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

- Il DNA ricombinante è DNA

da fonti differenti, di
solito da specie differenti,

che si combina insieme nel laboratorio

per ampi usi nella ricerca
clinica e scientifica.

Il DNA ricombinante consiste
di un gene di interesse,

qui l'insulina da un organismo donatore

inseriti in un vettore,

DNA autoreplicamente
da un altro organismo,

come un virus o un plasmide,
un piccolo pezzo circolare

di DNA batterico.

Il DNA da ciascuna risorsa
è per prima cosa tagliato

da endonucleasi di restrizione,

enzimi che tagliano il DNA
in una sequenza precisa.

Il DNA del donatore è collegato
a quello del DNA vettore,

con l'aiuto di enzimi DNA ligasi.

Il DNA ricombinante si inserisce
in un organismo ospite,

batteri o lievito, dove si replica,

creando molte copie del gene di interesse.

Questo gene può essere
studiato più facilmente

o, come con l'insulina,

trasformato in proteina usando
il meccanismo cellulare.

Khan, Suliman, Muhammad Wajid Ullah, Rabeea Siddique, Ghulam Nabi, Sehrish Manan, Muhammad Yousaf e Hongwei Hou. 2016. "Ruolo della tecnologia del DNA ricombinante per migliorare la vita". Giornale internazionale di genomica 2016. [<a href="https://doi.org/10.1155/2016/2405954" target="__blank">Sorgente</a>]

Matsumura, Ichiro. 2015. "Perché Johnny non può clonare: insidie comuni e soluzioni non così comuni". BioTechniques 59 (3): IV–XIII. [<a href="https://doi.org/10.2144/000114324" target="__blank">Sorgente</a>]

dna recombinante

<h4>Panoramica</h4>

Gli scienziati creano DNA ricombinante combinando il DNA di fonti diverse, spesso altre specie, in laboratorio. La clonazione del DNA consente ai ricercatori di studiare geni specifici inserendoli in cellule facilmente manipolate, come i batteri. Gli organismi che contengono DNA ricombinante sono noti come organismi geneticamente modificati (OGM). La tecnologia del DNA ricombinante produce organismi con nuovi geni che possono beneficiare la scienza, la medicina e l'agricoltura.

<h4>Come fanno gli scienziati a creare DNA ricombinante?</h4>

La creazione di DNA ricombinante comporta l'inserimento di un gene di interesse in un vettore, un veicolo che trasporta il DNA estraneo nelle cellule ospiti per la replicazione del DNA e l'espressione proteica. I vettori di clonazione più comunemente utilizzati sono plasmidi, piccoli pezzi circolari di DNA che si replicano indipendentemente dal DNA cromosomico dell'ospite.

Per creare DNA ricombinante, sia il DNA del donatore, compreso il gene di interesse, sia il vettore vengono tagliati in specifiche sequenze di nucleotidi, chiamate siti di restrizione, utilizzando enzimi di restrizione. L'enzima DNA ligase sigilla la spina dorsale zucchero-fosfato dove si connettono il gene di interesse e il plasmide.

Il risultato è una molecola di DNA ricombinante costituita da un vettore con un pezzo integrato di DNA del donatore, chiamato inserto. Uno scienziato può quindi introdurre questa molecola di DNA ibrido in un organismo ospite, tipicamente batteri o lieviti, dove si replica facilmente e rapidamente. Questo crea molte copie del gene di interesse, che è necessario per la ricerca scientifica e altre applicazioni. Il gene può anche essere trascritto e tradotto nella proteina desiderata, come l'insulina umana, utilizzando il meccanismo cellulare dell'ospite.

La creazione di DNA ricombinante è un processo imperfetto e spesso si verificano errori. Ad esempio, il vettore potrebbe chiudersi senza l'inserto o l'inserto potrebbe non essere corretto (ad esempio, all'indietro). Prima di utilizzare il DNA ricombinante per ulteriori studi, i ricercatori devono verificare la presenza di errori. Il sequenziamento dei nucleotidi può aiutare a identificare le colonie di batteri che trasportano plasmidi con l'inserto corretto.

Gli scienziati utilizzano il DNA ricombinante per studiare geni e proteine

La tecnologia del DNA ricombinante è particolarmente vantaggiosa quando uno scienziato ha bisogno di molte copie di un gene di interesse o di un prodotto proteico. Tuttavia, la ricerca di uno scienziato può richiedere un ulteriore livello di complessità, come il rilevamento o la purificazione della proteina desiderata. Per raggiungere questo obiettivo, un ricercatore può allegare un tag o un reporter, proteine utilizzate per identificare un prodotto genetico, alla proteina desiderata per creare un gene di fusione, o gene chimerico.

<h4>Applicazioni in Medicina e Agricoltura</h4>

Gli scienziati hanno usato per la prima volta la tecnologia del DNA ricombinante per produrre insulina umana nei batteri, con conseguente trattamento per il diabete. Da quella scoperta iniziale, i ricercatori hanno generato altre DLL ricombinanti per uso terapeutico. I batteri ricombinanti rendono l'ormone della crescita umano, una proteina necessaria per la normale crescita e sviluppo, per trattare i pazienti con carenza di ormone della crescita. Le cellule di mammiferi ricombinanti, derivate da esseri umani e criceti, producono il fattore VIII, una proteina necessaria per la normale coagulazione del sangue, per il trattamento di pazienti con emofilia. Evidentemente, la tecnologia del DNA ricombinante è un potente strumento per la produzione su larga scala di proteine essenziali.

I progressi agricoli nella tecnologia del DNA ricombinante influenzano anche il benessere umano. Ad esempio, gli allevatori di mais hanno subito notevoli danni alle colture a causa del borer di mais europeo. In risposta, gli scienziati hanno isolato i geni da un batterio che abita il suolo (Bacillus thuringiensis (Bt) - per creare mais geneticamente modificato e resistente ai parassiti. Bacillus thuringiensis produce naturalmente proteine tossiche per alcuni insetti, ma non per gli esseri umani, le piante o altri animali. L'introduzione del mais Bt resistente ai parassiti ha migliorato le rese delle colture e ridotto l'uso di pesticidi chimici. Tali applicazioni agricole migliorano la qualità e la quantità dell'approvvigionamento alimentare globale.

DNA recombinante

Overview

Scientists create recombinant DNA by combining DNA from different sources&#8212;often, other species&#8212;in the laboratory. DNA cloning allows ...

Education

Didattica

JoVE Core

Molecular Biology

Biologia Molecolare

Unit 2

Unità 2

Studying DNA and RNA

Studiare il DNA e l'RNA

KEY TERMS AND CONCEPTS

TERMINI E CONCETTI CHIAVE

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

Le video animazioni di JoVE Core Molecular Biology, organizzate in video lezioni, illustrano i processi molecolari cellulari in maniera chiara e concisa. Inoltre, nei video di "scienziati in azione", i concetti scientifici vengono applicati e dimostrati da ricercatori attraverso esperimenti in laboratorio.

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

Molecular Cloning

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Genome Editing

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DNA Isolation

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Restriction Enzymes

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DNA Agarose Gel Electrophoresis

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DNA Gel Electrophoresis

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Separating Protein with SDS-PAGE

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Plasmid Purification

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Gel Purification

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PCR - Polymerase Chain Reaction

Real Time RT-PCR

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RACE - Rapid Amplification of cDNA Ends

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PCR: The Polymerase Chain Reaction

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bacterial-transformation-the-heat-shock-method

Bacterial Transformation: The Heat Shock Method

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Bacterial Transformation: Electroporation

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An Introduction to Transfection

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Labeling DNA Probes

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Southern Blot

Agarose gel electrophoresis is very useful in separating DNA fragments by size. Running a DNA ladder containing fragments of the known length alongside ...

DNA Microarrays

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An Overview of Gene Expression

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Expression Profiling with Microarrays

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Complementary DNA

FISH - Fluorescent In-situ Hybridization

Fluorescence in situ hybridization, or FISH, was developed in the early 1980s and has quickly become one of the most widely used techniques in ...

Sanger Sequencing

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Maxam-Gilbert Sequencing

In the same year as the discovery of the Sanger sequencing method, another group of scientists, Allan Maxam and Walter Gilbert, demonstrated their ...

Next-generation Sequencing

The first human genome sequencing project cost $2.7 billion and was declared complete in 2003, after 15 years of international cooperation and ...

Chromatin Immunoprecipitation- ChIP

Chromatin immunoprecipitation, or ChIP, is an antibody-based technique used to identify sites on DNA that bind to transcription factors of interest or ...

RNA-seq

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Synthetic Biology

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Genome Annotation and Assembly

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Ribosome Profiling

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The ELISA Method

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Transgenic Plants

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Transgenic Organisms

Gene Therapy

Reproductive Cloning

CRISPR

Experimental RNAi

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Reporter Genes

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In-situ Hybridization

In situ hybridization (ISH) is a technique used to detect and localize specific DNA or RNA molecules in cells, tissue, or tissue sections using a labeled ...

Obtain, evaluate, and communicate information on how molecular biotechnology may be used in a variety of fields.
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Karyotyping

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Polytene Chromosomes

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Euchromatin

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Heterochromatin

X and Y Chromosomes

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The Ratio of X Chromosome to Autosomes

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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

Whole Body Regeneration

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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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