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

- [Verteller] Het doel van PCR, polymerasekettingreactie, is het versterken van een genetische sequentie. In dit voorbeeld zal een gen van belang worden geamplificeerd uit gezuiverd DNA. Om de amplificatie uit te voeren, is een polymerase vereist om het nieuwe DNA te synthetiseren.

Een primer, een kort stuk enkelstrengs DNA die homologie deelt met het gen van belang, is ook nodig. Tenslotte worden enkele nucleotiden genaamd deoxynucleotide trifosfaten, of dNTP's, gebruikt om de nieuwe streng te maken. De eerste stap in PCR is het verwarmen van het mengsel, die het DNA denatureert.

Vervolgens wordt de reactie afgekoeld en zullen de primers aan hun homoloog gebied gloeien. Eenmaal gebonden, wordt de reactie verhit tot een optimale temperatuur voor het polymerase. Het polymerase herkent vervolgens het primer-DNA-complex en begint de synthese van de nieuwe streng, met behulp van de dNTP's in de oplossing.

De reactie wordt vervolgens verhit en verloopt zoals beschreven in de aanvullende cycli. Na de derde cyclus zijn er in totaal acht exemplaren van het gen. Na de vierde cyclus, zestien exemplaren. En na de vijfde cyclus, tweeëndertig exemplaren. Het aantal exemplaren blijft exponentieel groeien. Na dertig cycli zijn er iets meer dan een miljard exemplaren.

pcr - polymerase chain reaction

<h4>Overzicht</h4>

De polymerasekettingreactie, of PCR, is een veelgebruikte techniek voor het kopiëren van DNA-segmenten. Dankzij exponentiële amplificatie kan PCR binnen slechts enkele uren miljoenen of miljarden DNA-kopieën produceren. Bij een PCR-reactie amplificeert een hittebestendig DNA-polymerase-enzym het oorspronkelijke DNA door een reeks temperatuurveranderingen in een geautomatiseerde machine die een thermocycler wordt genoemd.

<h4>PCR is een veelzijdige methode die een revolutie teweeg heeft gebracht in de moleculaire biologie</h4>

Kary Mullis ontwikkelde PCR in 1983, waarvoor hij in 1993 de Nobelprijs voor de Scheikunde ontving. Omdat het een relatief snelle, goedkope en nauwkeurige manier is om een DNA-sequentie te kopiëren, werd PCR een hulpmiddel van onschatbare waarde voor talloze toepassingen, waaronder moleculair klonen, genmutagenese, detectie van pathogenen, analyse van genexpressie, DNA-kwantificering en -sequencing, en diagnose van genetische ziekten.

<h4>De PCR-reactiecyclus</h4>

PCR bootst het natuurlijke DNA-replicatieproces na dat in cellen plaatsvindt. Het reactiemengsel omvat een sjabloon-DNA-sequentie die moet worden gekopieerd, een paar korte DNA-moleculen die primers worden genoemd, vrije DNA-bouwstenen die deoxynucleotide-trifosfaten (dNTP's) worden genoemd, en een gespecialiseerd DNA-polymerase-enzym.

PCR omvat een reeks stappen bij hoge temperaturen, waarvoor een DNA-polymerase-enzym nodig is dat bij dergelijke temperaturen functioneel is. Het meest gebruikte DNA-polymerase is Taq-polymerase, genoemd naar Thermus Aquaticus, de bacterie waaruit het polymerase aanvankelijk werd geïsoleerd. DNA-polymerase is niet in staat een DNA-molecuul helemaal opnieuw te synthetiseren, of de novo. In plaats daarvan voegt DNA-polymerase zich toe aan korte DNA-moleculen, primers genoemd, die via complementaire basenparing aan de DNA-sjabloon binden. De primers bieden een gratis 3’ hydroxylgroep waaraan DNA-polymerase nieuwe dNTP's kan hechten. Er zijn vier soorten dNTP's in een PCR, één voor elk nucleotide in het DNA-molecuul: dATP, dCTP, dGTP en dTTP.

Elke PCR-cyclus bestaat uit drie stappen: denaturatie, gloeien en DNA-synthese.

<ol>
	<li>Denaturatie. De PCR-cyclus wordt gestart door het reactiemengsel tot een hoge temperatuur te verwarmen, waardoor de dubbele DNA-helix in twee strengen wordt gescheiden. Dit ‘smelten’ proces vindt meestal plaats bij een temperatuur van 90°C–100°C.</li>
	<li>Gloeien. Het reactiemengsel wordt snel afgekoeld (meestal tot 50°C-65°C), waardoor de twee primers kunnen binden aan hun complementaire sequenties op de template-DNA-strengen.</li>
	<li>DNA-synthese (primerextensie). Het reactiemengsel wordt opnieuw verwarmd, deze keer tot een temperatuur (meestal 60°C – 75°C) waarbij DNA-polymerase de primers kan verlengen door dNTP's toe te voegen die paren met de basen in de matrijsstreng.</li>
</ol>



Een typische PCR omvat 20-40 herhaalde cycli van deze drie stappen, die plaatsvinden in de thermocycler. Omdat het aantal DNA-moleculen in elke cyclus wordt verdubbeld, wordt het DNA exponentieel geamplificeerd.

<h4>Beperkingen van PCR</h4>

Als de wetenschapper een specifiek stuk van het genoom wil amplificeren, moet de wetenschapper ten minste een deel van de doel-DNA-sequentie kennen om geschikte primers te kunnen ontwerpen. Een ander potentieel probleem is de niet-specifieke koppeling van primers aan gedeeltelijk vergelijkbare DNA-sequenties, wat leidt tot amplificatie van niet-doel-DNA. Dit probleem kan worden beheerst door de reactieomstandigheden te optimaliseren. Omdat het een zeer gevoelige detectiemethode is, is PCR ook kwetsbaar voor besmetting, en zelfs sporen van verontreinigend DNA kunnen misleidende resultaten veroorzaken. De DNA-polymerasen die bij PCR worden gebruikt, kunnen gevoelig zijn voor fouten. Als er binnen de eerste paar cycli een mutatie plaatsvindt, zal het grootste deel van het geamplificeerde DNA de mutatie dragen.

Overview

The polymerase chain reaction, or PCR, is a widely used technique for copying segments of DNA. Due to exponential amplification, PCR can produce ...

Education

JoVE Core

Molecular Biology

Moleculaire biologie

Unit 2

Onderdeel 2

Studying DNA and RNA

Het bestuderen van DNA en RNA

KEY TERMS AND CONCEPTS

BELANGRIJKE TERMEN EN CONCEPTEN

PCR - Polymerase Kettingreactie

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

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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...

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Gene Chips for Comparing Cell Activity

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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 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 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.

Florida Department of Education

Life Science

SC.912.L.16

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Texas Essential Knowledge and Skills

BIO.7

BIO.7.D

myJoVE Corporation

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In-situ Hybridization

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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

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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

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BIO1.ETS2.3

PCR: DNA Amplification for Genetics Labs

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