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

Rekombinante DNA ist DNA aus verschiedenen Quellen, normalerweise verschiedene Spezies, die im Labor für weitverbreitete Anwendungen in der klinischen und wissenschaftlichen Forschung kombiniert werden.
 Rekombinante DNA besteht normalerweise aus einem Gen von Interesse, hier Insulin von einem Spenderorganismus, welches in einen Vektor inseriert ist, selbstreplizierende DNA von einem anderen Organismus, wie einem Virus oder einem Plasmid, einem kleinen kreisförmigen Stück bakterieller DNA.
 Die DNA von jeder Quelle wird zuerst durch Restriktionsendonukleasen geschnitten, Enzyme, die DNA an einer bestimmten Sequenz spalten.
 Die Spender-DNA wird mit Hilfe des Enzyms DNA-Ligase an die Enden der Vektor-DNA gebunden.
 Die rekombinante DNA wird dann in einen Wirtsorganismus, ein Bakterium oder eine Hefe, inseriert, wo sie repliziert wird, wodurch viele Kopien des interessierenden Gens erzeugt werden.
 Das Gen kann dann leichter untersucht oder, wie im Fall von Insulin, unter Verwendung der Zellmaschinerie des Wirts in Protein übersetzt werden.

Khan, Suliman, Muhammad Wajid Ullah, Rabeea Siddique, Ghulam Nabi, Sehrish Manan, Muhammad Yousaf und Hongwei Hou. 2016. "Die Rolle der rekombinanten DNA-Technologie zur Verbesserung des Lebens." Internationales Journal für Genomik 2016. [<a href="https://doi.org/10.1155/2016/2405954" target="__blank">Quelle</a>]

Matsumura, Ichiro. 2015. "Warum Johnny nicht klonen kann: Häufige Fallstricke und nicht so häufige Lösungen." BioTechniques 59 (3): IV–XIII. [<a href="https://doi.org/10.2144/000114324" target="__blank">Quelle</a>]

rekombinante dna

<h4>Überblick</h4>
 
 Wissenschaftler stellen rekombinante DNA durch das Verbinden von DNA-Stücken von verschiedenen Quellen, meist verschiedenen Arten, im Labor her. Die DNA Klonierung ermöglicht den Forschern bestimmte Gene zu untersuchen, durch die Insertion in leicht manipulierbare Zellen, wie Bakterien. Organismen, die rekombinante DNA enthalten, sind als genetische veränderte Organismen (GVO) bekannt. Die rekombinante DNA-Technologie führt zu Organismen mit neuen Genen, die in der Wissenschaft, der Medizin, und der Landwirtschaft nützlich sein können.
 
 <h4>Wie stellen Wissenschaftler rekombinante DNA her?</h4>
 
 Bei der Herstellung von rekombinanter DNA wird das gewünschte Gen in einen Vektor eingefügt. Der Vektor ist ein Vehikel, der den Transport von fremder DNA in die Wirtszellen zur DNA-Replikation und Proteinexpression ermöglicht. Am meisten werden Plasmide als Klonierungsvektoren verwendet. Das sind kleine zirkulare DNA-Stücke, die sich unabhängig von der chromosomalen Wirts-DNA replizieren.
 
 Um rekombinante DNA herzustellen, müssen die DNA, die das gewünschte Gen enthält und der Vektor mit Restriktionsenzyme an einer bestimmten Nukleotid-Sequenz, der Restriktionsschnittstelle, geschnitten werden. Ein Enzyme, die DNA-Ligase, ligiert dann das Zucker-Phosphate-Grundgerüst, wo sich des gewünschte Gen und das Plasmid verbinden.
 
 Das Ergebnis ist dann ein rekombinantes DNA-Molekül, das aus einem Vektor mit einem integrierten neuen DNA-Stück, einem Insert, besteht. Ein Wissenschaftler kann dann diesen DNA-Hybrid in einen neuen Wirtsorganismus einführen, normalerweise Bakterien, wo es einfach und schnell vervielfältigt wird. Dadurch entstehen viele Kopien des gewünschten Gens, was notwendig für die wissenschaftliche Forschung und andere Anwendungen ist. Das Gen kann dann auch in der Wirtszelle transkribiert und übersetzt werden wobei das gewünschte Protein produzieret wird, wie zum Beispiel Insulin.
 
 Die Herstellung von rekombinanter DNA ist kein perfekter Prozess, da viele Fehler passieren. Zum Beispiel, der Vektor kann sich auch ohne das Insert schließen oder das Insert wurde verkehrt verbunden (umgekehrt). Daher müssen die Wissenschaftler bevor sie die DNA nutzen, sie auf Richtigkeit überprüfen. 
 Die Sequenzierung der Nukleotide kann dabei helfen die Bakterienkolonien zu identifizieren, die das Plasmid mit dem richtigen Insert haben.
 
 Wissenschaftler nutzen rekombinante DNA um Gene und Proteine zu untersuchen
 
 Die rekombinante DNA-Technologe ist besonders nützlich wenn ein Forscher viele Kopien eines gewünschten Gens oder Proteins benötigt. Es ist jedoch möglich, dass die Forschung des Wissenschaftlers komplexer ist, und die Reinigung oder die Detektion des Proteins benötigt. Um dies zu ermöglichen kann der Forscher einen Protein-tag oder Reporter an das gewünschte Protein fügen. Das sind Proteine die genutzt werden, um das Genprodukt zu lokalisieren. Dabei entsteht ein Fusionsgen oder ein chimärisches Gen.
 
 <h4>Die Anwendung in der Medizin und Landwirtschaft</h4>
 
 Wissenschaftler haben die rekombinante DNA-Technologie als erstes für die Produktion von menschlichem Insulin aus Bakterien genutzt, wodurch eine Therapie für Diabetes entstand. Seit dieser ersten Entdeckung haben Wissenschaftler verschiedene andere rekombinante DNAs für therapeutische Zwecke entwickelt. Rekombinante Bakterien produzieren das menschliche Wachstumshormon, Somatropin, welches ein Protein ist, dass für das normale Wachstum und Entwicklung benötigt wird. Es wird genutzt, um Patienten mit einem Mangel des Wachstumshormons zu behandeln. Rekombinante Säugetierzellen, die von Menschen und Hamstern stammen, produzieren den Faktor VIII. Dies ist ein Protein, das für die normale Blutgerinnung zur Behandlung von Patienten mit Hämophilie benötigt wird. Offensichtlich ist die rekombinante DNA-Technologie ein leistungsstarkes Werkzeug für die Produktion essenzieller Proteine in großem Maßstab.
 
 Landwirtschaftliche Fortschritte in der rekombinanten DNA-Technologie wirken sich auch auf das menschliche Wohlergehen aus. So erlitten Maisbauern früher beispielsweise erhebliche Ernteschäden durch den Maiszünsler, einen Schädling. Als Reaktion darauf isolierten Wissenschaftler Gene aus einem bodenbewohnenden Bakterium, demBacillus thuringiensis (Bt), um genetisch veränderten, schädlingsresistenten Mais zu erzeugen. Bacillus thuringiensis produziert auf natürliche Weise Proteine, die für bestimmte Insekten, aber nicht für Menschen, Pflanzen oder andere Tiere giftig sind. Die Einführung von schädlingsresistentem Bt-Mais verbesserte die Ernteerträge und verringerte den Einsatz von chemischen Pestiziden. Solche landwirtschaftlichen Anwendungen der DNA-Technologie können die Qualität und Quantität der globalen Nahrungsmittelversorgung verbessern.

Rekombinante DNA

Überblick

Wissenschaftler stellen rekombinante DNA her, indem sie DNA aus verschiedenen Quellen – oft von anderen Spezies – im Labor kombinieren. Die DNA-Klonierung ermöglicht ...

Education

Lehre

JoVE Core

Molecular Biology

Molekularbiologie

Unit 2

Einheit 2

Studying DNA and RNA

Untersuchung von DNA und RNA

KEY TERMS AND CONCEPTS

KERNBEGRIFFE UND KONZEPTE

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 Molecular Biology veranschaulicht die molekularen Prozesse der Zelle in prägnanten animierten Video-Lektionen. In Videos von Wissenschaftlern in Aktion wird die Anwendung dieser Konzepte zusätzlich durch reale Experimente in modernen Laboren demonstriert.

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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Karyotyping

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Investigate means by which karyotypes are utilized in diagnostic medicine.
EXPLANATION: Engineers incorporate growing scientific knowledge in order to increase benefits to humans and decrease cost and risks. Karyotyping technology and its use in medical diagnosis can complement the standards from LS3: Hereditary: Inheritance, and Variation of Traits, as the students are learning about chromosomal organization of genomic information that determines one's phenotype. Analyze a large set of karyotypes to identify common patterns. Identify variations from the most commonly observed patterns and correlate them with patient phenotypes to propose a cause and effect relationship, discussing limitations of correlative data interpretation.

BIO1.ETS2.2

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Analyze scientific and ethical arguments to support the pros and cons of applications of a specific biotechnology technique such as stem cell usage, in vitro fertilization, or genetically modified organisms.
EXPLANATION: The utilization of new technologies in any field of science is dependent on both economic and social factors. In addition to evaluating these factors, scientists must also consider long-term consequences that may not be initially apparent. The emphasis should be on the construction of a rationale argument that supports a position on the use of an application with ethical and social impact. Students should begin to appreciate the differences in ethical values that exist, and recognize that discussion of these values is imperative as knowledge and technology continue to advance, even when resolutions of differences can be rare. After investigation of a specific biotechnology application, students can write a position paper and/or participate in a classroom debate.

BIO1.ETS2.3

Recombinant DNA and Gene Cloning

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