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

Almost every cell in the body has the same DNA, but different cell types, such as neurons and muscle cells, express different genes because only certain genes are transcribed into messenger RNA, or mRNA, in each cell. In the laboratory, mRNAs can be used as a template to synthesize complementary DNA, cDNA, to study gene expression. A common method is to extract RNA from cells, then isolate the mRNA from other types of RNA, like ribosomal RNA or transfer RNA, by running the sample over a column of beads with stretches of thymine nucleotides attached.

These bind to the poly-A tail, a chain of adenine nucleotides specifically present on the 3-prime ends of eukaryotic mRNA. The other types of RNA do not bind and are washed away.

After the mRNA is isolated, a poly-T primer is bound to the poly-A tail, providing a starting point for reverse transcriptase enzymes to transcribe a single-stranded cDNA from the mRNA. Chemicals, such as RNase enzymes, are then added to degrade the RNA.

DNA polymerase enzymes are then used to synthesize a strand complementary to the cDNA, resulting in double-stranded cDNA, which can be inserted into a bacterial or viral vector and used in molecular biology research.

Módl się, Leslie A. "Rewolucja biotechnologiczna: PCR i zastosowanie odwrotnej transkryptazy do klonowania genów wyrażonych przez geny." Edukacja przyrodnicza 1, nr 1 (2008): 94. [<a href="https://www.nature.com/scitable/topicpage/the-biotechnology-revolution-pcr-and-the-use-553" target="__blank">Źródło</a>]

uzupełniające dna

Complementary DNA

<h4>Przegl&#x105;d</h4>

Tylko geny, kt&#xF3;re ulegaj&#x105; transkrypcji do informacyjnego RNA (mRNA), s&#x105; aktywne lub ulegaj&#x105; ekspresji. Naukowcy mog&#x105; zatem wyodr&#x119;bni&#x107; mRNA z kom&#xF3;rek, aby zbada&#x107; ekspresj&#x119; gen&#xF3;w w r&#xF3;&#x17C;nych kom&#xF3;rkach i tkankach. Naukowiec przekszta&#x142;ca mRNA w komplementarny DNA (cDNA) poprzez odwrotn&#x105; transkrypcj&#x119;. Poniewa&#x17C; mRNA nie zawiera intron&#xF3;w (region&#xF3;w niekoduj&#x105;cych) i innych sekwencji regulacyjnych, cDNA &#x2014; w przeciwie&#x144;stwie do genomowego DNA &#x2014; umo&#x17C;liwia tak&#x17C;e badaczom bezpo&#x15B;rednie okre&#x15B;lenie sekwencji aminokwas&#xF3;w peptydu kodowanego przez gen.

<h4>Synteza cDNA</h4>

cDNA mo&#x17C;na wytworzy&#x107; kilkoma metodami, ale powszechnym sposobem jest najpierw ekstrakcja ca&#x142;kowitego RNA z kom&#xF3;rek, a nast&#x119;pnie izolacja mRNA z bardziej dominuj&#x105;cych typ&#xF3;w &#x2014; RNA transferowego (tRNA) i rybosomalnego (rRNA). Dojrza&#x142;y eukariotyczny mRNA ma ogon poli(A) &#x2014; ci&#x105;g nukleotyd&#xF3;w adeninowych &#x2014; dodany do jego ko&#x144;ca 3&#x2019; koniec, podczas gdy inne typy RNA nie. Dlatego ci&#x105;g nukleotyd&#xF3;w tyminowych (oligo-dT) mo&#x17C;na przy&#x142;&#x105;czy&#x107; do pod&#x142;o&#x17C;a, takiego jak kolumna lub kulki magnetyczne, w celu specyficznego sparowania zasad z ogonami poli(A) mRNA. Podczas wychwytywania mRNA z ogonem poli(A) inne typy RNA s&#x105; wyp&#x142;ukane.

Nast&#x119;pnie do wytworzenia cDNA z mRNA wykorzystuje si&#x119; odwrotn&#x105; transkryptaz&#x119; &#x2014; enzym polimeraz&#x119; DNA z retrowirus&#xF3;w. Poniewa&#x17C;, podobnie jak wi&#x119;kszo&#x15B;&#x107; polimeraz DNA, odwrotna transkryptaza mo&#x17C;e dodawa&#x107; nukleotydy tylko do 3&#x2019; ko&#x144;cu &#x142;a&#x144;cucha, dodaje si&#x119; starter poli(T), aby zwi&#x105;za&#x107; si&#x119; z ogonem poli(A) i zapewni&#x107; punkt wyj&#x15B;cia do syntezy cDNA. Ni&#x107; cDNA ko&#x144;czy si&#x119; p&#x119;tl&#x105; typu spinka do w&#x142;os&#xF3;w. RNA jest nast&#x119;pnie degradowany &#x2014; zwykle poprzez dzia&#x142;anie alkaliami lub enzymami RNazy &#x2014; pozostawiaj&#x105;c jednoniciowy cDNA nienaruszony.

Druga ni&#x107; DNA komplementarna do cDNA jest nast&#x119;pnie syntetyzowana przez polimeraz&#x119; DNA &#x2014; cz&#x119;sto wykorzystuj&#x105;c jako starter p&#x119;tl&#x119; w kszta&#x142;cie spinki do w&#x142;os&#xF3;w pierwszej nici cDNA lub naci&#x119;ty fragment mRNA.

Powsta&#x142;y dwuniciowy cDNA mo&#x17C;na wstawi&#x107; do wektor&#xF3;w bakteryjnych lub wirusowych i sklonowa&#x107; przy u&#x17C;yciu standardowych technik biologii molekularnej. Bibliotek&#x119; cDNA&#x2014;reprezentuj&#x105;c&#x105; wszystkie mRNA w kom&#xF3;rkach lub tkankach b&#x119;d&#x105;cych przedmiotem zainteresowania&#x2014;mo&#x17C;na r&#xF3;wnie&#x17C; skonstruowa&#x107; na potrzeby dodatkowych bada&#x144;.

Uzupełniające 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

what is genetic engineering?

How Scientists Edit DNA for New Traits

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,...

dna isolation

Steps in DNA Extraction

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recombinant dna technology

Recombinant DNA in Cloning and GMOs

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PCR Cycle for DNA Amplification

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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...

antibiotic selection

Antibiotic Resistance in Cloning

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Creating Transgenic Plants and Animals

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in-vitro mutagenesis (gene knockouts)

Knockout Mice and Gene Replacement

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crispr

CRISPR-Cas9 Gene Editing in Action

CRISPR-Cas9 is a genome editing tool that lets scientists change DNA at a specific location. It can add, remove, or rearrange genetic material. Researchers study it because it may help treat genetic disorders such as hemophilia and sickle cell anemia. CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats, and Cas9 is a CRISPR-associated protein 9. The basic system includes a Cas9 endonuclease, which is an enzyme that cuts DNA, and a small RNA that guides Cas9 to the...

gene therapy

Gene Delivery for Serious Diseases

Gene therapy is a technique used to treat serious disease by adding a gene to a person’s cells. The added gene may be a healthy copy of a mutated gene. It may also be a different gene that inactivates or compensates for the disease-causing gene. One example is severe combined immunodeficiency, or SCID, caused by a mutation in the gene for adenosine deaminase. In that case, a working copy of the gene can be inserted so the patient’s cells make the enzyme again. This can cure the disease in some...

embryonic stem cells

How Embryonic Stem Cells Become Specialized

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adult stem cells

Adult Stem Cells in the Small Intestine

Adult stem cells help maintain tissues by replacing old or damaged cells. These undifferentiated cells divide to make more stem cells or progenitor cells, which then become mature, specialized cells. Small groups of adult stem cells are found in many tissues, including the bone marrow, brain, skin, and gut. The small intestine shows this process clearly. Its lining, called the epithelium, is renewed continuously by adult stem cells. It is also the fastest replaced tissue in the human body,...

induced pluripotent stem cells

Reprogramming Mature Cells into Stem Cells

Reprogramming mature cells into stem cells lets scientists turn specialized cells back into an unspecialized state. These cells are called induced pluripotent stem cells, or iPSCs. Like embryonic stem cells, iPSCs are pluripotent, which means they can form all cell types. To make iPSCs, researchers start with mature cells such as skin fibroblasts or blood cells. The cells are grown in culture, and genes for several transcription factors are delivered with a viral vector. The cells then make...

reproductive cloning

Cloning Animals with SCNT

Reproductive cloning can produce a genetically identical copy of an entire organism. In adult animals, this is usually done with somatic cell nuclear transfer, or SCNT. Another way clones can form is by splitting an early embryo, which is similar to how identical twins arise naturally. In SCNT, an egg cell is taken from an animal and its nucleus is removed. This creates an enucleated egg. A somatic cell, which is any body cell that is not a sex cell, is then taken from the animal to be cloned.

genomics

Genomics and DNA Sequencing

Genomics studies the full set of genetic material in an organism. In humans, that genome includes the DNA carried in 23 pairs of chromosomes in the nucleus, along with mitochondrial DNA. Genomics looks at both coding DNA and non-coding DNA, which helps scientists study how living things are built and how they differ. This field is useful in many areas of biology and medicine. Scientists use genomics to build phylogenetic trees, improve crop productivity and sustainability, help with criminal...

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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