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

Rekombinowane DNA to DNA z różnych źródeł, zwykle różnych gatunków, połączone razem w laboratorium w celu szerokiego zastosowania w badaniach klinicznych i naukowych.

Rekombinowane DNA zwykle składa się z genu będącego przedmiotem zainteresowania, w tym przypadku insuliny z organizmu dawcy wprowadzonego do wektora, samoreplikującego się DNA z innego organizmu, takiego jak wirus lub plazmid, mały okrągły fragment bakteryjnego DNA. DNA z każdego źródła jest najpierw cięte przez endonukleazy restrykcyjne, enzymy, które rozszczepiają DNA w określonej sekwencji.

DNA dawcy jest łączone z końcami wektorowego DNA za pomocą enzymu ligazy DNA.

Rekombinowane DNA jest następnie wprowadzane do organizmu gospodarza, bakterii lub drożdży, gdzie jest replikowane, tworząc wiele kopii interesującego genu. Gen ten może być wtedy łatwiej badany lub, jak w przypadku insuliny, przekształcany w białko za pomocą maszynerii komórkowej gospodarza.

Khan, Suliman, Muhammad Wajid Ullah, Rabeea Siddique, Ghulam Nabi, Sehrish Manan, Muhammad Yousaf i Hongwei Hou. 2016. "Rola technologii rekombinacji DNA w poprawie życia." Międzynarodowy Dziennik Genomiki 2016. [<a href="https://doi.org/10.1155/2016/2405954" target="__blank">Źródło</a>]

Matsumura, Ichiro. 2015. "Dlaczego Johnny nie może klonować: powszechne pułapki i nie tak powszechne rozwiązania." BioTechniques 59 (3): IV–XIII. [<a href="https://doi.org/10.2144/000114324" target="__blank">Źródło</a>]

rekombinowane dna

<h4>Przegląd</h4>

Naukowcy tworzą rekombinowany DNA, łącząc w laboratorium DNA z różnych źródeł — często innych gatunków—Klonowanie DNA umożliwia badaczom badanie określonych genów poprzez wstawianie ich do komórek, którymi można łatwo manipulować, takich jak bakterie. Organizmy zawierające rekombinowany DNA nazywane są organizmami modyfikowanymi genetycznie (GMO). Technologia rekombinacji DNA pozwala uzyskać organizmy z nowymi genami, które mogą przynieść korzyści nauce, medycynie i rolnictwu.

<h4>Jak naukowcy tworzą rekombinowane DNA?</h4>

Tworzenie rekombinowanego DNA obejmuje wstawienie interesującego genu do wektora — nośnika przenoszącego obcy DNA do komórek gospodarza w celu replikacji DNA i ekspresji białka. Najczęściej stosowanymi wektorami do klonowania są plazmidy, małe okrągłe fragmenty DNA, które replikują się niezależnie od chromosomalnego DNA gospodarza.

Aby utworzyć rekombinowany DNA, zarówno DNA dawcy, w tym gen będący przedmiotem zainteresowania, jak i wektor wycina się w określonych sekwencjach nukleotydowych — zwanych miejscami restrykcyjnymi — przy użyciu enzymów restrykcyjnych. Enzym Ligaza DNA uszczelnia szkielet cukrowo-fosforanowy w miejscu połączenia genu będącego przedmiotem zainteresowania i plazmidu.

Rezultatem jest rekombinowana cząsteczka DNA składająca się z wektora ze zintegrowanym fragmentem DNA dawcy — zwana wstawką. Naukowiec może następnie wprowadzić tę hybrydową cząsteczkę DNA do organizmu gospodarza — zazwyczaj bakterii lub drożdży — gdzie łatwo i szybko się replikuje. Tworzy to wiele kopii interesującego genu, które są niezbędne do badań naukowych i innych zastosowań. Gen może również podlegać transkrypcji i translacji na pożądane białko — takie jak ludzka insulina — przy użyciu maszynerii komórkowej gospodarza.

Tworzenie rekombinowanego DNA jest procesem niedoskonałym i często zdarzają się błędy. Na przykład wektor może zamknąć się bez wkładki lub wkładka może być nieprawidłowa (np. do tyłu). Przed wykorzystaniem rekombinowanego DNA do dalszych badań badacze muszą sprawdzić, czy nie występują w nim błędy. Sekwencjonowanie nukleotydów może pomóc w identyfikacji kolonii bakterii zawierających plazmidy z odpowiednią wstawką.

Naukowcy wykorzystują rekombinowane DNA do badania genów i białek

Technologia rekombinacji DNA jest szczególnie korzystna, gdy naukowiec potrzebuje wielu kopii interesującego genu lub produktu białkowego. Jednak badania naukowca mogą wymagać dodatkowego poziomu złożoności, takiego jak wykrycie lub oczyszczenie pożądanego białka. Aby osiągnąć ten cel, badacz może dołączyć znacznik lub reporter — białka używane do identyfikacji produktu genu — do pożądanego białka w celu utworzenia genu fuzyjnego, czyli genu chimerycznego.

<h4>Zastosowania w medycynie i rolnictwie</h4>

Naukowcy po raz pierwszy zastosowali technologię rekombinacji DNA do produkcji ludzkiej insuliny w bakteriach, co umożliwiło leczenie cukrzycy. Od czasu tego wstępnego odkrycia badacze wygenerowali inne rekombinowane DNA do zastosowań terapeutycznych. Rekombinowane bakterie wytwarzają ludzki hormon wzrostu — białko niezbędne do prawidłowego wzrostu i rozwoju — w celu leczenia pacjentów z niedoborem hormonu wzrostu. Rekombinowane komórki ssaków pochodzące od ludzi i chomików wytwarzają czynnik VIII — białko wymagane do prawidłowego krzepnięcia krwi — w celu leczenia pacjentów chorych na hemofilię. Najwyraźniej technologia rekombinacji DNA jest potężnym narzędziem do produkcji niezbędnych białek na dużą skalę.

Postępy w rolnictwie w technologii rekombinacji DNA również wpływają na dobrostan człowieka. Na przykład rolnicy zajmujący się kukurydzą ponieśli znaczne szkody w uprawach z powodu szkodnika omacnicy prosowianki. W odpowiedzi naukowcy wyizolowali geny z bakterii żyjącej w glebie — Bacillus thuringiensis (Bt) — aby stworzyć genetycznie zmodyfikowaną, odporną na szkodniki kukurydzę. Bacillus thuringiensis w naturalny sposób wytwarza białka, które są toksyczne dla niektórych owadów, ale nie dla ludzi, roślin ani innych zwierząt. Wprowadzenie odpornej na szkodniki kukurydzy Bt poprawiło plony i zmniejszyło użycie chemicznych pestycydów. Takie zastosowania rolnicze poprawiają jakość i ilość globalnej podaży żywności.

Rekombinowane DNA

Przegląd

Naukowcy tworzą rekombinowane DNA, łącząc DNA z różnych źródeł – często innych gatunków – w laboratorium. Klonowanie DNA pozwala ...

Education

JoVE Core

Molecular Biology

Biologia molekularna

Unit 2

Jednostka 2

Studying DNA and RNA

Badanie DNA i RNA

KEY TERMS AND CONCEPTS

Kluczowe terminy i pojęcia

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 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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Genome Editing

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Restriction Enzyme Digests

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DNA Isolation

DNA isolation protocols can be fast and straightforward or complex and time-consuming depending on the type and quality of DNA required for further ...

Restriction Enzymes

Restriction enzymes are bacterial enzymes used to cut DNA in a sequence-specific manner. To cleave DNA, they bind to specific palindromic sequences called ...

DNA Agarose Gel Electrophoresis

Agarose gel electrophoresis is a laboratory technique commonly used to separate DNA fragments by size. However, it can also be used to isolate and purify ...

DNA Gel Electrophoresis

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Separating Protein with SDS-PAGE

Sodium Dodecyl Sulfate Poly-Acrylamide Gel Electrophoresis, or SDS-PAGE, is a widely-used technique for separating mixtures of proteins based on their ...

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Gel Purification

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PCR - Polymerase Chain Reaction

Real Time RT-PCR

Real-time reverse transcription-polymerase chain reaction, or Real-time RT-PCR, is an analytical tool used to determine the expression level of target ...

RACE - Rapid Amplification of cDNA Ends

Rapid Amplification of cDNA Ends, or RACE, is one of the most effective methods to obtain a full-length cDNA from an mRNA sequence between a known ...

PCR: The Polymerase Chain Reaction

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bacterial-transformation-the-heat-shock-method

Bacterial Transformation: The Heat Shock Method

Transformation is the process that occurs when a cell ingests foreign DNA from its surroundings. Transformation can occur in nature in certain types of ...

Bacterial Transformation: Electroporation

The term &#x201C;transformation&#x201D; refers cellular ingestion of foreign DNA.  In nature, transformation can occur in certain types of bacteria.  In ...

An Introduction to Transfection

Transfection is the process of inserting genetic material, such as DNA and double stranded RNA, into mammalian cells. The insertion of DNA into a cell ...

Labeling DNA Probes

DNA probes are fragments of DNA labeled with a reporter tag to enable their detection or purification. The resulting labeled DNA probes can then hybridize ...

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

Microarrays are high-throughput and relatively inexpensive assays that can be automated to analyze large quantities of data at a time. They are used in ...

An Overview of Gene Expression

Gene expression is the complex process where a cell uses its genetic information to make functional products. This process is regulated at multiple ...

Expression Profiling with Microarrays

Microarrays are important tools for profiling gene expression, and are based on complementary binding between probes that are attached to glass chips and ...

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

DNA sequencing is a fundamental technique that is routinely used in the biological sciences. This method can be applied to a range of questions at ...

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

RNA sequencing, or RNA-Seq, is a high-throughput sequencing technology used to study the transcriptome of a cell. Transcriptomics helps to interpret the ...

Synthetic Biology

Synthetic biology is an interdisciplinary science that involves using principles from disciplines such as engineering, molecular biology, cell biology, ...

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

Ribosome profiling or ribo-sequencing is a deep sequencing technique that produces a snapshot of active translation in a cell. It selectively sequences ...

The ELISA Method

An enzyme-linked immunosorbent assay (ELISA) is typically performed to detect the presence and/or amount of a target protein of interest within an ...

Transgenic Plants

Recombinant DNA technology called transgenesis is often used to add a foreign gene or remove a detrimental gene from an organism. Such genetically ...

Transgenic Organisms

Gene Therapy

Reproductive Cloning

CRISPR

Experimental RNAi

RNA interference (RNAi) is a cellular mechanism that inhibits gene expression by suppressing its transcription or activating the RNA degradation process. ...

Reporter Genes

Reporter genes are a type of&#160;protein-coding gene&#160;that are often tagged to a gene of interest. Once inside a target cell, reporter genes usually ...

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.
EXPLANATION: The goal is to help students appreciate that the basic science knowledge being studied can be manipulated and used to design useful tools for further scientific investigations, medical treatments, agricultural yields, etc. A detailed understanding of any technical procedures is not expected, but rather a "big picture" view of concepts the technology utilizes and the applications the technology is being used for. These technologies may be best investigated along with the standards from LS1 From Molecules to Organisms: Structure and Functions, as the students are learning about DNA and protein. Comparative DNA and protein sequence analysis is also a large part of current phylogenic research and could accompany LS4 Biological Change: Unity and Diversity. Students may also consider the pros and cons of biotechnical applications. Investigation of techniques utilized could come from field trips to witness the use of these techniques in practice, lab exercises, virtual labs, simulation activities, interviews with professionals, etc. Molecular techniques could include: PCR, electrophoresis, restriction enzyme digestion of DNA, DNA sequencing, plasmid-based transformation, transfection, etc. These techniques are used in fields of medicine, agriculture, biomedical engineering, forensic science, etc.

Karyotyping

Lampbrush Chromosomes

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Polytene Chromosomes

Polytene chromosomes are giant interphase chromosomes with several DNA strands placed side by side. They were discovered in the year 1881 by Balbiani in ...

Euchromatin

The extent of chromatin compaction can be studied by staining chromatin using specific DNA binding dyes. Under the microscope, the dense-compacted regions ...

Heterochromatin

X and Y Chromosomes

Among mammals, the gender of an organism is determined by the sex chromosomes. Humans have two sex chromosomes, X and Y. Every human diploid cell has 22 ...

The Y Chromosome Determines Maleness

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The Ratio of X Chromosome to Autosomes

In most organisms, sex is determined by the ratio of X and Y chromosomes. However, in some organisms, such as Drosophila and C.elegans, sex is determined ...

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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