Complementary DNA

"CDNA" redirects here. For other uses, seeCDNA (disambiguation).

For the general property of complementarity in molecular biology, seeComplementarity (molecular biology).For complementation tests used in genetics research, seeComplementation (genetics).

Output from acDNAmicroarrayused in testing

Ingenetics,complementary DNA(cDNA) isDNAthat was reverse transcribed (viareverse transcriptase) from an RNA (e.g.,messenger RNAormicroRNA). cDNA exists in bothsingle-strandedand double-stranded forms and in both natural and engineered forms.

In engineered forms, it often is a copy (replicate) of the naturally occurring DNA from any particular organism's natural genome; the organism's own mRNA was naturally transcribed from its DNA, and the cDNA is reverse transcribed from the mRNA, yielding a duplicate of the original DNA. Engineered cDNA is often used toexpressa specificproteinin a cell that does not normally express that protein (i.e.,heterologousexpression), or to sequence or quantify mRNA molecules using DNA based methods (qPCR, RNA-seq). cDNA that codes for a specific protein can be transferred to a recipient cell for expression as part ofrecombinant DNA,often bacterial or yeast expression systems.cDNA is also generated to analyzetranscriptomicprofiles in bulk tissue, single cells, or single nuclei in assays such asmicroarrays,qPCR,andRNA-seq.

In natural forms, cDNA is produced byretroviruses(such asHIV-1,HIV-2,simian immunodeficiency virus,etc.) and then integrated into the host's genome, where it creates aprovirus.

The termcDNAis also used, typically in abioinformaticscontext, to refer to an mRNA transcript's sequence, expressed as DNA bases (deoxy-GCAT) rather than RNA bases (GCAU).

Patentability of cDNA was a subject of a 2013US Supreme Courtdecision inAssociation for Molecular Pathology v. Myriad Genetics, Inc.As a compromise, the Court declared, thatexons-only cDNA is patent-eligible, whereas isolated sequences of naturally occurringDNAcomprisingintronsare not.

Synthesis

RNAserves as a template for cDNA synthesis.In cellular life, cDNA is generated by viruses and retrotransposons for integration of RNA into targetgenomic DNA.In molecular biology, RNA is purified from source material after genomic DNA, proteins and other cellular components are removed. cDNA is then synthesized throughin vitroreverse transcription.

RNA purification

RNA is transcribed from genomic DNA in host cells and isextractedby firstlysingcells then purifying RNA utilizing widely-used methods such as phenol-chloroform, silica column, and bead-based RNA extraction methods.Extraction methods vary depending on the source material. For example, extracting RNA from plant tissue requires additional reagents, such as polyvinylpyrrolidone (PVP), to remove phenolic compounds, carbohydrates, and other compounds that will otherwise render RNA unusable.To remove DNA and proteins, enzymes such as DNase and Proteinase K are used for degradation.Importantly, RNA integrity is maintained by inactivating RNases with chaotropic agents such as guanidinium isothiocyanate, sodium dodecyl sulphate (SDS), phenol or chloroform. Total RNA is then separated from other cellular components and precipitated with alcohol. Various commercial kits exist for simple and rapid RNA extractions for specific applications.Additional bead-based methods can be used to isolate specific sub-types of RNA (e.g.mRNAandmicroRNA) based on size or unique RNA regions.

Reverse transcription

First-strand synthesis

Using a reverse transcriptase enzyme and purified RNA templates, one strand of cDNA is produced (first-strand cDNA synthesis). The M-MLV reverse transcriptase from the Moloney murine leukemia virus is commonly used due to its reducedRNase Hactivity suited for transcription of longer RNAs.The AMV reverse transcriptase from the avian myeloblastosis virus may also be used for RNA templates with strong secondary structures (i.e. high melting temperature).cDNA is commonly generated from mRNA for gene expression analyses such asRT-qPCRandRNA-seq.mRNA is selectively reverse transcribed using oligo-dTprimers that are the reverse complement of thepoly-adenylatedtail on the 3' end of all mRNA. The oligo-dT primer anneals to the poly-adenylated tail of the mRNA to serve as a binding site for the reverse transcriptase to begin reverse transcription. An optimized mixture of oligo-dT andrandom hexamerprimers increases the chance of obtaining full-length cDNA while reducing 5' or 3' bias.Ribosomal RNAmay also be depleted to enrich both mRNA and non-poly-adenylated transcripts such as somenon-coding RNA.

Second-strand synthesis

The result of first-strand syntheses, RNA-DNA hybrids, can be processed through multiple second-strand synthesis methods or processed directly in downstream assays.An early method known as hairpin-primed synthesis relied on hairpin formation on the 3' end of the first-strand cDNA to prime second-strand synthesis. However, priming is random and hairpin hydrolysis leads to loss of information. The Gubler and Hoffman Procedure uses E. Coli RNase H to nick mRNA that is replaced with E. ColiDNA PolymeraseI and sealed with E. ColiDNA Ligase.An optimization of this procedure relies on low RNase H activity of M-MLV to nick mRNA with remaining RNA later removed by adding RNase H after DNA Polymerase translation of the second-strand cDNA. This prevents lost sequence information at the 5' end of the mRNA.

Applications

Complementary DNA is often used ingene cloningor asgene probesor in the creation of acDNA 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 asexpressed sequence tags.

With amplification of DNA sequences viapolymerase chain reaction(PCR) 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 the 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.

cDNA is also used to study gene expression via methods such as RNA-seq orRT-qPCR.For sequencing, RNA must be fragmented due to sequencing platform size limitations. Additionally, second-strand synthesized cDNA must be ligated with adapters that allow cDNA fragments to be PCR amplified and bind to sequencing flow cells. Gene-specific analysis methods commonly use microarrays and RT-qPCR to quantify cDNA levels via fluorometric and other methods.

On 13 June 2013, theUnited States Supreme Courtruled in the case ofAssociation for Molecular Pathology v. Myriad Geneticsthat while naturally occurring genes cannot bepatented,cDNA is patent-eligible because it does not occur naturally.

Viruses and retrotransposons

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.

An example of this first step from viral RNA to cDNA can be seen in the HIV cycle of infection. Here, the host cell membrane becomes attached to the virus' lipid envelope which allows the viral capsid with two copies of viral genome RNA to enter the host. The cDNA copy is then made through reverse transcription of the viral RNA, a process facilitated by the chaperone CypA and a viral capsid associated reverse transcriptase.

cDNA is also generated byretrotransposonsin eukaryotic genomes. Retrotransposons are mobile genetic elements that move themselves within, and sometimes between, genomes via RNA intermediates. This mechanism is shared with viruses with the exclusion of the generation of infectious particles.

See also

• cDNA library– Type of DNA library
• cDNA microarray– Collection of microscopic DNA spots attached to a solid surfacePages displaying short descriptions of redirect targets
• RNA-Seq– Lab technique in cellular biology
• Real-time polymerase chain reaction– Laboratory technique of molecular biology (RT-qPCR)

References

Mark D. Adams et al. "Complementary DNA Sequencing: Expressed Sequence Tags and Human Genome Project."Science (American Association for the Advancement of Science)252.5013 (1991): 1651–1656. Web.

Philip M. Murphy, and H. Lee Tiffany. "Cloning of Complementary DNA Encoding a Functional Human Interleukin-8 Receptor."Science (American Association for the Advancement of Science)253.5025 (1991): 1280–1283. Web.

External links

• H-Invitational Database
• Functional Annotation of the Mouse database
• Complementary DNA tool

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