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

Rekombinant DNA farklı kaynaklardan, genelde farklı türlerden alınıp klinik ve bilimsel araştırmalarda yaygın kullanım için laboratuvarda birleştirilen DNA'dır.
 Rekombinant DNA genellikle istenilen bir genin, burada bağışçı organizmadan gelen insülinin, başka bir organizmadan gelen vektöre kendi kendini yenileyen DNA'ya bağlanmasıdır.
 Örneğin bir virüse veya plazmide yani küçük, halkasal bakteriyel DNA molekülüne.
Her iki kaynaktan gelen DNA önce restriksiyon endonükleazlarla kesilir.
 Bunlar DNA'ya belli bir dizilimle yapışan enzimlerdir.
 Bağışçı DNA vektör DNA'nın uçlarına enzim DNA ligaz yardımıyla yapışır.
Rekombinant DNA konak organizmaya sokulur, bir bakteri veya mantara.
 Orada türeyerek istenilen genin birçok kopyası elde edilir.
 Böylece gen daha kolay çalışılır ya da insülinde olduğu gibi konağın hücresel mekanizmasını kullanarak proteine çevrilir.

Khan, Suliman, Muhammed Wajid Ullah, Rabeea Siddique, Ghulam Nabi, Sehrish Manan, Muhammed Yousaf ve Hongwei Hou. 2016. "Yaşamı İyileştirmede Rekombinant DNA Teknolojisinin Rolü." Uluslararası Genomik Dergisi 2016. [<a href="https://doi.org/10.1155/2016/2405954" target="__blank">Kaynak</a>]

Matsumura, Ichiro. 2015. "Johnny Neden Klonlayamıyor: Yaygın Tuzaklar ve Çok Yaygın Olmayan Çözümler." BioTechniques 59 (3): IV–XIII. [<a href="https://doi.org/10.2144/000114324" target="__blank">Kaynak</a>]

rekombinant dna

<h4>Genel Bakış</h4>

Bilim adamları, laboratuvarda farklı kaynaklardan gelen DNA'yı birleştirerek rekombinant DNA oluştururlar. DNA klonlama, araştırmacıların, bakteriler gibi kolayca manipüle edilen hücrelere ekleyerek belirli genleri incelemelerine olanak tanır. Rekombinant DNA içeren organizmalar, genetiği değiştirilmiş organizmalar (GDO'lar) olarak bilinir. Rekombinant DNA teknolojisi bilime, tıbba ve tarıma fayda sağlayabilecek yeni genlere sahip organizmalar üretir.

<h4>Bilim Adamları Rekombinant DNA'yı Nasıl Oluşturur?</h4>

Rekombinant DNA'nın yaratılması, ilgilenilen bir genin bir vektöre, DNA replikasyonu ve protein ekspresyonu için konakçı hücrelere yabancı DNA taşıyan bir araç eklenmesini içerir. En yaygın olarak kullanılan klonlama vektörleri, konağın kromozomal DNA'sından bağımsız olarak çoğalan plazmidler, küçük dairesel DNA parçalarıdır.

Rekombinant DNA oluşturmak için, hem ilgili gen de dahil olmak üzere verici DNA hem de vektör, kısıtlama enzimleri kullanılarak kısıtlama siteleri adı verilen spesifik nükleotid dizilerinde kesilir. DNA ligaz enzimi, ilgi konusu genin ve plazmitin bağlandığı şeker-fosfat omurgasını mühürler.

Sonuç, ek adı verilen entegre bir verici DNA parçasına sahip bir vektörden oluşan rekombinant bir DNA molekülüdür. Bir bilim adamı daha sonra bu hibrit DNA molekülünü, kolayca ve hızlı bir şekilde çoğaldığı bir konakçı organizmaya (tipik olarak bakteri veya maya) sokabilir. Bu, bilimsel araştırma ve diğer uygulamalar için gerekli olan ilgi konusu genin birçok kopyasını oluşturur. Gen aynı zamanda, konağın hücresel mekanizması kullanılarak insan insülini gibi istenen proteine ​​kopyalanabilir ve çevrilebilir.

Rekombinant DNA oluşturmak kusurlu bir süreçtir ve sıklıkla hatalar meydana gelir. Örneğin, vektör ekleme olmadan kapanabilir veya ekleme yanlış (örneğin geriye doğru) olabilir. Araştırmacılar, daha ileri çalışmalar için rekombinant DNA'yı kullanmadan önce hataları kontrol etmelidir. Nükleotid dizilimi, doğru eklenti ile plazmit taşıyan bakteri kolonilerinin belirlenmesine yardımcı olabilir.

 Bilim İnsanları Genleri ve Proteinleri İncelemek İçin Rekombinant DNA Kullanıyor 

Rekombinant DNA teknolojisi, bir bilim insanının ilgilenilen bir genin veya bir protein ürününün birçok kopyasına ihtiyacı olduğunda özellikle avantajlıdır. Bununla birlikte, bir bilim adamının araştırması, istenen proteinin saptanması veya saflaştırılması gibi ek bir karmaşıklık düzeyi gerektirebilir. Bu amaca ulaşmak için, bir araştırmacı, bir füzyon geni veya kimerik gen oluşturmak için istenen proteine ​​bir gen ürününü tanımlamak için kullanılan bir etiket veya raportör ve proteinler ekleyebilir.

<h4>Tıp ve Tarımdaki Uygulamalar</h4>

Bilim adamları ilk olarak bakterilerde insan insülini üretmek için rekombinant DNA teknolojisini kullandı ve sonuçta diyabet tedavisi sağladı. Bu ilk keşiften bu yana, araştırmacılar terapötik kullanım için başka rekombinant DNA'lar ürettiler. Rekombinant bakteriler, büyüme hormonu eksikliği olan hastaları tedavi etmek için normal büyüme ve gelişme için gerekli bir protein olan insan büyüme hormonunu yapar. İnsanlardan ve hamsterlerden türetilen rekombinant memeli hücreleri, hemofili hastalarını tedavi etmek için normal kan pıhtılaşması için gerekli bir protein olan Faktör VIII'i üretir. Açıkça görülüyor ki, rekombinant DNA teknolojisi, temel proteinlerin büyük ölçekli üretimi için güçlü bir araçtır.

Rekombinant DNA teknolojisindeki tarımsal gelişmeler, insan refahını da etkiliyor. Örneğin, mısır çiftçileri, zararlı Avrupa mısır kurdu nedeniyle önemli ölçüde mahsul hasarına uğradı. Buna yanıt olarak bilim adamları, genetik olarak modifiye edilmiş, haşere dirençli mısır oluşturmak için toprakta yaşayan bir bakteriden Bacillus thuringiensis (Bt) — genleri izole etti. Bacillus thuringiensis doğal olarak insanlar, bitkiler veya diğer hayvanlar için değil, bazı böcekler için toksik olan proteinler üretir. Zararlılara dirençli Bt mısırının piyasaya sürülmesi mahsul verimini artırdı ve kimyasal böcek ilacı kullanımını azalttı. Bu tür tarımsal uygulamalar, küresel gıda tedarikinin kalitesini ve miktarını artırıyor.

Rekombinant DNA

Genel bakış

Bilim adamları, laboratuvarda farklı kaynaklardan (genellikle diğer türlerden) gelen DNA'yı birleştirerek rekombinant DNA oluştururlar. DNA klonlama izin verir ...

Education

JoVE Core

Molecular Biology

Moleküler Biyoloji

Unit 2

Ünite 2

Studying DNA and RNA

DNA ve RNA Çalışmaları

KEY TERMS AND CONCEPTS

TEMEL TERİMLER VE KAVRAMLAR

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 Moleküler Biyoloji, hücrenin moleküler süreçlerini açık ve özlü animasyonlu video dersleriyle sergiler. Buna ek olarak, bilim insanı-eylem videolarında, bu kavramların uygulaması modern laboratuvarlarda gerçek deneylerle gösterilmektedir.

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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Investigate means by which karyotypes are utilized in diagnostic medicine.
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BIO1.ETS2.3

Recombinant DNA and Gene Cloning

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