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

組み替えDNAとは 通常異なる種からの、異なる発生源を持つDNAが 研究所で結合したDNAで 臨床研究や科学的研究など様々な目的で行われます 組み替えDNAは通常関心の対象となる遺伝子で構成され ここではドナー生物からインシュリンが 媒介生物に挿入され 他の生物からの自己増殖するDNAとして ウィルスやバクテリアDNAの小さな円状の一部である プラスミドなどが使用されます 各発生源からのDNAを最初に 切り取るのは制限酵素 DNAを特定の間隔で切り取る酵素です ドナーDNAは媒介DNAの末端と結合し DNA連結酵素がそれを助けます 組み替えDNAはその後宿主生物に挿入されます 細菌や酵母などが宿主にあたり、増殖すると 関心の対象である遺伝子のコピーを大量に作成します こうしてより容易な遺伝子研究が可能となり インシュリンの場合は 宿主の細胞内機関を利用してタンパク質に翻訳されます

カーン、スリマン、ムハンマド・ワジド・ウラー、ラビーア・シディク、グラム・ナビ、セリシュ・マナン、ムハンマド・ユサフ、ホンウェイ・ホウ2016.「生活を改善するための組換えDNA技術の役割」 International Journal of Genomics 2016年。[<a href="https://doi.org/10.1155/2016/2405954" target="__blank">ソース</a>]

松村 一郎.2015.「ジョニーがクローンを作成できない理由:一般的な落とし穴とそれほど一般的ではない解決策」 BioTechniques 59(3):IV–XIII。[<a href="https://doi.org/10.2144/000114324" target="__blank">ソース</a>]

組換えdna

<h4>概要</h4>
 
 科学者は、実験室で異なるソース（多くの場合、他の種）からのDNAを組み合わせて、組み換えDNAを作成します。DNAクローニングにより、研究者は特定の遺伝子をバクテリアのような操作しやすい細胞に挿入して研究できます。組換えDNAを含む生物は、遺伝子組換え生物（GMO）として知られています。組換えDNA技術は、新しい遺伝子を持つ生物を作り出し、科学、医学、農業に貢献しています。
 
 <h4>組換えDNAはどのようにして作られるのでしょうか</h4>？
 
 組換えDNAを作るには、目的の遺伝子をベクターに挿入します。ベクターとは、外来のDNAを宿主の細胞に運び、DNAの複製やタンパク質の発現を行うための道具です。最も一般的に使用されているクローニングベクターはプラスミドで、宿主の染色体DNAとは独立して複製を行う小さな円形のDNAです。
 
 組換えDNAを作るには、目的の遺伝子を含むドナーDNAとベクターの両方を、制限酵素を使って特定のヌクレオチド配列（制限部位と呼ばれる）で切断します。そして、DNAリガーゼという酵素が、目的の遺伝子とプラスミドをつなぐ糖-リン酸の骨格を結合させます。
 
 その結果、インサートと呼ばれるドナーDNAの一部が組み込まれたベクターからなる組換えDNA分子ができあがります。科学者はこのハイブリッドDNA分子を宿主生物（主にバクテリアや酵母）に導入し、容易かつ迅速に複製させます。このようにして、科学的研究やその他の用途に必要な、目的の遺伝子の多数のコピーが作られます。また、この遺伝子は、宿主の細胞機構を利用して、ヒトインスリンなどの目的のタンパク質に転写・翻訳されます。
 
 組換えDNAの作成は不完全なプロセスであり、エラーが発生することがよくあります。例えば、ベクターがインサートなしで閉じてしまったり、インサートが間違っていたり（例えば、逆向き）することがあります。組換えDNAをさらに研究に使用する前に、研究者はエラーをチェックしなければなりません。ヌクレオチド配列を調べることで、正しいインサートを持つプラスミドを持つバクテリアのコロニーを特定できます。
 
 遺伝子やタンパク質の研究に組換えDNAを使用する科学者たち組換えDNAを使用する科学者たち
 
 組換えDNA技術は、科学者が目的の遺伝子やタンパク質生成物の多数のコピーを必要とする場合に、特に有利な技術です。しかし、研究者の研究では、目的のタンパク質の検出や精製など、さらに複雑なレベルが必要になる場合があります。この目的を達成するために、研究者は目的のタンパク質にタグやレポーター（遺伝子産物を特定するためのタンパク質）を結合させ、融合遺伝子やキメラ遺伝子を作成できます。
 
 <h4>医学・農学への応用</h4>
 
 科学者たちは、組換えDNA技術を使ってバクテリアでヒトのインスリンを生産し、糖尿病の治療薬を初めて開発しました。この最初の発見以来、研究者たちは、治療用に他の組換えDNAを生成してきました。組換えバクテリアは、正常な成長と発達に必要なタンパク質であるヒト成長ホルモンを作り、成長ホルモン欠乏症の患者を治療します。また、ヒトやハムスターから採取した組換え哺乳類細胞は、血液凝固に必要なタンパク質である第VIII因子を産生し、血友病の治療に用いられます。組換えDNA技術は、必須タンパク質を大量に生産するための強力なツールであることは間違いないです。
 
 組換えDNA技術による農業の進歩は、人間の福利厚生にも影響を与えます。農業分野では、組換えDNA技術は人間の生活にも影響を与えています。例えば、トウモロコシ農家はヨーロッパのコーンボーラーという害虫のために大きな被害を受けました。そこで科学者たちは、土壌中のバクテリアであるバチルス・チューリンゲンシス(Bt)の遺伝子を分離し、遺伝子組み換えによって害虫に強いトウモロコシを作りました。害虫に強いBtコーンの導入により、作物の収穫量が向上し、化学農薬の使用量が減少しました。このような農業利用は、世界の食糧供給の質と量を向上させます。

組換えDNA

概要

科学者は、実験室でさまざまなソース(多くの場合、他の種)からのDNAを組み合わせて、組換えDNAを作成します。DNAクローニングは...

Education

JoVE Core

Molecular Biology

分子生物学

Unit 2

ユニット2

Studying DNA and RNA

DNAとRNAの研究

KEY TERMS AND CONCEPTS

主要用語と概念

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 分子生物学は、明確で簡潔なアニメーション ビデオ レッスンを通じて細胞の分子プロセスを紹介します。さらに、科学者の実際のビデオでは、これらの概念の応用が現代の研究室での実際の実験を通じて実証されています。

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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bacterial-transformation-the-heat-shock-method

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Southern Blot

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Maxam-Gilbert Sequencing

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Next-generation Sequencing

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Chromatin Immunoprecipitation- ChIP

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RNA-seq

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Synthetic Biology

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Genome Annotation and Assembly

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Transgenic Organisms

Gene Therapy

Reproductive Cloning

CRISPR

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Reporter Genes

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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

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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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