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

- [Instructor] ADN recombinante
es ADN de fuentes diferentes

normalmente de especies diferentes

combinadas en el laboratorio
para una amplia gama de usos

en investigación clínica y científica.

El ADN recombinante suele
componerse de un gen de interés,

en este caso la insulina
de un organismo donante

insertada en un vector,

el ADN autorreplicante de otro organismo,

como un virus o un plásmido,

un pequeño fragmento
circular de ADN bacteriano.

El ADN de cada fuente se corta primero

por endonucleasas de restricción,

enzimas que dividen el ADN
en una secuencia concreta.

El ADN donante se une a
los extremos del ADN vector

con la ayuda de la enzima ADN ligasa.

El ADN recombinante se inserta entonces

en un organismo anfitrión,
una bacteria o levadura,

donde se replica, creando muchas
copias del gen de interés.

Así el gen puede ser
estudiado con mayor facilidad

o, como en el caso de la insulina,

ser convertido en proteína

usando la maquinaria celular del huésped.

Khan, Suliman, Muhammad Wajid Ullah, Rabeea Siddique, Ghulam Nabi, Sehrish Manan, Muhammad Yousaf y Hongwei Hou. 2016. "El papel de la tecnología del ADN recombinante para mejorar la vida". Revista Internacional de Genómica 2016. [<a href="https://doi.org/10.1155/2016/2405954" target="__blank">Fuente</a>]

Matsumura, Ichiro. 2015. "Por qué Johnny no puede clonar: trampas comunes y soluciones no tan comunes". BioTechniques 59 (3): IV-XIII. [<a href="https://doi.org/10.2144/000114324" target="__blank">Fuente</a>]

adn recombinante

<h4>Descripción general</h4> Los científicos crean ADN recombinante combinando ADN de diferentes fuentes,a menudo, otras especies, en el laboratorio. La clonación de ADN permite a los investigadores estudiar genes específicos insertándolos en células fácilmente manipulables, como las bacterias. Los organismos que contienen ADN recombinante se conocen como organismos genéticamente modificados (OGM). La tecnología del ADN recombinante produce organismos con nuevos genes que pueden beneficiar a la ciencia, la medicina y la agricultura. <h4>¿Cómo crean los científicos ADN recombinante?</h4> La creación de ADN recombinante implica insertar un gen de interés en un vector,un vehículo que transporta ADN extraño a las células huésped para la replicación del ADN y la expresión de proteínas. Los vectores de clonación más utilizados son los plásmidos, pequeños trozos circulares de ADN que se replican independientemente del ADN cromosómico del huésped. Para crear ADN recombinante, tanto el ADN del donante, incluido el gen de interés, como el vector se cortan en secuencias de nucleótidos específicas,llamadas sitios de restricción, utilizando enzimas de restricción. La enzima ADN ligasa sella la columna vertebral de azúcar-fosfato donde se conectan el gen de interés y el plásmido. El resultado es una molécula de ADN recombinante que está formada por un vector con una pieza integrada de ADN del donante, llamada inserto. Luego, un científico puede introducir esta molécula de ADN híbrida en un organismo huésped,normalmente bacterias o levaduras, donde se replica fácil y rápidamente. Esto crea muchas copias del gen de interés, que es necesario para la investigación científica y otras aplicaciones. El gen también puede transcribirse y traducirse a la proteína deseada,como la insulina humana, utilizando la maquinaria celular del huésped. La creación de ADN recombinante es un proceso imperfecto y con frecuencia se producen errores. Por ejemplo, el vector puede cerrarse sin la inserción o la inserción puede ser incorrecta (por ejemplo, al revés). Antes de utilizar el ADN recombinante para futuros estudios, los investigadores deben comprobar si hay errores. La secuenciación de nucleótidos puede ayudar a identificar colonias de bacterias que transportan plásmidos con el inserto correcto. Los científicos utilizan ADN recombinante para estudiar genes y proteínas La tecnología del ADN recombinante es particularmente ventajosa cuando un científico necesita muchas copias de un gen de interés o de un producto proteico. Sin embargo, la investigación de un científico puede requerir un nivel adicional de complejidad, como la detección o purificación de la proteína deseada. Para lograr este objetivo, un investigador puede adjuntar una etiqueta o indicador,proteínas utilizadas para identificar un producto genético, a la proteína deseada para crear un gen de fusión o gen quimérico. <h4>Aplicaciones en Medicina y Agricultura</h4> Los científicos utilizaron por primera vez la tecnología del ADN recombinante para producir insulina humana en bacterias, lo que dio como resultado un tratamiento para la diabetes. Desde ese descubrimiento inicial, los investigadores han generado otros ADN recombinantes para uso terapéutico. Las bacterias recombinantes producen la hormona del crecimiento humano, una proteína necesaria para el crecimiento y desarrollo normales, para tratar a pacientes con deficiencia de hormona del crecimiento. Las células recombinantes de mamíferos, derivadas de humanos y hámsteres, producen factor VIII, una proteína necesaria para la coagulación sanguínea normal, para tratar a pacientes con hemofilia. Evidentemente, la tecnología del ADN recombinante es una poderosa herramienta para la producción a gran escala de proteínas esenciales. Los avances agrícolas en la tecnología del ADN recombinante también impactan el bienestar humano. Por ejemplo, los agricultores de maíz sufrieron daños sustanciales a sus cosechas debido a la plaga del barrenador europeo del maíz. En respuesta, los científicos aislaron genes de una bacteria que habita en el suelo,Bacillus thuringiensis (Bt), para crear maíz genéticamente modificado y resistente a las plagas. Bacillus thuringiensis produce naturalmente proteínas que son tóxicas para ciertos insectos, pero no para los humanos, las plantas u otros animales. La introducción de maíz Bt resistente a plagas mejoró el rendimiento de los cultivos y disminuyó el uso de pesticidas químicos. Estas aplicaciones agrícolas mejoran la calidad y cantidad del suministro mundial de alimentos.

ADN recombinante

Visión general

Los científicos crean ADN recombinante combinando ADN de diferentes fuentes, a menudo, de otras especies, en el laboratorio. La clonación de ADN permite ...

Education

Educación

JoVE Core

Molecular Biology

Biología Molecular

Unit 2

Unidad 2

Studying DNA and RNA

Estudio del ADN y el ARN

KEY TERMS AND CONCEPTS

TÉRMINOS Y CONCEPTOS CLAVE

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 Biología Molecular presenta los procesos moleculares de la célula a través de lecciones claras y concisas de animaciones audiovisuales. Además, en los vídeos de científicos en acción, se muestran aplicaciones de estos conceptos mediante experimentos reales en laboratorios especializados.

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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Southern Blot

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Sanger Sequencing

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Maxam-Gilbert Sequencing

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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

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RNA-seq

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Synthetic Biology

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Genome Annotation and Assembly

The genome refers to all of the genetic material in an organism. It can range from a few million base pairs in microbial cells to several billion base ...

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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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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