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

重组DNA是来自不同来源 的DNA，它们在 实验室中结合在一起，可广泛用于 临床和科学研究。 重组DNA通常由目的基因组成， 此处是来自供体生物体的胰岛素插入载体中， 来自另一生物体的自我复制DNA， 例如病毒或质粒，是一小段环状 细菌DNA。 首先通过限制性核酸内切酶切割 每种来源的DNA，这些核酸是一种以特定 序列切割DNA的酶。
在酶DNA连接酶的帮助下， 供体DNA与载体DNA的末端连接。 然后将重组DNA插入宿主生物， 细菌或酵母中，在其中进行复制， 从而产生许多目的基因的拷贝。 然后可以更容易地研究该基因， 或者像使用胰岛素一样，使用宿主的细胞机制 将其转化成蛋白质。

汗、苏利曼、穆罕默德·瓦吉德·乌拉、拉比亚·西迪克、古拉姆·纳比、塞里什·马南、穆罕默德·优素福和侯宏伟。2016. "重组 DNA 技术在改善生活方面的作用"。国际基因组学杂志 2016。[<a href="https://doi.org/10.1155/2016/2405954" target="__blank">来源</a>]

松村一郎。2015. "为什么约翰尼不能克隆:常见陷阱和不那么常见的解决方案。"生物技术 59 （3）:IV-XIII。[<a href="https://doi.org/10.2144/000114324" target="__blank">来源</a>]

重组dna

“科学家通过将来自不同来源的DNA（通常是其它物种的DNA）在实验室中合成重组DNA。DNA克隆使研究人员能够通过将特定基因插入易于操作的细胞（如细菌）中来进行研究。含有重组DNA的生物体被称为转基因生物体（gmos）。重组DNA技术生产出具有新基因的有机体，这些基因可以造福于科学、医学和农业。”

<h4>科学家如何制造重组DNA？</h4>
 
构建重组DNA需要将兴趣基因插入载体中，载体是将外源DNA携带到宿主细胞中进行DNA复制和蛋白质表达的载体。最常用的克隆载体是质粒，即独立于宿主染色体DNA进行复制的小环状DNA片段。
 
为了产生重组DNA，供体DNA（包括兴趣基因）和载体都被用限制性内切酶切割成特定的核苷酸序列，称为限制性位点。DNA连接酶在兴趣基因和质粒连接处封闭糖磷酸骨架。
 
结果是一个重组DNA分子，由一个载体和一个完整的供体DNA片段组成，称为插入物。科学家可能会将这种杂交DNA分子引入宿主生物体，通常是细菌或酵母，在那里它很容易快速复制。这就产生了许多兴趣基因的拷贝，这是科学研究和其他应用所必需的。该基因也可以通过宿主的细胞机制转录并翻译成所需的蛋白质，如人类胰岛素。
 
创建重组DNA是一个不完善的过程，而且经常会出现错误。例如，向量可以在没有插入的情况下闭合，或者插入可能不正确（例如，向后）。在使用重组DNA进行进一步研究之前，研究人员必须检查错误。核苷酸测序有助于识别携带正确插入质粒的细菌菌落。
 
<h4>科学家利用重组DNA研究基因和蛋白质</h4>
 
当科学家需要兴趣的基因或蛋白质产品的多个拷贝时，重组DNA技术尤其有利。然而，科学家的研究可能需要额外的复杂程度，例如检测或纯化所需的蛋白质。为了实现这一目标，研究人员可以将用于识别基因产品的标签或报告蛋白附加到他们所需的蛋白质上，以创建融合基因或嵌合基因。
 
<h4>在医药和农业中的应用</h4>
 
科学家首先利用重组DNA技术在细菌中产生人胰岛素，从而治疗糖尿病。自从最初的发现，研究人员已经产生了其它用于治疗的重组DNA。重组细菌使人生长激素（一种正常生长发育所需的蛋白质）用于治疗生长激素缺乏症患者。重组哺乳动物细胞，来源于人类和仓鼠，产生治疗血友病患者所需的凝血因子viii。显然，重组DNA技术是大规模生产必需蛋白质的有力工具。
 
农业上重组DNA技术的进步也影响到人类的福祉。例如，由于欧洲玉米螟的危害，玉米农民遭受了巨大的农作物损失。作为回应，科学家们从一种土壤细菌苏云金芽孢杆菌（Bt）中分离出基因，创造出转基因的、抗虫的玉米。苏云金芽孢杆菌自然产生对某些昆虫有毒的蛋白质，而不是人类、植物或其他动物。抗虫Bt玉米的引入提高了作物产量，减少了化学农药的使用。这种农业应用提高了全球粮食供应的质量和数量。

重组DNA

概述

科学家通过在实验室中将来自不同来源（通常是不同物种）的DNA进行组合，从而构建重组DNA。DNA克隆技术使得……

Education

教育

JoVE Core

Molecular Biology

分子生物学

Unit 2

第二单元

Studying DNA and RNA

研究 DNA 和 RNA

KEY TERMS AND CONCEPTS

关键术语与概念

重组 DNA

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 分子生物学通过清晰简洁的动画视频课程展示了细胞的分子过程。此外，在科学家在行动的视频中，这些概念的应用是通过现代实验室中的真实实验演示的。

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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Complementary DNA

FISH - Fluorescent In-situ Hybridization

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Sanger Sequencing

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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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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

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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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Euchromatin

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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

Recombinant DNA and Gene Cloning

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