View source for Biology:Complementary DNA

{{Short description|Single-stranded DNA synthesized from  RNA}}

[[File:Cdnaarray.jpg|thumb|right|250px|Output from a '''cDNA''' [[Biology:DNA microarray|microarray]] used in testing]]

In [[Biology:Genetics|genetics]], '''complementary DNA''' ('''cDNA''') is [[Biology:DNA|DNA]] synthesized from a single-stranded RNA (e.g., messenger RNA ([[Biology:MRNA|mRNA]]) or [[Biology:MicroRNA|microRNA]] (miRNA)) template in a reaction catalyzed by the enzyme [[Biology:Reverse transcriptase|reverse transcriptase]].<ref>{{Citation |last=Hastings |first=P. J. |title=Complementary DNA (cDNA) |date=2001-01-01 |url=https://www.sciencedirect.com/science/article/pii/B0122270800002536 |encyclopedia=Encyclopedia of Genetics |pages=433 |editor-last=Brenner |editor-first=Sydney |place=New York |publisher=Academic Press |language=en |isbn=978-0-12-227080-2 |access-date=2022-11-29 |editor2-last=Miller |editor2-first=Jefferey H.}}</ref> cDNA is often used to [[Biology:Gene expression|express]] a specific [[Biology:Protein|protein]] in a cell that does not normally express that protein (i.e., [[Biology:Heterologous|heterologous]] expression), 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, often bacterial or yeast expression systems. cDNA is also generated to analyze [[Biology:Transcriptome|transcriptomic]] profiles in bulk tissue, single cells, or single nuclei in assays such as [[Biology:Microarray|microarray]]s, [[Biology:Real-time polymerase chain reaction|qPCR]], and [[Biology:RNA-Seq|RNA-seq]].

cDNA is also produced naturally by [[Biology:Retrovirus|retrovirus]]es (such as HIV-1, HIV-2, [[Biology:Simian immunodeficiency virus|simian immunodeficiency virus]], etc.) and then integrated into the host's genome, where it creates a [[Biology:Provirus|provirus]].<ref name="CroyNotes1998">{{cite web|last1=Croy|first1=Ron|title=Molecular Genetics II - Genetic Engineering Course (Supplementary notes)|url=http://dwb4.unl.edu/Chem/CHEM869N/CHEM869NLinks/www.dur.ac.uk/~dbl0www/Staff/Croy/cDNAfigs.htm|website=Durham University durham.ac.uk; 20 April 1998|access-date=4 February 2015|archive-url=https://web.archive.org/web/20020824023822/http://www.dur.ac.uk/~dbl0www/Staff/Croy/cDNAfigs.htm|archive-date=24 August 2002}}</ref> 

The term ''cDNA'' is also used, typically in a [[Biology:Bioinformatics|bioinformatics]] context, 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 2013 US Supreme Court decision in Association for Molecular Pathology v. Myriad Genetics, Inc. As a compromise, the Court declared, that exons-only cDNA is patent-eligible, whereas isolated sequences of naturally occurring [[Biology:DNA|DNA]] comprising introns are not. 

== Synthesis ==
[[Biology:RNA|RNA]] serves as a template for cDNA synthesis.<ref>{{Cite journal|last=Ying|first=Shao-Yao|date=1 July 2004|title=Complementary DNA libraries|journal=Molecular Biotechnology|language=en|volume=27|issue=3|pages=245–252|doi=10.1385/MB:27:3:245|pmid=15247497|s2cid=25600775|issn=1559-0305}}</ref> In cellular life, cDNA is generated by viruses and retrotransposons for integration of RNA into target [[Biology:Genomic DNA|genomic DNA]]. In molecular biology, RNA is purified from source material after genomic DNA, proteins and other cellular components are removed. cDNA is then synthesized through ''[[In vitro|in vitro]]'' [[Biology:Reverse transcriptase|reverse transcription]].<ref>{{cite web|title=5 Steps to Optimal cDNA Synthesis - US|url=https://www.thermofisher.com/us/en/home/life-science/pcr/reverse-transcription/5steps-cDNA.html|website=www.thermofisher.com|language=en|access-date=12 May 2020}}</ref>

=== RNA purification ===
RNA is transcribed from genomic DNA in host cells and is [[Biology:RNA extraction|extracted]] by first [[Biology:Lysis|lysing]] cells then purifying RNA utilizing widely-used methods such as phenol-chloroform, silica column, and bead-based RNA extraction methods.<ref>{{Cite journal|last1=Tavares|first1=Lucélia|last2=Alves|first2=Paula M.|last3=Ferreira|first3=Ricardo B.|last4=Santos|first4=Claudia N.|date=6 January 2011|title=Comparison of different methods for DNA-free RNA isolation from SK-N-MC neuroblastoma|journal=BMC Research Notes|volume=4|issue=1|pages=3|doi=10.1186/1756-0500-4-3|issn=1756-0500|pmc=3050700|pmid=21211020 |doi-access=free }}</ref> 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.<ref>{{cite journal|title=Improved and Convenient Method of RNA Isolation From Polyphenols and Polysaccharide Rich Plant Tissues|last1=R|first1=Kansal|last2=K|first2=Kuhar|date=December 2008|language=en|pmid=19245182|last3=I|first3=Verma|last4=Rn|first4=Gupta|last5=Vk|first5=Gupta|last6=Kr|first6=Koundal|journal=Indian Journal of Experimental Biology|volume=46|issue=12|pages=842–5}}</ref> To remove DNA and proteins, enzymes such as DNase and Proteinase K are used for degradation.<ref>{{Cite journal|title=Methods of RNA Purification. All Ways (Should) Lead to Rome|last1=I|first1=Vomelová|last2=Z|first2=Vanícková|date=2009|language=en|pmid=20163774|last3=A|first3=Sedo|journal=Folia Biologica|volume=55|issue=6|pages=243–51}}</ref> 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.<ref>{{Cite journal|last1=Sellin Jeffries|first1=Marlo K.|last2=Kiss|first2=Andor J.|last3=Smith|first3=Austin W.|last4=Oris|first4=James T.|date=14 November 2014|title=A comparison of commercially-available automated and manual extraction kits for the isolation of total RNA from small tissue samples|journal=BMC Biotechnology|volume=14|issue=1|pages=94|doi=10.1186/s12896-014-0094-8|issn=1472-6750|pmc=4239376|pmid=25394494 |doi-access=free }}</ref> Additional bead-based methods can be used to isolate specific sub-types of RNA ......(e.g. [[Biology:Messenger RNA|mRNA]] and [[Biology:MicroRNA|microRNA]]) based on size or unique RNA regions.<ref>{{Cite web|title=mRNA Isolation with Dynabeads in 15 minutes - US|url=https://www.thermofisher.com/us/en/home/life-science/dna-rna-purification-analysis/napamisc/mrna-isolation-dynabeads.html|website=www.thermofisher.com|language=en|access-date=20 May 2020}}</ref><ref>{{cite journal|last1=Gaarz|first1=Andrea|last2=Debey-Pascher|first2=Svenja|last3=Classen|first3=Sabine|last4=Eggle|first4=Daniela|last5=Gathof|first5=Birgit|last6=Chen|first6=Jing|last7=Fan|first7=Jian-Bing|last8=Voss|first8=Thorsten|last9=Schultze|first9=Joachim L.|last10=Staratschek-Jox|first10=Andrea|date=May 2010|title=Bead Array–Based microRNA Expression Profiling of Peripheral Blood and the Impact of Different RNA Isolation Approaches|journal=The Journal of Molecular Diagnostics |volume=12|issue=3|pages=335–344|doi=10.2353/jmoldx.2010.090116|issn=1525-1578|pmc=2860470|pmid=20228267}}</ref>

=== 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 reduced [[Biology:Ribonuclease H|RNase H]] activity suited for transcription of longer RNAs.<ref>{{Citation|last1=Haddad|first1=Fadia|title=Reverse Transcription of the Ribonucleic Acid: The First Step in RT-PCR Assay|date=2010|work=RT-PCR Protocols: Second Edition|pages=261–270|editor-last=King|editor-first=Nicola|series=Methods in Molecular Biology|publisher=Humana Press|language=en|doi=10.1007/978-1-60761-629-0_17|isbn=978-1-60761-629-0|last2=Baldwin|first2=Kenneth M.|volume=630|pmid=20301003}}</ref> 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).<ref>{{cite web|title=Reverse Transcriptase & cDNA Overview & Applications|url=https://www.goldbio.com/articles/article/Reverse-Transcriptase-cDNA-Overview-Applications|last=Martin|first=Karen|website=Gold Biotechnology|access-date=20 May 2020}}</ref> cDNA is commonly generated from mRNA for gene expression analyses such as [[Biology:Real-time polymerase chain reaction|RT-qPCR]] and [[Biology:RNA-Seq|RNA-seq]].<ref>{{Cite web|title=qPCR, Microarrays or RNA Sequencing - What to Choose?|url=https://www.biosistemika.com/blog/qpcr-microarrays-rna-sequencing-choose-one/|date=10 August 2017|website=BioSistemika|language=en-US|access-date=20 May 2020}}</ref> mRNA is selectively reverse transcribed using oligo-d[[Chemistry:Thymine|T]] primers that are the reverse complement of the [[Biology:Polyadenylation|poly-adenylated]] tail 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 and [[Biology:Random hexamer|random hexamer]] primers increases the chance of obtaining full-length cDNA while reducing 5' or 3' bias.<ref>{{Cite web|title=cDNA Synthesis {{!}} Bio-Rad|url=https://www.bio-rad.com/featured/en/cdna-synthesis.html|website=www.bio-rad.com|access-date=28 May 2020}}</ref> [[Biology:Ribosomal RNA|Ribosomal RNA]] may also be depleted to enrich both mRNA and non-poly-adenylated transcripts such as some [[Biology:Non-coding RNA|non-coding RNA]].<ref>{{Cite journal|last1=Herbert|first1=Zachary T.|last2=Kershner|first2=Jamie P.|last3=Butty|first3=Vincent L.|last4=Thimmapuram|first4=Jyothi|last5=Choudhari|first5=Sulbha|last6=Alekseyev|first6=Yuriy O.|last7=Fan|first7=Jun|last8=Podnar|first8=Jessica W.|last9=Wilcox|first9=Edward|last10=Gipson|first10=Jenny|last11=Gillaspy|first11=Allison|date=15 March 2018|title=Cross-site comparison of ribosomal depletion kits for Illumina RNAseq library construction|journal=BMC Genomics|volume=19|issue=1|pages=199|doi=10.1186/s12864-018-4585-1|issn=1471-2164|pmc=6389247|pmid=29703133 |doi-access=free }}</ref>

==== 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.<ref>{{cite web|title=cDNA Synthesis System|url=http://tools.thermofisher.com/content/sfs/manuals/18267.pdf |archive-url=https://web.archive.org/web/20181222122300/http://tools.thermofisher.com/content/sfs/manuals/18267.pdf |archive-date=2018-12-22 |url-status=live|last=Invitrogen|website=Thermofisher|access-date=27 May 2020}}</ref><ref>{{Cite journal|last1=Agarwal|first1=Saurabh|last2=Macfarlan|first2=Todd S.|last3=Sartor|first3=Maureen A.|last4=Iwase|first4=Shigeki|date=21 January 2015|title=Sequencing of first-strand cDNA library reveals full-length transcriptomes|journal=Nature Communications|language=en|volume=6|issue=1|page=6002|doi=10.1038/ncomms7002|pmid=25607527|pmc=5054741|bibcode=2015NatCo...6.6002A|issn=2041-1723}}</ref> 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. Coli [[Biology:DNA polymerase I|DNA Polymerase]] I and sealed with E. Coli [[Biology:DNA ligase|DNA 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 in gene cloning or as gene probes or in the creation of a [[Biology:CDNA library|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 (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., [[Biology:Intron|intron]]s). Partial sequences of cDNAs are often obtained as [[Biology:Expressed sequence tag|expressed sequence tag]]s.

With amplification of DNA sequences via [[Biology:Polymerase chain reaction|polymerase 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 or [[Biology:Real-time polymerase chain reaction|RT-qPCR]].<ref>{{Cite journal|date=December 1996|title=Use of a cDNA microarray to analyse gene expression patterns in human cancer|url=https://www.nature.com/articles/ng1296-457|journal=Nature Genetics|language=en|volume=14|issue=4|pages=457–460|doi=10.1038/ng1296-457|pmid=8944026|issn=1546-1718|last1=Derisi|first1=J.|last2=Penland|first2=L.|last3=Brown|first3=P. O.|last4=Bittner|first4=M. L.|last5=Meltzer|first5=P. S.|last6=Ray|first6=M.|last7=Chen|first7=Y.|last8=Su|first8=Y. A.|last9=Trent|first9=J. M.|s2cid=23091561}}</ref><ref>{{Cite journal|last1=White|first1=Adam K.|last2=VanInsberghe|first2=Michael|last3=Petriv|first3=Oleh I.|last4=Hamidi|first4=Mani|last5=Sikorski|first5=Darek|last6=Marra|first6=Marco A.|last7=Piret|first7=James|last8=Aparicio|first8=Samuel|last9=Hansen|first9=Carl L.|date=2011-08-23|title=High-throughput microfluidic single-cell RT-qPCR|journal=Proceedings of the National Academy of Sciences|language=en|volume=108|issue=34|pages=13999–14004|doi=10.1073/pnas.1019446108|issn=0027-8424|pmid=21808033|pmc=3161570|bibcode=2011PNAS..10813999W|doi-access=free}}</ref><ref>{{Cite journal|last1=Hrdlickova|first1=Radmila|last2=Toloue|first2=Masoud|last3=Tian|first3=Bin|date=January 2017|title=RNA-Seq methods for transcriptome analysis|journal=Wiley Interdisciplinary Reviews. RNA|volume=8|issue=1|pages=e1364|doi=10.1002/wrna.1364|issn=1757-7004|pmc=5717752|pmid=27198714}}</ref> 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, the United States Supreme Court ruled in the case of ''Association for Molecular Pathology v. Myriad Genetics'' that while naturally occurring genes cannot be [[Social:Patent|patent]]ed, cDNA is patent-eligible because it does not occur naturally.<ref>{{cite news|last=Liptak|first=Adam|date=13 June 2013|title=Supreme Court Rules Human Genes May Not Be Patented|newspaper={{wipe|The New York Times}}|url=https://www.nytimes.com/2013/06/14/us/supreme-court-rules-human-genes-may-not-be-patented.html |archive-url=https://ghostarchive.org/archive/20220101/https://www.nytimes.com/2013/06/14/us/supreme-court-rules-human-genes-may-not-be-patented.html |archive-date=2022-01-01 |url-access=limited|access-date=14 June 2013}}{{cbignore}}</ref>

==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.<ref>{{Cite journal|last1=Altfeld|first1=Marcus|last2=Gale|first2=Michael Jr.|date=1 June 2015|title=Innate immunity against HIV-1 infection|journal=Nature Immunology|language=en|volume=16|issue=6|pages=554–562|doi=10.1038/ni.3157|pmid=25988887|s2cid=1577651|issn=1529-2908|doi-access=free}}</ref>

cDNA is also generated by [[Biology:Retrotransposon|retrotransposon]]s in 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.<ref>{{Cite journal|last1=Havecker|first1=Ericka R.|last2=Gao|first2=Xiang|last3=Voytas|first3=Daniel F.|date=2004-05-18|title=The diversity of LTR retrotransposons|journal=Genome Biology|volume=5|issue=6|pages=225|doi=10.1186/gb-2004-5-6-225|issn=1474-760X|pmc=463057|pmid=15186483 |doi-access=free }}</ref><ref>{{Cite journal|last1=Cordaux|first1=Richard|last2=Batzer|first2=Mark A.|date=October 2009|title=The impact of retrotransposons on human genome evolution|journal=Nature Reviews Genetics|language=en|volume=10|issue=10|pages=691–703|doi=10.1038/nrg2640|pmid=19763152|pmc=2884099|issn=1471-0064}}</ref>

==See also==
* {{annotated link|Biology:CDNA library}}
* {{annotated link|Biology:RNA-Seq}}
* {{annotated link|Biology:Real-time polymerase chain reaction}} (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.
{{Reflist}}

== External links ==
* [https://web.archive.org/web/20100527204607/http://www.h-invitational.jp/ H-Invitational Database]
* [https://web.archive.org/web/20181102155217/http://fantom.gsc.riken.jp/ Functional Annotation of the Mouse database]
* [http://www.bugaco.com/calculators/dna_reverse_complement.php Complementary DNA tool]
{{Nucleic acids}}
* http://news.icecric.com/today-match-prediction/


{{DEFAULTSORT:Complementary Dna}}
[[Category:DNA]]

{{Sourceattribution|Complementary DNA}}