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double-stranded cDNA

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出典(authority):フリー百科事典『ウィキペディア（Wikipedia）』「2019/04/01 07:36:43」(JST)

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相補的DNA（そうほてきDNA、complementary DNA）は、mRNA から逆転写酵素を用いた逆転写反応によって合成された二本鎖 DNA。一般には「相補的」を意味する英語、complementary の頭文字をとって、cDNA と省略される。遺伝子の上でタンパク質に翻訳される領域の配列が開始コドンから終止コドンまで一続きに含まれているため、タンパク質の一次構造（アミノ酸配列）を解明する出発点として、また人工的にタンパク質を発現させる目的でも単離される。

ヒトを始めとする真核生物では、遺伝子はゲノム上にコードされているものの、多くはそこから転写された mRNA前駆体がスプライシングを受けてイントロンが除去されるまでは蛋白質に翻訳されない情報も含んでいる。

そこで、スプライシング済みの成熟mRNA から cDNA を合成すればイントロンを含まない状態の遺伝子（塩基配列）を知ることができることから、遺伝子のクローニングに広く利用されている。

また、mRNA はスプライシング以外にも RNAエディティングという加工が起こるため、対応するゲノムDNA と cDNA の比較を行うことによって、エディティングサイトの特定が可能となる。

cDNA合成を逆転写酵素によって行ったのち、

λファージベクターやプラスミドにクローニングし、cDNAライブラリーを作製。標識プロープや抗体などにより cDNA ライブラリーをスクリーニングして、特定遺伝子の cDNA を単離する。
PCR法により、既知の塩基配列の情報をもとに、特定配列を増幅する（RT-PCRという）。それを直接解析するか、プラスミド等にクローニングした後、解析を行う。この方法を工夫して、未知の 5'側配列、3'側配列を増幅するのに、RACE法（Rapid Amplification of cDNA end) と呼ばれる cDNA増幅法がある。
サブトラクション法などによって、未知であるが、遺伝子発現量に差がある cDNA のみを増幅し、それを単離する。

といった解析が行われる。

cDNAの合成法

cDNA の合成には、(1) 全RNA（リボソームRNA、mRNAを含む）、または (2) polyA-RNA（mRNA）を鋳型とする方法がある。

また、逆転写酵素の反応開始には、RNA上に反応の開始となるプライマーをアニールさせ、3'から5’方向にcDNAを合成する必要がある。プライマーには、

polyA構造を狙ったオリゴdTプライマーにより mRNA の 3'側から選択的に cDNA を作る。
既知の cDNA配列に特異的な遺伝子特異的なプライマーを用いる。
ランダムプライマー（通常、6塩基から8塩基程度の長さのものが使われる）を用いて、mRNA のすべての領域から cDNA合成を開始させる。

といった方法がある。

cDNAの合成に用いられる逆転写酵素の種類としては、AMV（トリmyeloblastosisウイルス）、あるいは MoMuLV（モロニーマウス白血病ウイルス）由来の酵素が用いられることが多い。従来は、AMV由来の酵素が熱安定性が高かったため、mRNA の高次構造の存在を回避する手段として用いられていたが、近年、MoMuLV由来酵素の改良が進み、熱安定性の高い変異型酵素が市販されるようになったので、多くの研究者によって利用されている。なお、ある種の熱耐性型 DNAポリメラーゼ（PCR に用いられる）には、RNA を鋳型としてDNAを合成する逆転写酵素活性を有するものがあり、それを利用する方法もある。

逆転写酵素反応は、mRNA が高次構造を取った場合に伸長しない。また、5'側から cDNA の相補鎖（mRNA と同じ方向をした DNA鎖）を作る際に、5'側にできるヘアピン構造を利用するため、本当の 5'末端が失われる。前者の解決法には、mRNA の熱変性や、上記に記載した高温度での逆転写反応が利用される。後者の解決法には、ピロリン酸ホスファターゼを利用した方法などがあり、理化学研究所の完全長cDNA塩基配列決定プロジェクトで利用されている cDNAライブラリーは、このような方法で作製されたものである。

EST

EST とは expressed sequence tag の略であり、遺伝子転写産物 (RNA) の一部（普通5'末端か3'末端）に当たる短い配列で、転写産物の“目印”として使われる。cDNAライブラリーからランダムに多数のクローンを選び、それから簡単なシークエンシングによって長さ 500 - 800ヌクレオチド程度の配列を決定したものが EST であり、それらをまとめてデータベースを作製し利用する。まとめる方法としては ESTクラスタリングという手法が用いられる。特定の遺伝子の解析から断片的な配列が判明した場合、ESTデータベースからそれに対応する EST を探索し、これを手がかりにして段階的に RNA 全体の配列を得ることができる。現在はゲノムプロジェクトの補助的技術（あるいは ESTプロジェクト）として盛んに用いられている。また EST は DNAマイクロアレイ用のプローブとして用いることもできる。最近では初めから完全長cDNA（RNA全長に相当する cDNA）を得て解析する技術も進歩しつつあるが、EST は依然として遺伝子解析における有用な技術である。

FANTOM

Functional Annotation of Mouse for the RIKEN full-length cDNA clones

http://fantom.gsc.riken.jp/jp/

FANTOMは、理化学研究所のマウスゲノム百科事典プロジェクトで収集された完全長cDNAのアノテーション（機能注釈）を行うことを目的に、林崎良英博士が中心となり2000年に結成された国際研究コンソーシアムです。その役割はトランスクリプトーム解析の分野を軸に発展・拡大してきました。また、プロジェクトの研究対象は、ゲノムの転写産物という「要素」の理解から、転写制御ネットワークという「システム」つまり「生命体のシステム」の理解へ、より高次の階層に向けて着実に進められています。

マイクロアレイ

「DNAマイクロアレイ」を参照

GeneChips

SAGE

遺伝子解析法 SAGE は(Serial Analysis of Gene Expression)から英語の頭字語をとった略称。遺伝子転写産物 (RNA) を逆転写した相補的DNA(cDNA)の一部(10-20bp)を識別用配列(5-10bp?)をはさんで直列に連結したものをDNAシーケンサーで読み取ることにより、多くの転写産物の発現量を調べることができる。相補的DNAの一部を網羅的にシーケンシングする手法としては、SAGE法を改良したSuperSAGE, オリゴキャップ法と併用したCAGE、MPSS, HiCEP等がある。

詳細は「SAGE法」を参照

外部リンク

The NCBI Handbook, Part 3, Chapter 21

この項目は、生物学に関連した書きかけの項目です。この項目を加筆・訂正などしてくださる協力者を求めています（プロジェクト:生命科学／Portal:生物学）。

wiki en

[Wiki en表示]

Output from a cDNA microarray used in testing

In genetics, complementary DNA (cDNA) is DNA synthesized from a single-stranded RNA (e.g., messenger RNA (mRNA) or microRNA) template in a reaction catalyzed by the enzyme 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 (i.e., heterologous expression), they will transfer the cDNA that codes for the protein to the recipient cell. cDNA is also produced naturally by retroviruses (such as HIV-1, HIV-2, simian immunodeficiency virus, etc.) and then integrated into the host's genome, where it creates a provirus.^[1]

The term cDNA is also used, typically in a bioinformatics context, to refer to an mRNA transcript's sequence, expressed as DNA bases (GCAT) rather than RNA bases (GCAU).

cDNA is derived from mRNA, so it contains only exons but no introns.

Synthesis

Although there are several methods for doing so, cDNA is most often synthesized from mature (fully spliced) 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 (A, U, G and C) to their DNA complements (T, A, C and G, respectively).

To obtain eukaryotic cDNA whose introns have been removed:

A eukaryotic cell transcribes the DNA (from genes) into RNA (pre-mRNA).
The same cell processes the pre-mRNA strands by removing introns, and adding a poly-A tail and 5’ Methyl-Guanine cap (this is known as post-transcriptional modification)
This mixture of mature mRNA strands is extracted from the cell. The poly-A tail of the post-transcriptional 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 requires this double-stranded segment as a primer to start its operation.)
Reverse transcriptase is added, along with deoxynucleotide triphosphates (A, T, G, C). 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 (ssDNA) 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 (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 as expressed sequence tags (EST).

With amplification of DNA sequences via 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.

On June 13, 2013, the United States Supreme Court ruled in the case of Association for Molecular Pathology v. Myriad Genetics that while naturally occurring human genes cannot be patented, cDNA is patent eligible because it does not occur naturally.^[2]

Viruses

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 DNA 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 though reverse transcription of the viral RNA, a process facilitated by the chaperone CypA and a viral capsid associated reverse transcriptase.^[3]

References

^ Croy, Ron. "Molecular Genetics II - Genetic Engineering Course (Supplementary notes)". Durham University durham.ac.uk; 20 April 1998. Archived from the original on 2002-08-24. Retrieved 4 February 2015.
^ Liptak, Adam (13 June 2013). "Supreme Court Rules Human Genes May Not Be Patented". The New York Times. Retrieved 14 June 2013.
^ Altfeld, Marcus; Jr, Michael Gale (2015-06-01). "Innate immunity against HIV-1 infection". Nature Immunology. 16 (6): 554–562. doi:10.1038/ni.3157. ISSN 1529-2908.

External links

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

UpToDate Contents

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English Journal

Development of an ultrasensitive aptasensor for the detection of aflatoxin B1.

Guo X1, Wen F2, Zheng N3, Luo Q4, Wang H2, Wang H2, Li S2, Wang J5.Author information 1Ministry of Agriculture Laboratory of Quality & Safety Risk Assessment for Dairy Products (Beijing), Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, PR China; College of Animal Science and Technology, Xinjiang Agricultural University, Urumchi 830000, PR China; Ministry of Agriculture-Milk and Dairy Product Inspection Center (Beijing), Beijing 100193, PR China.2Ministry of Agriculture Laboratory of Quality & Safety Risk Assessment for Dairy Products (Beijing), Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, PR China; Ministry of Agriculture-Milk and Dairy Product Inspection Center (Beijing), Beijing 100193, PR China.3Ministry of Agriculture Laboratory of Quality & Safety Risk Assessment for Dairy Products (Beijing), Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, PR China; Ministry of Agriculture-Milk and Dairy Product Inspection Center (Beijing), Beijing 100193, PR China; State Key Laboratory of Animal Nutrition, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, PR China. Electronic address: wangjiaqinmqc@126.com.4College of Animal Science and Technology, Xinjiang Agricultural University, Urumchi 830000, PR China.5Ministry of Agriculture Laboratory of Quality & Safety Risk Assessment for Dairy Products (Beijing), Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, PR China; Ministry of Agriculture-Milk and Dairy Product Inspection Center (Beijing), Beijing 100193, PR China; State Key Laboratory of Animal Nutrition, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, PR China.AbstractContamination of feed and food by aflatoxin B1 (AFB1), one of the most toxic of the mycotoxins, is a global concern. To prevent food safety scares, and avoid subsequent economic losses due to the recall of contaminated items, methods for the rapid, sensitive and specific detection of AFB1 at trace levels are much in demand. In this work, a simple, ultrasensitive, and reliable aptasensor is described for the detection of AFB1. An AFB1 aptamer was used as a molecular recognition probe, while its complementary DNA played a role as a signal generator for amplification by real-time quantitative polymerase chain reaction (PCR). Under optimal conditions, a wide linear detection range (5.0×10(-5) to 5.0ngmL(-1)) was achieved, with a high sensitivity (limit of detection (LOD)=25fgmL(-1)). In addition, the proposed aptasensor exhibited excellent specificity for AFB1 compared with eight other mycotoxins, with no obvious Ct value change. This aptasensor can also be used in quantifying AFB1 levels in Chinese wildrye hay samples and infant rice cereal samples, demonstrating satisfactory recoveries in the range of 88-127% and 94-119%, respectively. This detection technique has a significant potential for high-throughput, quantitative determination of mycotoxin levels in a large range of feeds and foods.
Biosensors & bioelectronics.Biosens Bioelectron.2014 Jun 15;56:340-4. doi: 10.1016/j.bios.2014.01.045. Epub 2014 Feb 2.
Contamination of feed and food by aflatoxin B1 (AFB1), one of the most toxic of the mycotoxins, is a global concern. To prevent food safety scares, and avoid subsequent economic losses due to the recall of contaminated items, methods for the rapid, sensitive and specific detection of AFB1 at trace l
PMID 24549114

The study of marine corrosion of copper alloys in chlorinated condenser cooling circuits: The role of microbiological components.

Carvalho ML1, Doma J2, Sztyler M3, Beech I2, Cristiani P4.Author information 1RSE SpA, Ricerca sul Sistema Energetico, Dipartimento Ambiente e Sviluppo Sostenibile, Via Rubattino 54, 20134 Milano, Italy.2University of Portsmouth, School of Pharmacy and Biomedical Sciences, St Michael's Building, White Swan Road, Portsmouth PO1 2DT, UK; University of Oklahoma, Department of Botany and Microbiology, Institute for Energy and the Environment, 770 Van Vleet Oval, Norman, OK 73019, USA.3University of Portsmouth, School of Pharmacy and Biomedical Sciences, St Michael's Building, White Swan Road, Portsmouth PO1 2DT, UK.4RSE SpA, Ricerca sul Sistema Energetico, Dipartimento Ambiente e Sviluppo Sostenibile, Via Rubattino 54, 20134 Milano, Italy. Electronic address: pierangela.cristiani@rse-web.it.AbstractThe present paper reports the on-line monitoring of corrosion behavior of the CuNi 70:30 and Al brass alloys exposed to seawater and complementary offline microbiological analyses. An electrochemical equipment with sensors specifically set for industrial application and suitable to estimate the corrosion (by linear polarization resistance technique), the biofilm growth (by the BIOX electrochemical probe), the chlorination treatment and other physical-chemical parameters of the water has been used for the on-line monitoring. In order to identify and better characterize the bacteria community present on copper alloys, tube samples were collected after a long period (1year) and short period (2days) of exposition to treated natural seawater (TNSW) and natural seawater (NSW). From the collected samples, molecular techniques such as DNA extraction, polymerase chain reaction (PCR), denaturing gradient gel electrophoresis (DGGE) and identification by sequencing were performed to better characterize and identify the microbial biodiversity present in the samples. The monitoring data confirmed the significant role played by biofouling deposition against the passivity of these Cu alloys in seawater and the positive influence of antifouling treatments based on low level dosages. Molecular analysis indicated biodiversity with the presence of Marinobacter, Alteromonas and Pseudomonas species.
Bioelectrochemistry (Amsterdam, Netherlands).Bioelectrochemistry.2014 Jun;97:2-6. doi: 10.1016/j.bioelechem.2013.12.005. Epub 2013 Dec 24.
The present paper reports the on-line monitoring of corrosion behavior of the CuNi 70:30 and Al brass alloys exposed to seawater and complementary offline microbiological analyses. An electrochemical equipment with sensors specifically set for industrial application and suitable to estimate the corr
PMID 24411305

The essential roles of core binding factors CfRunt and CfCBFβ in hemocyte production of scallop Chlamys farreri.

Yue F1, Zhou Z2, Wang L3, Sun R1, Jiang Q1, Yi Q1, Zhang T1, Song L4.Author information 1Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, 7 Nanhai Rd., Qingdao 266071, China; University of Chinese Academy of Sciences, Beijing 100049, China.2Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, 7 Nanhai Rd., Qingdao 266071, China.3Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, 7 Nanhai Rd., Qingdao 266071, China. Electronic address: wanglingling@ms.qdio.ac.cn.4Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, 7 Nanhai Rd., Qingdao 266071, China. Electronic address: lshsong@ms.qdio.ac.cn.AbstractCore binding factor (CBF) is a family of heterodimeric transcription factors composed of a DNA-binding CBFα subunit and a non-DNA-binding CBFβ subunit, which plays critical roles in regulating hematopoiesis, osteogenesis and neurogenesis. In the present study, two genes encoding Runt (designed as CfRunt) and CBFβ (designed as CfCBFβ) were cloned and characterized from scallop Chlamys farreri. The full-length cDNA of CfRunt and CfCBFβ consists of 2128bp and 1729bp encoding a predicted polypeptide of 530 and 183 amino acids with a conserved Runt domain and CBFβ domain, respectively. Electrophoretic mobility shift assay demonstrated that the recombinant CfRunt protein (rCfRunt) exhibited solid ability to bind specific DNA, whereas rCfCBFβ could remarkably increase the DNA-binding affinity of rCfRunt. The mRNA transcripts of CfRunt and CfCBFβ could be detected in all tested tissues, especially in hemocytes, heart, hepatopancreas or muscle. After bacterial challenge, the circulating total hemocyte count (THC) of scallop reduced to the lowest level at 6h (P<0.05), and then it recovered gradually to the control level at 48-96h, while the mRNA expressions of CfRunt and CfCBFβ were significant up-regulated between 6 and 48h (P<0.05). After CfRunt gene was silenced by RNA interference, the hemocyte renewal rate and circulating THC both decreased significantly (P<0.05). However, following the RNA interference of CfRunt, the mRNA expression of CfRunt was significantly induced (P<0.05) and the attenuated hemocyte renewal rate and circulating THC could be repaired partially by LPS stimulation in the CfRunt-silenced scallops. The results collectively indicated that CfRunt and CfCBFβ, as conserved transcription factors, played essential roles in regulating hemocyte production of scallop.
Developmental and comparative immunology.Dev Comp Immunol.2014 Jun;44(2):291-302. doi: 10.1016/j.dci.2014.01.008. Epub 2014 Jan 21.
Core binding factor (CBF) is a family of heterodimeric transcription factors composed of a DNA-binding CBFα subunit and a non-DNA-binding CBFβ subunit, which plays critical roles in regulating hematopoiesis, osteogenesis and neurogenesis. In the present study, two genes encoding Runt (designed as
PMID 24462835

Japanese Journal

Expression levels of domestic cDNA cassettes integrated in the nuclear genomes of various Chlamydomonas reinhardtii strains

Kong Fantao,Yamasaki Tomohito,Ohama Takeshi
Journal of bioscience and bioengineering 117(5), 613-616, 2014-05
NAID 40020068693

Cloning of a cDNA Encoding the Gly m Bd 28K Precursor and Its Vacuole Transport in Tobacco BY2 Suspension-Cultured Cells

YUMIOKA-ITO Hitomi,MISAKI Ryo,YOKORO Miyuki [他]
Journal of nutritional science and vitaminology 60(2), 129-139, 2014-04
NAID 40020056157

Peptidomic Analysis of Antimicrobial Peptides in Skin Secretions of Amolops mantzorum

Hu Yuhong,Yu Zhijun,Xu Shiqi [他]
Zoological science 31(3), 143-151, 2014-03
NAID 40020028989

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