Name: from mrna to cdna for gene studies
Uploaded: 2026-08-26T08:20:09.925Z
Duration: 2 min 25 s
Channel: JoVE

Fast jede Zelle im K&#xF6;rper hat die gleiche DNA, aber verschiedene Zelltypen wie Neuronen und Muskelzellen exprimieren unterschiedliche Gene, da in jeder Zelle nur bestimmte Gene in Messenger-RNA oder mRNA transkribiert werden.
 Im Labor k&#xF6;nnen mRNAs als Matrize zur Synthese komplement&#xE4;rer DNA (cDNA) zur Untersuchung der Genexpression verwendet werden.
 Eine &#xFC;bliche Methode besteht darin, RNA aus Zellen zu extrahieren und dann die mRNA aus anderen RNA-Typen wie ribosomaler RNA oder Transfer-RNA zu isolieren, indem die Probe &#xFC;ber eine K&#xFC;gelchens&#xE4;ule mit anhaftenden Thyminnukleotid-Abschnitten gef&#xFC;hrt wird.
 Diese binden an den Poly(A)-Schwanz, eine Kette von Adeninnukleotiden, die spezifisch an den 3 prim&#xE4;ren Enden der eukaryotischen mRNA vorhanden sind.
 Die anderen RNA-Typen binden nicht und werden weggesp&#xFC;lt.
 Nachdem die mRNA isoliert wurde, wird ein Poly(T)-Primer an den Poly(A)-Schwanz gebunden, was einen Ausgangspunkt f&#xFC;r reverse Transkriptaseenzyme darstellt, um einen Einzelstrang in cDNA aus der mRNA zu transkribieren.
 Chemikalien wie RNase-Enzyme werden dann zugesetzt, um die RNA abzubauen.
 DNA-Polymeraseenzyme werden dann verwendet, um einen zur cDNA komplement&#xE4;ren Strang zu synthetisieren, was zu doppelstr&#xE4;ngiger cDNA f&#xFC;hrt, die in einen bakteriellen oder viralen Vektor inseriert und in der molekularbiologischen Forschung verwendet werden kann.
 cDNA - bakterieller Vektor

Pray, Leslie A. "Die biotechnologische Revolution: PCR und die Verwendung von reverser Transkriptase zum Klonen exprimierter Gene." Nature Education 1, Nr. 1 (2008): 94. [<a href="https://www.nature.com/scitable/topicpage/the-biotechnology-revolution-pcr-and-the-use-553" target="__blank">Quelle</a>]

komplementäre dna

Complementary DNA

<h4>&#xDC;berblick</h4>
 
 Nur Gene, die in Boten-RNAs (mRNA) umgesetzt werden, sind aktiv oder werden exprimiert. Wissenschaftler k&#xF6;nnen daher die mRNA aus Zellen extrahieren, um die Genexpression in verschiedenen Zellen und Geweben zu untersuchen. Dabei wird transkribiert der Wissenschaftler die mRNA in komplement&#xE4;re DNA (cDNA) durch die reverse Transkription. Da die mRNA keine Introns (nicht-kodierende Regionen) und andere regulatorische Sequenzen enth&#xE4;lt, erlaubt die cDNAauch die direkte Bestimmung der Aminos&#xE4;uresequenz des vom Gen kodierten Peptids. Dies ist bei der &#x2014;im genomischen DANN nicht m&#xF6;glich.
 
 <h4>Die cDNA-Synthese</h4>
 
 cDNA kann durch verschiedene Methoden erzeugt werden, aber der meist verwendete Weg ist es die gesamte RNA aus den Zellen zu isolieren und dann die mRNA von den am h&#xE4;ufigsten vorkommenden Typen, der Transfer-RNA (tRNA) oder der ribosomalen RNA (rRNA) zu trennen. Reife eukaryotische mRNA hat einen poly(A)-Schwanz. Das ist eine Reihe von Adenin-Nukleotiden, die sich an dem 3&#x2019; Ende befinden, w&#xE4;hrend andere RNA-Typen diese nicht besitzen. Daher kann eine Reihe von Thymin-Nukeotiden (Oligo-dTs) an ein Substrat wie eine S&#xE4;ule oder magnetische Beads angeh&#xE4;ngt werden, um spezifisch mit den poly(A)-Schw&#xE4;nzen der mRNA ein Basenpaar zu bilden. W&#xE4;hrend die mRNA mit dem poly(A)-Schwanz gebunden wird, k&#xF6;nnen die anderen RNA-Typen weggewaschen werden.
 
 Als n&#xE4;chstes wird die Reverse Transkriptase, eine DNA-Polymerase-Enzym aus Retroviren, verwendet um die mRNA in cDNA umzuschreiben. Da die Reverse Transkriptase, wie die meisten DNA-Polymerasen, nur Nukleotide an das 3&#x2019; Ende hinzuf&#xFC;gen kann, wird ein Poly(T)-Primer verwendet, der den Poly(A)-Schwanz bindet und somit als Startpunkt f&#xFC;r die cDNA-Synthese fungiert. Der cDNA-Strang endet in einer Haarnadelschleife. Die RNA wird dann &#xFC;blicherweise mit Hilfe einer Alkalibehandlung oder RNase-Enzymen abgebaut, wobei die einzelstr&#xE4;ngige cDNA intakt bleibt.
 
 Ein zweiter, zur cDNA komplement&#xE4;rer DNA-Strang wird dann durch eine DNA-Polymerase synthetisiert. Dies findet oft unter Verwendung der Haarnadelschleife des ersten cDNA-Stranges oder eines St&#xFC;ckes der mRNA als Primer statt.
 
 Die resultierende doppelstr&#xE4;ngige cDNA kann in bakterielle oder virale Vektoren eingef&#xFC;gt und mit den &#xFC;blichen molekularbiologischen Techniken geklont werden. Eine cDNA-Bibliothek, welche alle mRNAs von bestimmten Zellen oder Geweben darstellt, kann auch f&#xFC;r zus&#xE4;tzliche Forschung hergestellt werden.

Komplementäre DNA

Overview

Only genes that are transcribed into messenger RNA (mRNA) are active, or expressed. Scientists can, therefore, extract the mRNA from cells to ...

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from mrna to cdna for gene studies

Complementary DNA, or cDNA, is made from messenger RNA (mRNA) so scientists can study which genes are active in cells and tissues. Only genes that are transcribed into mRNA are expressed. By extracting mRNA from cells, researchers can compare gene expression across samples.

cDNA is produced by reverse transcription. This process copies mRNA into DNA, which is useful because mRNA does not contain introns, or non-coding regions, and other regulatory sequences. As a result, cDNA can help researchers determine the amino acid sequence of the peptide encoded by a gene, unlike genomic DNA.

To make cDNA, scientists often start by extracting total RNA from cells. They then isolate mRNA from the more abundant transfer RNA (tRNA) and ribosomal RNA (rRNA). Mature eukaryotic mRNA has a poly(A) tail, which is a chain of adenine nucleotides added to the 3' end. Other RNA types do not have this feature.

Because of the poly(A) tail, oligo-dT molecules can be attached to a column or magnetic beads to capture mRNA by base pairing. The other RNA molecules are washed away. Next, reverse transcriptase, a DNA polymerase enzyme from retroviruses, builds cDNA from the mRNA template. A poly(T) primer binds to the poly(A) tail and gives the enzyme a starting point, since it adds nucleotides only to the 3' end of a chain.

The first cDNA strand ends in a hairpin loop. The RNA is then degraded, often with alkali treatment or RNase enzymes, and the single-stranded cDNA remains. A second DNA strand is then synthesized by DNA polymerase. It may use the hairpin loop of the first strand or a nicked piece of mRNA as a primer. The final double-stranded cDNA can be inserted into bacterial or viral vectors and cloned with standard molecular biology methods. Researchers can also build a cDNA library that represents the mRNAs found in a specific cell or tissue sample.

From mRNA to cDNA for Gene Studies

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Genetic engineering changes an organism’s DNA to add useful traits. Scientists use it in bacteria, plants, animals, and even viruses for research, medicine, agriculture, and industry. It can bring major benefits, but it also raises ethical concerns about humans and the food supply. This work is possible because DNA has the same basic structure and genetic code across life. That shared code lets scientists modify DNA in several ways. One method is CRISPR/Cas9, often called molecular scissors,...

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complementary dna

Complementary DNA, or cDNA, is made from messenger RNA (mRNA) so scientists can study which genes are active in cells and tissues. Only genes that are transcribed into mRNA are expressed. By extracting mRNA from cells, researchers can compare gene expression across samples. cDNA is produced by reverse transcription. This process copies mRNA into DNA, which is useful because mRNA does not contain introns, or non-coding regions, and other regulatory sequences. As a result, cDNA can help...

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genomics

Genomics and DNA Sequencing

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key terms and concepts

Learn how scientists isolate mRNA, use reverse transcription, and make cDNA for gene expression studies, cloning, and cDNA libraries in cells and tissues.

From mRNA to cDNA for Gene Studies | JoVE K12

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From mRNA to cDNA for Gene Studies

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