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

Prawie każda komórka w ciele ma to samo DNA, ale różne typy komórek, takie jak neurony i komórki mięśniowe, wyrażają różne geny, ponieważ tylko niektóre geny są transkrybowane na informacyjny RNA lub mRNA w każdej komórce. W laboratorium mRNA można wykorzystać jako matrycę do syntezy komplementarnego DNA, cDNA, do badania ekspresji genów. Powszechną metodą jest ekstrakcja RNA z komórek, a następnie wyizolowanie mRNA z innych typów RNA, takich jak rybosomalny RNA lub transfer RNA, poprzez przeciągnięcie próbki przez kolumnę kulek z przyłączonymi odcinkami nukleotydów tyminy.

Wiążą się one z ogonem poli-A, łańcuchem nukleotydów adeninowych specyficznie obecnych na 3-pierwszych końcach eukariotycznego mRNA. Inne typy RNA nie wiążą się i są wypłukiwane.

Po wyizolowaniu mRNA starter poli-T jest wiązany z ogonem poli-A, zapewniając punkt wyjścia dla enzymów odwrotnej transkryptazy do transkrypcji jednoniciowego cDNA z mRNA. Substancje chemiczne, takie jak enzymy RNazy, są następnie dodawane w celu degradacji RNA.

Enzymy polimerazy DNA są następnie wykorzystywane do syntezy nici komplementarnej do cDNA, w wyniku czego powstaje dwuniciowe cDNA, które można wprowadzić do wektora bakteryjnego lub wirusowego i wykorzystać w badaniach biologii molekularnej.

Módl się, Leslie A. "Rewolucja biotechnologiczna: PCR i zastosowanie odwrotnej transkryptazy do klonowania genów wyrażonych przez geny." Edukacja przyrodnicza 1, nr 1 (2008): 94. [<a href="https://www.nature.com/scitable/topicpage/the-biotechnology-revolution-pcr-and-the-use-553" target="__blank">Źródło</a>]

komplementarne dna

<h4>Przegląd</h4>

Tylko geny, które ulegają transkrypcji do informacyjnego RNA (mRNA), są aktywne lub ulegają ekspresji. Naukowcy mogą zatem wyodrębnić mRNA z komórek, aby zbadać ekspresję genów w różnych komórkach i tkankach. Naukowiec przekształca mRNA w komplementarny DNA (cDNA) poprzez odwrotną transkrypcję. Ponieważ mRNA nie zawiera intronów (regionów niekodujących) i innych sekwencji regulacyjnych, cDNA — w przeciwieństwie do genomowego DNA — umożliwia także badaczom bezpośrednie określenie sekwencji aminokwasów peptydu kodowanego przez gen.

<h4>Synteza cDNA</h4>

cDNA można wytworzyć kilkoma metodami, ale powszechnym sposobem jest najpierw ekstrakcja całkowitego RNA z komórek, a następnie izolacja mRNA z bardziej dominujących typów — RNA transferowego (tRNA) i rybosomalnego (rRNA). Dojrzały eukariotyczny mRNA ma ogon poli(A) — ciąg nukleotydów adeninowych — dodany do jego końca 3’ koniec, podczas gdy inne typy RNA nie. Dlatego ciąg nukleotydów tyminowych (oligo-dT) można przyłączyć do podłoża, takiego jak kolumna lub kulki magnetyczne, w celu specyficznego sparowania zasad z ogonami poli(A) mRNA. Podczas wychwytywania mRNA z ogonem poli(A) inne typy RNA są wypłukane.

Następnie do wytworzenia cDNA z mRNA wykorzystuje się odwrotną transkryptazę — enzym polimerazę DNA z retrowirusów. Ponieważ, podobnie jak większość polimeraz DNA, odwrotna transkryptaza może dodawać nukleotydy tylko do 3’ końcu łańcucha, dodaje się starter poli(T), aby związać się z ogonem poli(A) i zapewnić punkt wyjścia do syntezy cDNA. Nić cDNA kończy się pętlą typu spinka do włosów. RNA jest następnie degradowany — zwykle poprzez działanie alkaliami lub enzymami RNazy — pozostawiając jednoniciowy cDNA nienaruszony.

Druga nić DNA komplementarna do cDNA jest następnie syntetyzowana przez polimerazę DNA — często wykorzystując jako starter pętlę w kształcie spinki do włosów pierwszej nici cDNA lub nacięty fragment mRNA.

Powstały dwuniciowy cDNA można wstawić do wektorów bakteryjnych lub wirusowych i sklonować przy użyciu standardowych technik biologii molekularnej. Bibliotekę cDNA—reprezentującą wszystkie mRNA w komórkach lub tkankach będących przedmiotem zainteresowania—można również skonstruować na potrzeby dodatkowych badań.

Komplementarne DNA

Przegląd

Tylko geny, które są transkrybowane na informacyjny RNA (mRNA), są aktywne lub ulegają ekspresji. Naukowcy mogą zatem wyodrębnić mRNA z komórek, aby ...

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

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 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.

Texas Essential Knowledge and Skills

BIO.7

BIO.7.D

myJoVE Corporation

Molecular Cloning

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An Overview of Genetic Engineering

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Recombineering and Gene Targeting

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Genome Editing

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Recombinant DNA

DNA Ligation Reactions

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Restriction Enzyme Digests

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DNA Isolation

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Restriction Enzymes

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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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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 ...

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

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Euchromatin

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Heterochromatin

X and Y Chromosomes

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The Ratio of X Chromosome to Autosomes

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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

Whole Body Regeneration

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stem-cell-therapy-for-tissue-regeneration

Stem Cell Therapy for Tissue Regeneration

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Introduction to Nuclear Reprogramming

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Methods of Nuclear Reprogramming

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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

Making cDNA from RNA Templates

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