Name: pcr: dna amplification for genetics labs
Uploaded: 2021-04-07T12:12:00.000Z
Duration: 1 min 32 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

PCR - Polymerase Chain Reaction

Das Ziel der PCR (Polymerase-Kettenreaktion) ist die Amplifikation einer genetischen Sequenz.
 In diesem Beispiel wird ein interessierendes Gen aus gereinigter DNA amplifiziert.
 Um die Amplifikation durchzuführen, ist eine Polymerase erforderlich, um die neue DNA zu synthetisieren.
 Ein Primer, ein kurzes Stück einzelsträngiger DNA, das Homologie zu dem interessierenden Gen aufweist, wird ebenfalls benötigt.
 Schließlich werden einzelne Nukleotide, sogenannte Desoxynukleotidtriphosphate oder dNTPs, verwendet, um den neuen Strang herzustellen.
 Der erste Schritt bei der PCR besteht darin, die Mischung zu erhitzen, wodurch die DNA denaturiert wird.
 Als nächstes wird die Reaktion abgekühlt und die Primer werden an ihren homologen Bereich angelagert.
 Einmal gebunden, wird die Reaktion auf eine für die Polymerase optimale Temperatur erhitzt.
 Die Polymerase erkennt dann den Primer-DNA-Komplex und beginnt die Synthese des neuen Strangs unter Verwendung der dNTPs in der Lösung.
 Die Reaktion wird dann erhitzt und verläuft über weitere Zyklen wie beschrieben.
 Nach dem dritten Zyklus gibt es insgesamt acht Kopien des Gens.
 Nach dem vierten Zyklus 16 Exemplare, und dem fünften Zyklus, 32 Exemplare.
 Die Anzahl der Kopien wird weiterhin exponentiell zunehmen.
 Nach 30 Zyklen gibt es etwas mehr als 1 Milliarde Exemplare.

Garibyan, Lilit und Nidhi Avashia. "Forschungstechniken leicht gemacht: Polymerase-Kettenreaktion (PCR)." The Journal of Investigative Dermatology 133, Nr. 3 (März 2013): e6. [<a href="https://doi.org/10.1038/jid.2013.1" target="__blank">Quelltext</a>]

Pray, Leslie A. "Die biotechnologische Revolution: PCR und die Verwendung von reverser Transkriptase zum Klonen exprimierter Gene." Nature Education 1, Nr. 1 (2008): 94. [<a href="https://www.nature.com/scitable/topicpage/the-biotechnology-revolution-pcr-and-the-use-553" target="__blank">Quelle</a>]

pcr - polymerase-kettenreaktion

<h4>Überblick</h4>
 
 Die Polymerase-Kettenreaktion (englisch polymerase chain reaction, PCR) ist eine weit verbreitete Methode für das Kopieren von DNA-Abschnitten. Aufgrund der exponentiellen Amplifikation kann die PCR innerhalb weniger Stunden Millionen oder Milliarden von DNA-Kopien herstellen. In einer PCR-Reaktion amplifiziert ein hitzebeständiges DNA-Polymerase-Enzym die ursprüngliche DNA mit Hilfe einer Reihe von Temperaturänderungen in dem Thermocycler.
 
 <h4>Die PCR ist eine vielseitige Methode, die die Molekularbiologie revolutionierte</h4>
 
 Kary Mullis entwickelte die PCR im Jahr 1983. Dafür erhielt er im Jahr 1993 den Nobelpreis für Chemie. Als relativ schnelle, kostengünstige und präzise Methode für das Kopieren einer DNA-Sequenz wurde PCR zu einem unschätzbaren Werkzeug für zahlreiche Anwendungen. Dazu gehören beispielsweise das molekulare Klonen, die Mutagenese, der Nachweis von Krankheitserregern, die Analyse der Genexpression, die Quantifizierung und Sequenzierung von DNA sowie die Diagnose genetischer Krankheiten.
 
 <h4>Der PCR-Reaktionszyklus</h4>
 
 PCR ahmt den natürlichen DNA-Replikationsprozess nach, der in Zellen abläuft. Das Reaktionsgemisch enthält eine zu kopierende Original-DNA-Sequenz (Template), ein Paar kurze DNA-Moleküle (die Primer genannt werden), freie DNA-Bausteine, die Desoxynukleotidtriphosphate (die dNTPs), und ein spezialisiertes DNA-Polymerase-Enzym.
 
 PCR umfasst eine Reihe von Schritten bei hohen Temperaturen. Daher wird ein DNA-Polymerase-Enzym gebraucht, das bei solchen Temperaturen aktiv ist. Die am häufigsten verwendete DNA-Polymerase ist die Taq-Polymerase, benannt nach Thermus aquaticus, dem Bakterium, aus dem die Polymerase isoliert wurde. Die DNA-Polymerase ist nicht dazu in der Lage, ein DNA-Molekül von Grund auf oder neu zu synthetisieren (de novo). Stattdessen fügt die DNA-Polymerase an die kurzen DNA-Moleküle, die Primer, an. Diese binden sich durch komplementäre Basenpaarung an die Original-DNA. Die Primer haben eine freie 3’-Hydroxylgruppe, an welche die DNA-Polymerase neue dNTPs binden kann. Es gibt vier Arten von dNTPs in einer PCR-Reaktion, eines für jedes Nukleotid im DNA-Molekül: dATP, dCTP, dGTP und dTTP.
 
 Der PCR-Zyklus besteht aus drei Schritten: Denaturierung, Annealing und DNA-Synthese.
 
 <ol>
 <li>Denaturierung. Der PCR-Zyklus beginnt mit dem Erhitzen des Reaktionsgemisches auf eine hohe Temperatur. Dabei wird die DNA-Doppelhelix in zwei Stränge aufgetrennt. Dieser “Schmelz”-Prozess findet normalerweise bei Temperaturen zwischen 90°C–100°C statt.</li>
 <li>Annealing. Das Reaktionsgemisch wird schnell abgekühlt (normalerweise auf 50°C–65°C, wodurch die beiden Primer an ihre komplementären Sequenzen auf den Template-DNA-Strängen binden können.</li>
 <li>DNA-Synthese (Primer-Verlängerung). Das Reaktionsgemisch wird erneut erhitzt, diesmal auf eine Temperatur (normalerweise 60°C–75°C, die es der DNA-Polymerase erlaubt, die Primer durch das Hinzufügen von dNTPs, die sich mit den Basen der Template-DNA paaren, zu verlängern.</li>
 </ol>
 
 Eine typische PCR beinhaltet 20-40 wiederholte Zyklen dieser drei Schritte, die im Thermocycler stattfinden. Da sich die Anzahl der DNA-Moleküle in jedem Zyklus verdoppelt, wird die DNA exponentiell vervielfältigt.
 
 <h4>Grenzen der PCR-Methode</h4>
 
 Wenn ein Wissenschaftler einen bestimmten Abschnitt des Genoms amplifizieren will, muss er zumindest einen Teil der gewünschten DNA-Sequenz kennen, um einen geeigneten Primer zu entwerfen. Ein weiteres potenzielles Problem ist die unspezifische Annealierung von Primern an teilweise ähnliche DNA-Sequenzen, welches zur Amplifikation von nicht gewünschten DNA-Sequenzen führt. Dieses Problem lässt sich durch die Optimierung der Reaktionsbedingungen kontrollieren. Da die PCR eine hochempfindliche Detektionsmethode ist, ist sie auch für Kontaminationen anfällig. Selbst Spuren kontaminierender DNA können zu irreführenden Ergebnissen führen. Zusätzlich, können die in der PCR verwendeten DNA-Polymerasen fehleranfällig sein. Kommt es innerhalb der ersten PCR-Zyklen zu einer Mutation, trägt der größte Teil der amplifizierten DNA diese Mutation.

PCR - Polymerase-Kettenreaktion

Überblick

Die Polymerase-Kettenreaktion (PCR) ist eine weit verbreitete Technik zum Kopieren von DNA-Segmenten. Aufgrund der exponentiellen Amplifikation kann die PCR ...

Education

Lehre

JoVE Core

Molecular Biology

Molekularbiologie

Unit 2

Einheit 2

Studying DNA and RNA

Untersuchung von DNA und RNA

KEY TERMS AND CONCEPTS

KERNBEGRIFFE UND KONZEPTE

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 topics. It helps students connect DNA structure with laboratory methods used to analyze genetic information.

PCR: DNA Amplification for Genetics Labs

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

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 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 PCR amplifies a specific DNA segment through heating, cooling, primers, and DNA polymerase in genetics and molecular biology labs.

PCR: DNA Amplification for Genetics Labs | JoVE K12

molecular biology

JoVE Core Molecular Biology veranschaulicht die molekularen Prozesse der Zelle in prägnanten animierten Video-Lektionen. In Videos von Wissenschaftlern in Aktion wird die Anwendung dieser Konzepte zusätzlich durch reale Experimente in modernen Laboren demonstriert.

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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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.
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

PCR: DNA Amplification for Genetics Labs

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