Complementary DNA
The cDNA ( complementary DNA English, German complementary DNA ) is a DNA that is synthesized by the enzyme reverse transcriptase of RNA ( such as mRNA and ncRNA ). Common applications for the cDNA in the molecular biology, transcriptome and in medical diagnostics.
Synthesis
With the aid of the enzyme reverse transcriptase can be prepared from the RNA complementary cDNA. This specific RNA-dependent DNA polymerase, as required, other DNA polymerases, a primer (a short, complementary DNA portion ) to the synthesis, which binds to the RNA. Most is used as a primer an oligo -dT nucleotide ( 15-25 Deoxythymidines ), which is complementary to the poly -A tail of eukaryotic mRNA, or random hexamer oligonucleotides used (consisting of six randomly assembled nucleotides ). But it may also include specific primers are used to selectively isolate a transcript. The product is now a cDNA strand, which is hybridized to the original RNA strand. The latter can now be removed with the enzyme RNase H. The further processing is prepared ( a primer), the complementary DNA strand to the existing cDNA single strand now by means of a DNA-dependent DNA polymerase. The primer can serve mRNA residues that have not been degraded by the RNase H. The result is a double-stranded cDNA. Since the original template was a processed RNA (which has, inter alia, the splicing already passed ), found on the cDNA unlike natural eukaryotic DNAs no introns. This fact is used for cloning of the cDNA into vectors such as plasmids and subsequent recombinant protein expression is important.
Applications and importance
About PCR cDNAs can reproduce itself. Using gene expression analysis can determine the expression rate of the underlying RNA, so that it is possible in the research and diagnostics differences in gene expression of various tissues or prove healthy to diseased tissue. Further cDNA is used in diagnostics for the detection of pathogens, such as HIV in the blood of patients.
By cloning and sequencing of the cDNA (as expressed sequence tags EST ) the sequence of the corresponding genes can be analyzed by the projection of the corresponding genome. The cDNA thus enables information to alternative splicing to win, that is, in which tissue or cell type occur which variant and where intron - exon boundaries are located in the gene. Further, from cDNA by means of the genetic code, the amino acid sequence of a protein can be derived and thus unique, a cDNA clone can be used for the expression of the corresponding protein ( recombinant protein expression).
CDNA library
, A cDNA library, and cDNA library is a collection of many cDNAs isolated from the mRNA of a particular cell or tissue and rewritten and represents, ideally, the total of all expressed genes, the transcriptome of the examined sample. CDNAs are cloned into the control plasmids which are propagated in bacteria. Such a cDNA library can then be used for example for screening procedures. A typical screening method is the study of gene expression using microarrays or SAGE. With these methods, different samples can be compared to the gene expression pattern in a short time. Also unknown interaction partners can be studied, for example in the yeast two -hybrid system.