Name: making cdna from rna templates
Uploaded: 2021-04-07T12:12:00.000Z
Duration: 1 min 44 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

Complementary DNA

신체의 거의 모든 세포가 동일한 DNA를 갖지만신경 세포나 근육 세포 같은 여러 세포 유형은다른 유전자를 발현하는데, 이유는 특정 유전자만이각 세포에서 mRNA로 전사되기 때문이다실험실에서 mRNA를 견본으로 이용하여상보적 DNA(cDNA)를 합성하는데유전자 발현 연구에 이용된다흔한 방법은 세포에서 RNA를 추출하고다른 RNA, 즉 리보솜 RNA나 전이 RNA에서mRNA를 분리하는데방법은 샘플을 티민 뉴클레오티드 줄이 부착된비드 기둥 위로 달리게 것이다이것들은 폴리(A) 꼬리에 결합이 되는데그 꼬리는 진핵 생물의 mRNA의 3' 끝에 있는아데닌 뉴클레오티디 체인이다RNA의 다른 유형은 결합이 안되고 씻겨 없어진다mRNA가 분리된 후에 폴리(T) 시발체가폴리(A) 꼬리에 결합되어역 전사 효소가 mRNA로부터 cDNA에 있는단일 가닥을 전사하는 시발점을 제공해준다Rnase 효소 같은 화학 물질이 첨가되어RNA가 분해된다그 다음 DNA 폴리메라아제 효소를 이용해서cDNA에 상보적인 가닥을 합성하여그 결과 이중 가닥 cDNA를 만들어 내는데이것을 박테리아나 바이러스 벡터에 삽입할 수 있고분자 생물학 연구에 이용할 수 있다

기도, 레슬리 A. "생명 공학 혁명 : PCR 및 발현 유전자를 복제하기위한 역 전사 효소의 사용." 자연 교육 1, no. 1 (2008) : 94. [<a href="https://www.nature.com/scitable/topicpage/the-biotechnology-revolution-pcr-and-the-use-553" target="__blank">출처</a>]

상보적 dna

<h4>개요</h4>
 
 오직 전령RNA(messenger RNA, 줄여서 mRNA)에 전사된 유전자만이 활성화되거나 발현됩니다. 따라서 과학자들은 여러 세포와 조직의 유전자 발현을 연구하기 위해 세포에서 mRNA를 추출할 수 있습니다. 이는 역전사(reverse transcription)를 통해 mRNA를 상보적 DNA(complementary DNA, 줄여서 cDNA; 상보 DNA)로 변환해 이뤄집니다. mRNA는 인트론(intron; 비암호화 영역) 및 기타 조절 서열(regulatory sequence)을 포함하지 않기 때문에, cDNA는 유전자가 암호화하는 펩타이드(peptide)의 아미노산 서열을 과학자들이 직접 확인할 수 있게 합니다.
 
 <h4>cDNA 합성</h4>
 
 cDNA는 여러 가지 방법으로 생성할 수 있지만, 일반적인 방법은 먼저 세포에서 총 RNA를 추출한 다음 mRNA를 세포에 mRNA보다 많이 있는 운반RNA(transfer RNA, 줄여서 tRNA)와 리보솜 RNA(ribosomal RNA, 줄여서 rRNA)에서 분리하는 것입니다. 진핵생물(eukaryote)의 성숙 mRNA(mature mRNA)는 3’ 말단에 아데닌(adenine) 뉴클레오타이드(nucleotide)의 사슬인 폴리 A 꼬리(poly A tail)를 가지고 있지만, 다른 유형의 RNA는 그렇지 않습니다. 따라서, 타이민(thymine; 티민) 뉴클레오타이드의 사슬(oligo-dTs)을 컬럼(column)이나 자기 비드(magnetic bead)와 같은 기질(substrate)에 결합시켜 이를 이용해 mRNA의 폴리 A 꼬리와 특이적으로 염기쌍을 이루게 만들 수 있습니다. 폴리 A 꼬리를 가진 mRNA가 포착되는 동안, 다른 유형의 RNA는 씻겨 내려갑니다.
 
 그다음, 역전사효소(reverse transcriptase; 레트로바이러스의 DNA 중합효소)를 mRNA로부터 cDNA를 생성하는 데 사용합니다. 역전사효소는 대부분의 DNA 중합효소(DNA polymerase; DNA 폴리머레이스)와 마찬가지로 사슬의 3’ 말단에만 뉴클레오타이드를 첨가할 수 있기 때문에 폴리 T 프라이머(primer; 시발체)를 추가해 cDNA 합성의 시발점을 제공합니다. cDNA 가닥은 헤어핀 구조(hairpin loop)로 끝납니다. 그런 다음 (일반적으로 알칼리 처리 또는 RNase(ribonuclease; 리보핵산가수분해효소) 효소를 사용해) RNA를 분해합니다. 단일 가닥 cDNA는 그대로 유지됩니다.
 
 그런 다음 cDNA에 상보 되는 두 번째 DNA 가닥을 DNA 중합효소로 합성하는데, 여기서 종종 첫 번째 cDNA 가닥의 헤어핀 구조 또는 mRNA의 잘린 조각(nicked piece)을 프라이머로 사용합니다.
 
 이렇게 얻은 이중 가닥 cDNA는 박테리아나 바이러스 벡터에 삽입되고 표준 분자 생물학 기술을 사용하여 복제될 수 있습니다. 관심 세포나 조직의 모든 mRNA를 나타내는 cDNA 라이브러리도 추가 연구를 위해 구성할 수 있습니다.

상보적 DNA

개요

전령 RNA(messenger RNA, mRNA)로 전사된 유전자만 활성화되거나 발현됩니다. 따라서 과학자들은 세포에서 mRNA를 추출하여 ...

Education

교육

JoVE Core

Molecular Biology

분자생물학

Unit 2

제2단원

Studying DNA and RNA

DNA와 RNA 연구

KEY TERMS AND CONCEPTS

주요 용어 및 개념

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.

Making cDNA from RNA Templates

recombinant 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...

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

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 complementary DNA, or cDNA, is made from an RNA template and how it helps scientists study gene expression in the lab.

Making cDNA from RNA Templates | JoVE K12

molecular biology

JoVE Core 분자생물학은 명확하고 간결한 애니메이션을 통해 분자 단계에서 일어나는 세포 과정을 설명합니다.또한, 과학자가 직접 실험을 진행하는 실사 영상을 통해 분자생물학 개념의 응용 사례를 보여줍니다.

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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BIO1.ETS2.3

Making cDNA from RNA Templates

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