Name: recombinant dna and gene cloning
Uploaded: 2021-04-07T12:11:59.000Z
Duration: 1 min 9 s
Channel: JoVE

K12 Standards

Tennessee Academic Standards

High School

Biology

links-among-engineering-technology-science-and-society

Links Among Engineering, Technology, Science, and Society

BIO1.ETS2.1

Recombinant DNA

- L'ADN recombinantest de l'ADN provenant de différentes sources,généralement différentes espècescombinées dans un laboratoirepour un usage généralisé dans la recherche cliniqueet scientifique. 
L'ADN recombinant consiste généralementen un gène intéressant, ici l'insuline,d'un organisme donneurinséré dans un vecteur,ADN autoreproducteur d'un autre organisme,comme un virus ou un plasmide,petit morceau circulaire d'ADN bactérien. 
L'ADN de chaque source est d'abord coupépar les endonucléases de restriction,des enzymes qui détachent l'ADN à une séquence précise. 
L'ADN donneur est joint aux extrémités de l'ADN vecteur,avec l'aide de l'enzyme ADN ligase. 
L'ADN recombinant est ensuite inséré dans un organisme hôte,une bactérie ou une levure, où il est reproduit,créant de nombreuses copies du gène intéressant. 
Le gène peut ensuite être plus facilement étudié ou,dans le cas de l'insuline, traduit en protéine,en utilisant la machinerie cellulaire de l'hôte.

Khan, Suliman, Muhammad Wajid Ullah, Rabeea Siddique, Ghulam Nabi, Sehrish Manan, Muhammad Yousaf et Hongwei Hou. 2016. « Rôle de la technologie de l’ADN recombinant pour améliorer la vie. » Revue internationale de génomique 2016. [<a href="https://doi.org/10.1155/2016/2405954 » target="__blank">Source</a>]

Matsumura, Ichiro. 2015. « Pourquoi Johnny ne peut pas cloner : pièges courants et solutions moins courantes. » BioTechniques 59 (3) : IV-XIII. [<a href="https://doi.org/10.2144/000114324 » target="__blank">Source</a>]

adn recombinant

<h4>Aperçu</h4>
 
 Les scientifiques créent de l’ADN recombinant en combinant en laboratoire l’ADN de sources différentes, souvent d’autres espèces. Le clonage d’ADN permet aux chercheurs d’étudier des gènes précis en les insérant dans des cellules qui se manipulent facilement, comme les bactéries. Les organismes qui contiennent de l’ADN recombinant sont connus sous le nom d’organismes génétiquement modifiés (OGM). La technologie de l’ADN recombinant produit des organismes avec de nouveaux gènes qui peuvent être bénéfiques à la science, à la médecine et à l’agriculture.
 
 <h4>Comment les scientifiques créent-ils de l’ADN recombinant ?</h4>
 
 La création d’ADN recombinant implique l’insertion d’un gène d’intérêt dans un vecteur — un véhicule qui transporte de l’ADN étranger dans les cellules hôtes pour la réplication de l’ADN et l’expression des protéines. Les vecteurs de clonage les plus couramment utilisés sont les plasmides, de petits morceaux circulaires d’ADN qui se répliquent indépendamment de l’ADN chromosomique de l’hôte.
 
 Pour créer de l’ADN recombinant, l’ADN du donneur, y compris le gène d’intérêt, et le vecteur sont coupés à des séquences nucléotidiques spécifiques, appelées sites de restriction, à l’aide d’enzymes de restriction. L’enzyme ADN ligase scelle le squelette sucre-phosphate à l’endroit où le gène d’intérêt et le plasmide se connectent. 
 
 Le résultat est une molécule d’ADN recombinant composée d’un vecteur avec un morceau intégré de l’ADN du donneur, appelé un insert. Un scientifique peut alors introduire cette molécule d’ADN hybride dans un organisme hôte, généralement des bactéries ou des levures, dans lesquelles elle se réplique facilement et rapidement. Cela crée de nombreuses copies du gène d’intérêt, ce qui est nécessaire pour la recherche scientifique et d’autres applications. Le gène peut également être transcrit et traduit dans la protéine désirée, comme l’insuline humaine, en utilisant la machinerie cellulaire de l’hôte. 
 
 La création d’ADN recombinant est un processus imparfait, et des erreurs se produisent souvent. Par exemple, le vecteur peut se fermer sans l’insert ou l’insert peut être incorrect (par exemple, inversé). Avant d’utiliser l’ADN recombinant pour d’autres études, les chercheurs doivent vérifier les erreurs. Le séquençage des nucléotides peut aider à identifier les colonies de bactéries qui portent des plasmides avec l’insert correct.
 
 Les scientifiques utilisent l’ADN recombinant pour étudier les gènes et les protéines
 
 La technologie de l’ADN recombinant est particulièrement avantageuse lorsqu’un scientifique a besoin de nombreuses copies d’un gène d’intérêt ou d’un produit protéique. Cependant, la recherche d’un scientifique peut exiger un niveau supplémentaire de complexité, comme la détection ou la purification de la protéine désirée. Pour atteindre cet objectif, un chercheur peut attacher un marqueur ou un rapporteur — des protéines utilisées pour identifier un produit génétique — à la protéine désirée afin de créer un gène de fusion ou un gène chimérique.
 
 <h4>Applications en médecine et en agriculture</h4>
 
 Les scientifiques ont d’abord utilisé la technologie de l’ADN recombinant pour produire de l’insuline humaine dans les bactéries, ce qui a conduit à un traitement pour le diabète. Depuis cette découverte initiale, les chercheurs ont généré d’autres ADN recombinants à des fins thérapeutiques. Les bactéries recombinantes fabriquent l’hormone de croissance humaine — une protéine nécessaire à la croissance et au développement normaux — pour traiter les patients souffrant d’une carence en hormone de croissance. Les cellules de mammifères recombinantes, dérivées de l’homme et des hamsters, produisent le facteur VIII — une protéine nécessaire à la coagulation normale du sang — pour traiter les patients atteints d’hémophilie. De toute évidence, la technologie de l’ADN recombinant est un outil puissant pour la production à grande échelle de protéines essentielles.
 
 Les progrès agricoles de la technologie de l’ADN recombinant ont également un impact sur le bien-être humain. Par exemple, les cultures de producteurs de maïs ont subi d’importants dégâts en raison de la pyrale du maïs, un insecte ravageur. En réponse, les scientifiques ont isolé des gènes d’une bactérie vivant dans le sol, Bacillus thuringiensis (Bt), pour créer du maïs génétiquement modifié et résistant aux insectes ravageurs. Bacillus thuringiensis produit naturellement des protéines qui sont toxiques pour certains insectes, mais pas pour les humains, les plantes ou d’autres animaux. L’introduction de maïs Bt résistant aux insectes ravageurs a amélioré les rendements des cultures et réduit l’utilisation de pesticides chimiques. Ces applications agricoles améliorent la qualité et la quantité de l’approvisionnement alimentaire mondial.

ADN recombinant

Aperçu

Les scientifiques créent de l’ADN recombinant en combinant de l’ADN provenant de différentes sources, souvent d’autres espèces, en laboratoire. Le clonage de l’ADN permet ...

Education

Enseignement

JoVE Core

Molecular Biology

Biologie moléculaire

Unit 2

Unité 2

Studying DNA and RNA

L'étude de l'ADN et de l'ARN

KEY TERMS AND CONCEPTS

TERMES ET CONCEPTS CLÉS

recombinant dna and gene cloning

Recombinant DNA and gene cloning are tools used to combine DNA from different sources. Scientists use them to isolate a gene, join it to a carrier DNA molecule, and make copies of that DNA.

A plasmid is one common carrier used in this process. A plasmid is a small ring of DNA found in bacteria, and it can carry the inserted gene into a host cell. Once inside the cell, the recombinant DNA can be copied as the cell grows.

This approach is important in biotechnology and genetic research. It helps scientists study genes, produce useful DNA copies, and work with DNA in a controlled way.

Recombinant DNA and Gene Cloning

recombinant dna

Recombinant DNA and gene cloning are tools used to combine DNA from different sources. Scientists use them to isolate a gene, join it to a carrier DNA molecule, and make copies of that DNA. A plasmid is one common carrier used in this process. A plasmid is a small ring of DNA found in bacteria, and it can carry the inserted gene into a host cell. Once inside the cell, the recombinant DNA can be copied as the cell grows. This approach is important in biotechnology and genetic research. It...

dna isolation

Genomic DNA Extraction Methods

Genomic DNA extraction methods separate DNA from proteins, RNA, and other cell material. The process usually follows four main steps. First, the cell structure is disrupted with mechanical or chemical methods to make a cell lysate. Then the DNA is protected from damage during processing. After that, the soluble DNA is separated from cell debris. Finally, the purified DNA is eluted, or washed out, for later use. DNA isolation can follow two main approaches: solution-based methods or solid-phase...

dna agarose gel electrophoresis

Purifying DNA Bands from Agarose Gels

Agarose gel electrophoresis can be used to purify DNA bands after separation by size. In gel extraction, the DNA fragment of interest is cut from the gel and cleaned before later use. This is especially useful in cloning, where pure DNA is needed before ligation, the joining step that links DNA pieces together. In cloning experiments, both the insert and vector DNA fragments are first cut with restriction endonucleases. These fragments often contain enzymes, salts, and other contaminants. Such...

labeling dna probes

DNA Probe Labels for Detecting Targets

DNA probes are short DNA fragments with a reporter tag attached so they can be detected or purified. They bind to matching nucleic acid sequences by complementary base-pairing. That binding helps researchers recover or identify specific regions of DNA. Several reporter tags are commonly used to label DNA probes. These include radioisotopes, fluorophores, and small molecule binding partners such as biotin or digoxigenin. The label choice depends on the probe’s intended use and on the source of...

southern blot

Southern Blot for DNA Detection

Southern blotting is a DNA detection method used after agarose gel electrophoresis separates DNA fragments by size. A DNA ladder with fragments of known length is run beside the sample so the fragment size can be estimated. The blot adds another step when scientists need to confirm the sequence identity of the DNA. First, the target DNA is cut into smaller pieces with a restriction endonuclease and run on an agarose gel. The DNA is then denatured, which means the double strands are separated...

dna microarrays

Gene Chips for Comparing Cell Activity

DNA microarrays are gene chips used to compare gene activity in different conditions. They are high-throughput assays, which means they can analyze large amounts of data at once. They are also relatively inexpensive and can be automated, making them useful for genome-wide studies. These chips are built on glass or silica slides. Probe molecules are covalently attached to the surface through surface functionalization. In many cases, the slides are prepared by chemisorption of silanes to silica...

complementary dna

Making cDNA from RNA Templates

Complementary DNA, or cDNA, is made from an RNA template. It is a DNA copy of RNA, so it can help students study gene expression in a lab setting. cDNA is useful when scientists want to work with genes after they have been turned on in a cell. Because it is copied from RNA, it reflects which genes were active at the time the RNA was collected. This process links RNA and DNA in a simple way. It also gives students a clear view of how genetic information can be copied for analysis.

fish - fluorescent in-situ hybridization

FISH for Chromosome and Pathogen Detection

FISH is a DNA and RNA detection method used in cytogenetics and medical testing. FISH stands for fluorescence in situ hybridization. It uses labeled probes that bind to complementary DNA or RNA sequences on a chromosome or inside a cell. The method was developed in the early 1980s and became widely used because it can show specific genetic targets with high detail. In the past, probes were made by cloning or by reverse transcription of a DNA template. Today, probe oligonucleotides can also be...

pcr - polymerase chain reaction

PCR: DNA Amplification for Genetics Labs

PCR is a DNA amplification method used in genetics labs. PCR stands for polymerase chain reaction. It copies a specific DNA segment so scientists can study small amounts of genetic material. The process uses repeated heating and cooling steps. These steps let the DNA strands separate, primers bind to the target region, and a DNA polymerase build new strands. With each cycle, the target DNA can increase in amount. PCR is a core technique in molecular biology and many school-level biology...

real time rt-pcr

Real-Time RT-PCR for Gene Expression Tracking

Real-time RT-PCR tracks gene expression by measuring how much target DNA is made during PCR. It starts with mRNA, which is converted into complementary DNA, or cDNA, by an enzyme called reverse transcriptase. The cDNA is then amplified by PCR so the target sequence can be measured. This method can be done in one tube as a single-step reaction or in two separate steps. During amplification, the amount of product is followed in real time with fluorescent dyes or sequence-specific probes linked...

race - rapid amplification of cdna ends

Finding cDNA Ends with RACE PCR

Rapid Amplification of cDNA Ends, or RACE, helps scientists capture the full-length cDNA from an mRNA sequence. It works by linking a known internal region to the unknown sequence at either the 5' end or the 3' end. The method is widely used when the missing end of a gene needs to be identified. RACE uses a gene-specific primer that binds to the known cDNA region. A hybrid primer then adds a set anchor sequence to the unknown end of the cDNA. After that, PCR amplifies the DNA segment between...

sanger sequencing

How Sanger Sequencing Reads DNA Fragments

Sanger sequencing reads DNA fragments by separating pieces of different lengths and decoding the order of bases. It is a key method in biology and is often used to study a cloned DNA fragment, a gene mutation, or even much larger genomes. Fredrick Sanger and his collaborators developed the chain-termination method in 1977. The method works by producing a mixture of DNA fragments with variable sizes. These fragments are then separated by capillary gel electrophoresis, which sorts them by length.

next-generation sequencing

DNA Sequencing Methods and Uses

DNA sequencing methods can read millions of DNA fragments at the same time. Next-generation sequencing made this process much faster and much less expensive than older methods. The first human genome project cost $2.7 billion and took 15 years. It was completed in 2003 after major international cooperation among research teams and funding agencies. To prepare DNA for next-generation sequencing, pure genomic DNA is first broken into smaller pieces. These pieces are used to build a sequencing...

rna-seq

RNA-Seq Workflow and Data Analysis

RNA sequencing, or RNA-Seq, is a high-throughput method for studying the transcriptome of a cell. The transcriptome is the full set of RNA molecules in a sample. Transcriptomics helps scientists understand how genomes work, how organisms develop, and how some diseases occur. Before RNA-Seq, researchers often used microarray methods and Sanger sequencing to study RNA. Microarrays had limited coverage and depended on already knowing the genome sequence. Sanger sequencing was low-throughput,...

genome annotation and assembly

How Genome Assemblies Are Updated

Genome assembly and genome annotation are key steps in genome analysis. Genome assembly puts DNA sequencing data back in the correct order to make a close copy of the original genome. Genome annotation then identifies functional parts on that assembled genome. Assembling a genome is not simple. In human populations, gene copy number can vary, and repeated DNA sequences can make the process harder. Even so, the physical location of human genes stays constant. Bacterial genomes can be more...

restriction enzymes

DNA Cutting Sites and Bacterial Defense

Restriction enzymes are bacterial proteins that cut DNA at specific sequence sites. They recognize palindromic DNA sequences, also called inverted repeats, and cut the DNA in a sequence-specific way. These sites are often found in regions with important functions, such as the origin of replication, gene operator sites, and transcription termination signals. Bacteria protect their own DNA by methylating these same sites. Methylation means adding chemical marks that keep the restriction enzyme...

maxam-gilbert sequencing

Chemical Cleavage DNA Sequencing

Chemical cleavage DNA sequencing uses specific chemicals to cut DNA at known sites. Allan Maxam and Walter Gilbert described this method in the same year that the Sanger sequencing method was discovered. It reads the DNA sequence by breaking purified DNA into fragments and then studying the fragment pattern. The method depends on chemicals that cleave DNA at specific bases. The resulting fragments are separated by electrophoresis, which is a way to sort DNA pieces by size using an electric...

key terms and concepts

Learn how recombinant DNA and gene cloning work by joining a gene to a plasmid and copying it in a host cell for biotechnology and research.

Recombinant DNA and Gene Cloning | JoVE K12

molecular biology

JoVE Core Biologie Moléculaire illustre les processus moléculaires au sein de la cellule grâce à des leçons vidéos claires et concises. Les applications de ces concepts sont ensuite démontrées grâce à des expériences concrètes en laboratoire.

Virginia  Standards of Learning

HIgh School

BIO.5

BIO.5.e

Alabama Course of Study Science

9-12

from-molecules-to-organisms-structures-and-processes

From Molecules to Organisms: Structures and Processes

9-12.B.1.2.c

Florida Department of Education

Life Science

SC.912.L.16

SC.912.L.16.12

Texas Essential Knowledge and Skills

BIO.7

BIO.7.D

myJoVE Corporation

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BIO1.ETS2.3

Recombinant DNA and Gene Cloning

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