RNA strand . The latter can now be broken down with the enzyme RNase H. In the further processing, the complementary DNA strand to the already existing cDNA single strand is synthesized by means of a DNA-dependent DNA polymerase (via a primer). Remnants of mRNA that have not yet been degraded by RNase H can serve as primers. The methods used today for second-strand synthesis are based on the work of Hiroto Okayama and Paul Berg at Stanford University and the further development of Ueli Gubler and Beth Hoffmann at Hoffmann-LaRoche . The result is a double-stranded cDNA. Since the original template was a processed RNA (which, among other things , has already gone through splicing ), there are no introns on the cDNA in contrast to natural eukaryotic DNAs. This fact is important for the cloning of the cDNA in vectors such as plasmids and subsequent recombinant protein expression .
Applications and meaning
CDNAs can be amplified via PCR . This reverse transcriptase polymerase chain reaction (RT-PCR) can also be carried out quantitatively as qPCR, for example as real-time quantitative PCR . With this variant of the gene expression analysis , the expression rate of the respective underlying RNA can be determined, so that differences in the gene expression of different tissues or healthy to diseased tissue can be detected in research and diagnostics . Furthermore, cDNA is used in diagnostics for the detection of pathogens, e.g. B. HIV in the blood of patients. CDNAs can also be used to generate microarrays . Starting point of the generation of fluorescent samples for the screening of DNA chips is also often the RT reaction. The serial analysis of gene expression (SAGE) is also based on the generation of, in this case short, cDNAs. A modification of RT-PCR, RACE-PCR, is used to identify full-length cDNAs .
By cloning and sequencing the cDNA (as expressed sequence tags , EST), the sequence of the corresponding genes can be analyzed by projecting them onto the corresponding genome . The cDNA also enables information on alternative splicing to be obtained, i.e. which variant occurs in which tissue or cell type and where intron - exon boundaries are located in the gene. Furthermore, the amino acid sequence of a protein can be clearly deduced from cDNA using the genetic code and a cDNA clone can thus be used to express the corresponding protein ( recombinant protein expression ).
cDNA library
A cDNA library , also called cDNA library , is a collection of many cDNAs that have been isolated and transcribed from the mRNA of a specific cell or tissue and ideally represents the entirety of all expressed genes, the transcriptome , of the examined sample. The cDNAs are usually cloned in the bacteriophage lambda , or plasmids , which are propagated in bacteria . Such a cDNA library can then e.g. B. used for screening procedures. A typical screening method for cDNA banks is the colony hybridization screening technique, in which radioactive DNA samples are used. The aim is, for example, to isolate a certain cDNA from an as yet unexplored organism or to discover gene families. A well-known example is research into homeobox proteins . If the cDNAs are cloned in expression vectors, this allows isolation with antibodies or on the basis of a property that can be detected biochemically or electrophysiologically . Unknown interaction partners can also be identified with expression banks, e.g. B. in the yeast two-hybrid system .
Web links
Individual evidence
- ↑ H. Okayama, P. Berg: High-efficiency cloning of full-length cDNA. In: Molecular and cellular biology. Volume 2, Number 2, February 1982, pp. 161-170, PMID 6287227 , PMC 369769 (free full text).
- ^ U. Gubler, BJ Hoffman: A simple and very efficient method for generating cDNA libraries. In: Genes. Vol. 25, Numbers 2-3, November 1983, pp. 263-269, PMID 6198242 .