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

- [Verteller] Bijna elke cel in het lichaam heeft hetzelfde DNA maar verschillende celtypes, zoals neuronen en spiercellen, uiten verschillende genen omdat alleen bepaalde genen overgebracht worden in messenger RNA of mRNA in elke cel.
 In het laboratorium kunnen mRNA&#x27;s gebruikt worden als template om complementair DNA, cDNA, te synthetiseren om genexpressie te bestuderen.
 Een gangbare methode is RNA extraheren van cellen, dan het mRNA isoleren van andere soorten RNA, zoals robisomaal RNA of transfer RNA, door het sample over een kralenkolom te laten lopen waaraan strengen thymine nucleotiden bevestigd zijn.
Deze binden met de poly(A)-staart, een keten van adenine nucleotiden specifiek aanwezig aan de drie uiteindes van eukaryotisch mRNA.
 De andere soorten RNA binden zich niet en worden weggespoeld.
 Nadat het mRNA ge&#xEF;soleerd is, wordt een poly(T)-primer gebonden aan de poly(A)-staart, en vormt een startpunt voor reverse-transcriptase enzymes om een enkelstrengs cDNA te translateren van het mRNA.
Stoffen zoals RNase enzymen worden dan toegevoegd om het RNA te degraderen.
 DNA polymerase enzymen worden dan gebruikt om een streng te synthetiseren, complementair met het cDNA wat resulteert in dubbelstrengs cDNA, dat ingevoegd kan worden in een bacteri&#xEB;le of virale vector en gebruikt wordt in moleculair biologie-onderzoek.

complementair dna

Complementary DNA

<h4>Overzicht</h4>

Alleen genen die worden getranscribeerd in messenger RNA (mRNA) zijn actief of komen tot expressie. Wetenschappers kunnen daarom het mRNA uit cellen halen om genexpressie in verschillende cellen en weefsels te bestuderen. De wetenschapper zet mRNA via omgekeerde transcriptie om in complementair DNA (cDNA). Omdat mRNA geen introns (niet-coderende regio&#x27;s) en andere regulerende sequenties bevat, stelt cDNA &#x2013; in tegenstelling tot genomisch DNA &#x2013; onderzoekers ook in staat om direct de aminozuursequentie te bepalen van het peptide dat door het gen wordt gecodeerd.

<h4>cDNA-synthese</h4>

cDNA kan op verschillende manieren worden gegenereerd, maar een gebruikelijke manier is om eerst totaal RNA uit cellen te extraheren en vervolgens het mRNA te isoleren van de meer overheersende typen&#x2013; transfer-RNA (tRNA) en ribosomaal (rRNA). Rijp eukaryoot mRNA heeft een poly(A)-staart &#x2013; een reeks adenine-nucleotiden &#x2013; toegevoegd aan de 3&#x2019;-kernen. eindigen, terwijl andere soorten RNA dat niet doen. Daarom kan een reeks thyminenucleotiden (oligo-dTs) worden vastgemaakt aan een substraat zoals een kolom of magnetische kralen, om specifiek basenparen te vormen met de poly(A)-staarten van mRNA. Terwijl mRNA met een poly(A)-staart wordt gevangen, worden de andere soorten RNA weggespoeld.

Vervolgens wordt reverse transcriptase &#x2013; een DNA-polymerase-enzym uit retrovirussen &#x2013; gebruikt om cDNA uit het mRNA te genereren. Omdat reverse transcriptase, zoals de meeste DNA-polymerasen, alleen nucleotiden kan toevoegen aan de 3&#x2019; aan het uiteinde van een keten wordt een poly(T)-primer toegevoegd om aan de poly(A)-staart te binden en zo een startpunt voor cDNA-synthese te verschaffen. De cDNA-streng eindigt in een haarspeldlus. Het RNA wordt vervolgens afgebroken &#x2013; gewoonlijk met alkalibehandeling of RNase-enzymen &#x2013; waardoor het enkelstrengige cDNA intact blijft.

Een tweede DNA-streng die complementair is aan het cDNA wordt vervolgens gesynthetiseerd door DNA-polymerase - vaak met behulp van de haarspeldlus van de eerste cDNA-streng of een ingekerfd stuk van het mRNA als primer.

Het resulterende dubbelstrengige cDNA kan in bacteri&#xEB;le of virale vectoren worden ingevoegd en worden gekloneerd met behulp van standaard moleculair biologische technieken. Er kan ook een cDNA-bibliotheek&#x2013;worden gebouwd die alle mRNA&#x27;s in de cellen of het weefsel van interesse&#x2013; vertegenwoordigt voor aanvullend onderzoek.

Complementair 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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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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