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

- [Instructeur] Recombinant DNA is DNA van verschillende bronnen, meestal verschillende soorten die worden gecombineerd in het laboratorium voor wĳdverspreid gebruik in klinisch en wetenschappelijk onderzoek. Recombinant DNA bestaat gewoonlijk uit een gen van belang, hier insuline, uit een donor-organisme dat in een vector wordt ingebracht, waardoor DNA van een ander organisme zichzelf repliceert, zoals een virus of een plasmide, een kleine cirkelvormig stukje bacterieel DNA. Het DNA van elke bron wordt eerst door restrictie-endonuclease gesneden, enzymen die het DNA op een bepaalde sequentie splitsen.

Het donor-dna wordt aan de uiteinden van het vector-DNA verbonden met de hulp van het enzym DNA-ligase. Het recombinant DNA wordt dan ingebracht in een gastorganisme, een bacterie of gist, waar het gerepliceerd wordt, en zo vele kopieën genereert van het betreffende gen. Het gen kan dan gemakkelijker bestudeerd worden, of, zoals in het geval van insuline, vertaald worden in een proteïne door de cellulaire machine van de gastheer.

recombinant dna

<h4>Overzicht</h4>



Wetenschappers creëren recombinant DNA door DNA uit verschillende bronnen – vaak andere soorten – in het laboratorium te combineren. Met DNA-klonering kunnen onderzoekers specifieke genen bestuderen door ze in gemakkelijk te manipuleren cellen, zoals bacteriën, te plaatsen. Organismen die recombinant DNA bevatten, staan bekend als genetisch gemodificeerde organismen (GGO's). Recombinant-DNA-technologie produceert organismen met nieuwe genen die de wetenschap, de geneeskunde en de landbouw ten goede kunnen komen.



<h4>Hoe creëren wetenschappers recombinant DNA?</h4>



Het creëren van recombinant DNA omvat het inbrengen van een interessant gen in een vector – een voertuig dat vreemd DNA naar gastheercellen transporteert voor DNA-replicatie en eiwitexpressie. De meest gebruikte kloonvectoren zijn plasmiden, kleine cirkelvormige stukjes DNA die zich onafhankelijk van het chromosomale DNA van de gastheer repliceren.

Om recombinant DNA te creëren, worden zowel het donor-DNA, inclusief het betreffende gen, als de vector geknipt op specifieke nucleotidesequenties – restrictieplaatsen genoemd – met behulp van restrictie-enzymen. Het enzym DNA-ligase sluit de suiker-fosfaat-ruggengraat af waar het gen van belang en het plasmide met elkaar verbinden.

Het resultaat is een recombinant DNA-molecuul dat bestaat uit een vector met een geïntegreerd stukje donor-DNA, een zogenaamde insert. Een wetenschapper kan dit hybride DNA-molecuul vervolgens in een gastheerorganisme introduceren – meestal bacteriën of gist – waar het zich gemakkelijk en snel vermenigvuldigt. Hierdoor ontstaan er veel kopieën van het betreffende gen, wat nodig is voor wetenschappelijk onderzoek en andere toepassingen. Het gen kan ook worden getranscribeerd en vertaald in het gewenste eiwit – zoals humane insuline – door gebruik te maken van de cellulaire machinerie van de gastheer.

Het creëren van recombinant DNA is een onvolmaakt proces en er komen vaak fouten voor. De vector kan bijvoorbeeld sluiten zonder de insertie of de insertie kan onjuist zijn (bijvoorbeeld achteruit). Voordat onderzoekers het recombinant DNA voor verder onderzoek kunnen gebruiken, moeten ze controleren op fouten. Nucleotidensequencing kan helpen bij het identificeren van bacteriekolonies die plasmiden dragen met de juiste insert.



<h4>Wetenschappers gebruiken recombinant DNA om genen en eiwitten te bestuderen</h4>



Recombinant-DNA-technologie is vooral voordelig wanneer een wetenschapper veel kopieën nodig heeft van een gen van interesse of een eiwitproduct. Het onderzoek van een wetenschapper kan echter een extra niveau van complexiteit vereisen, zoals de detectie of zuivering van het gewenste eiwit. Om dit doel te bereiken kan een onderzoeker een tag of reporter –eiwitten die worden gebruikt om een genproduct te identificeren– aan het gewenste eiwit hechten om een fusiegen of chimeer gen te creëren.



<h4>Toepassingen in de geneeskunde en de landbouw</h4>



Wetenschappers gebruikten voor het eerst recombinant-DNA-technologie om humane insuline in bacteriën te produceren, wat resulteerde in een behandeling voor diabetes. Sinds die eerste ontdekking hebben onderzoekers andere recombinante DNA's gegenereerd voor therapeutisch gebruik. Recombinante bacteriën maken menselijk groeihormoon – een eiwit dat nodig is voor normale groei en ontwikkeling – om patiënten met groeihormoondeficiëntie te behandelen. Recombinante zoogdiercellen, afkomstig van mensen en hamsters, produceren factor VIII – een eiwit dat nodig is voor een normale bloedstolling – om patiënten met hemofilie te behandelen. Het is duidelijk dat recombinant-DNA-technologie een krachtig hulpmiddel is voor de grootschalige productie van essentiële eiwitten.

De vooruitgang in de landbouw op het gebied van recombinant-DNA-technologie heeft ook invloed op het menselijk welzijn. Zo leden maïsboeren bijvoorbeeld aanzienlijke gewasschade als gevolg van de plaag van de Europese maïsboorder. Als reactie hierop isoleerden wetenschappers genen uit een in de bodem levende bacterie – Bacillus thuringiensis (Bt) – om genetisch gemodificeerde, ongediertebestendige maïs te creëren. Bacillus thuringiensis produceert van nature eiwitten die giftig zijn voor bepaalde insecten, maar niet voor mensen, planten of andere dieren. De introductie van pestresistente Bt-maïs verbeterde de gewasopbrengsten en verminderde het gebruik van chemische pesticiden. Dergelijke landbouwtoepassingen verbeteren de kwaliteit en kwantiteit van de mondiale voedselvoorziening.

Overview

Scientists create recombinant DNA by combining DNA from different sources&mdash;often, other species&mdash;in the laboratory. DNA cloning allows ...

Education

JoVE Core

Molecular Biology

Moleculaire biologie

Unit 2

Onderdeel 2

Studying DNA and RNA

Het bestuderen van DNA en RNA

KEY TERMS AND CONCEPTS

BELANGRIJKE TERMEN EN CONCEPTEN

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 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 showcases the molecular processes of the cell through clear and concise animated video lessons. Additionally, in scientist-in-action videos, these concepts' application is demonstrated through real experiments in modern laboratories.

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.

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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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