In genetics, complementary DNA is DNA synthesized from a messenger RNA template in a reaction
catalysed by the enzymes reverse transcriptase.
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, they will transfer the cDNA that codes for the protein to the recipient
cell.
cDNA is also produced naturally by retroviruses and then integrated into the host's genome
where it creates a provirus.
The term cDNA is also used, typically in a bioinformatics context, to refer to an mRNA
transcript's sequence, expressed as DNA bases rather than RNA bases.
Overview According to the central dogma of molecular
biology, when synthesizing a protein, a gene's DNA is transcribed into mRNA which is then
translated into protein.
One difference between eukaryotic and prokaryotic mRNA-coding genes is that eukaryotic genes
can contain introns which are non coding sequences, in contrast with exons, sequences that code
for mRNA.
During transcription, all intron RNA is cut from the RNA primary transcript and the remaining
pieces of the RNA primary transcript are spliced back together to become mRNA.
The mRNA code is then translated into an amino acid chain that constitutes the newly made
protein.
Prokaryotic genes have no introns, thus their RNA is not subject to cutting and splicing.
Often it is desirable to make prokaryotic cells express eukaryotic genes.
An approach one might consider is to add eukaryotic DNA directly into a prokaryotic cell, and
let it make the protein.
However, because eukaryotic DNA has introns, and prokaryotes lack the machinery for removing
introns from transcribed RNA, to make this approach work, all intron sequences must be
removed from eukaryotic DNA prior to transferring it into the host.
This 'intron-free' DNA is constructed using 'intron-free' mRNA as a template.
It is therefore a 'complementary' copy of the mRNA, and is thus called complementary
DNA.
To obtain expression of the protein encoded by the cDNA, prokaryotic regulatory sequences
would also be required.
Synthesis Though there are several methods for doing
so, cDNA is most often synthesized from mature mRNA using the enzyme reverse transcriptase.
This enzyme, which naturally occurs in retroviruses, operates on a single strand of mRNA, generating
its complementary DNA based on the pairing of RNA base pairs to their DNA complements.
To obtain eukaryotic cDNA whose introns have been removed:
A eukaryotic cell transcribes the DNA into RNA.
The same cell processes the pre-mRNA strands by removing introns, and adding a poly-A tail
and 5’ Methyl-Guanine cap 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.
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 is added, along with deoxynucleotide triphosphates.
This synthesizes one complementary strand of DNA hybridized to the original mRNA strand.
To synthesize an additional DNA strand, traditionally one would digest the RNA of the hybrid strand,
using an enzyme like RNase H, or through alkali digestion method.
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.
From the hairpin loop, a DNA polymerase can then use it as a primer to transcribe a complementary
sequence for the ss cDNA.
Now, you should be left with a double stranded cDNA with identical sequence as the mRNA of
interest.
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,
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.
Partial sequences of cDNAs are often obtained as expressed sequence tags.
With amplification of DNA sequences via polymerase chain reaction now commonplace, one will typically
conduct reverse transcription as an initial step, followed by PCR to obtain an exact sequence
of cDNA for intra-cellular expression.
This is achieved by designing sequence-specific DNA primers that hybridize to the 5' and 3'
ends of a cDNA region coding for a protein.
Once amplified, the sequence can be cut at each end with nucleases and inserted into
one of many small circular DNA sequences known as expression vectors.
Such vectors allow for self-replication inside cells, and potentially integration in the
host DNA.
They typically also contain a strong promoter to drive transcription of the target cDNA
into mRNA, which is then translated into protein.
On June 13, 2013, the United States Supreme Court ruled in Association for Molecular Pathology
v. Myriad Genetics that while human genes cannot be patented, cDNA can be.
Viruses Some viruses also use cDNA to turn their viral
RNA into mRNA.
The mRNA is used to make viral proteins to take over the host cell.
See also cDNA library
cDNA microarray RNA-Seq
References
External links H-Invitational Database
Functional Annotation of the Mouse database cDNA Animation
Complementary DNA tool