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

Почти каждая клетка в теле имеет одну и ту же ДНК, но разные типы клеток, такие как нейроны и мышечные клетки, экспрессируют различные гены потому что только некоторые гены транскрибируются в информационной РНК, или мРНК, в каждой клетке. В лаборатории можно использовать мРНК в качестве шаблона для синтеза комплементарной ДНК, кДНК, для изучения экспрессии генов. Общий метод извлечь РНК из клетки, затем изолировать мРНК от других типов РНК, как рибосомальная РНК или трансферная РНК, путём запуска образца над рядом бусин с присоединёнными растяжками нуклеотидов тимина.Они связаны с полимерным хвостом, цепочкой адениновых нуклеотидов специфически представленных на конце 3-штрих эукариотической мРНК. Другие типы РНК не связываются и смываются. После выделения мРНК, поли праймер связан с поли хвостовой частью, создавая отправную точку для ферментов обратной транскриптазы для транскрибирования одноцепочечной кДНК из мРНК.Химикаты, такие как фермент RNase добавляются, чтобы деградировать РНК. Затем используются ферменты ДНК-полимеразы, чтобы синтезировать цепочки, дополняющие кДНК приводя к двухцепочечной кДНК, которую можно вставить в бактериальный или вирусный вектор и использовать в исследованиях по молекулярной биологии.

Прей, Лесли А. "Биотехнологическая революция: ПЦР и использование обратной транскриптазы для клонирования экспрессированных генов." Nature Education 1, No1 (2008): 94. [<a href="https://www.nature.com/scitable/topicpage/the-biotechnology-revolution-pcr-and-the-use-553" target="__blank">Источник</a>]

Комплементарная ДНК

<h4>Обзор</h4>

Активны или выражены только гены, транскрибированные в РНК-мессенджер (мРНК). Таким образом, ученые могут извлекать мРНК из клеток для изучения экспрессии генов в различных клетках и тканях. Ученый преобразует мРНК в комплементарную ДНК (кДНК) с помощью обратной транскрипции. Поскольку мРНК не содержит интронов (некодирующих регионов) и других нормативных последовательностей, кДНК, в отличие от геномной ДНК, также позволяет исследователям напрямую определять аминокислотную последовательность пептида, кодируемого геном.

<h4>Синтез cDNA</h4>

cDNA может быть сгенерирован несколькими методами, но общий способ заключается в том, чтобы сначала извлечь общую РНК из клеток, а затем изолировать мРНК от более преобладающих типов- передачи РНК (тРНК) и рибосомной (рРНК). Зрелая эукариотическая мРНК имеет поли(А) хвост – вереницу нуклеотидов аденина – добавленную к ее 3' концу, в то время как другие типы РНК этого не делают. Таким образом, строка нуклеотидов тимина (олиго-дэ) может быть прикреплена к субстрату, такому как колонка или магнитные бусы, к специально базовой паре с поли(А) хвостами мРНК. В то время как мРНК с поли(А) хвост захвачен, другие типы РНК смываются.

Далее, обратная транскриптаза - фермент полимеразы ДНК из ретровирусов - используется для генерации кДНК из мРНК. Так как, как и большинство полимераз ДНК, обратная транскриптаза может добавить нуклеотиды только к 3 'конец цепи, поли (T) грунтовка добавляется, чтобы связать с поли (A) хвост, чтобы обеспечить отправную точку для синтеза кДНК. Цепь cDNA заканчивается в петле шпильки. РНК затем деградирует, как правило, при щелочной обработке или обрабоке RNase ферментов, оставляя одноцепочечную кДНК нетронутой.

Вторая цепь ДНК дополняет cDNA затем синтезируется полимеразы ДНК-часто с помощью шпильки петли первой цепи cDNA или заимствованным участком мРНК в качестве праймера.

В результате двухместный кДНК может быть вставлен в бактериальные или вирусные векторы и клонирован с использованием стандартных методов молекулярной биологии. Библиотека cDNA, представляющая все мРНК в клетках или тканях, представляющих интерес, также может быть построена для дополнительных исследований.

Обзор

Только гены, которые транскрибируются в матричную РНК (мРНК), являются активными или экспрессированными. Таким образом, ученые могут извлекать мРНК из клеток в ...

Education

JoVE Core

Molecular Biology

Молекулярная биология

Unit 2

Раздел 2

Studying DNA and RNA

Изучение ДНК и РНК

KEY TERMS AND CONCEPTS

ОСНОВНЫЕ ТЕРМИНЫ И ПОНЯТИЯ

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

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Gel Purification

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PCR - Polymerase Chain Reaction

Real Time RT-PCR

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RACE - Rapid Amplification of cDNA Ends

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PCR: The Polymerase Chain Reaction

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bacterial-transformation-the-heat-shock-method

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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

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An Overview of Gene Expression

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Expression Profiling with Microarrays

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FISH - Fluorescent In-situ Hybridization

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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

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Chromatin Immunoprecipitation- ChIP

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RNA-seq

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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

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The ELISA Method

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Transgenic Plants

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Gene Therapy

Reproductive Cloning

CRISPR

Experimental RNAi

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Reporter Genes

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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.
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Karyotyping

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

Making cDNA from RNA Templates

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