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

- [Istruttore] L'obiettivo della RCP,

reazione a catena della polimerasi

è di amplificare una sequenza genetica.

In questo esempio,

un gene di interesse sarà amplificato

dal DNA purificato.

Per eseguire l'amplificazione,

è necessaria una polimerasi
per sintetizzare il nuovo DNA.

Anche un primer, breve pezzo
di DNA a filamento singolo

che condivide l'omologia
con il gene di interesse,

è necessario.

Infine, singoli nucleotidi,

chiamati disossinucleotidi
trifosfati o dNTP,

sono usati per creare il nuovo filone.

Il primo passo nella PCR

è riscaldare la miscela,

che denatura il DNA.

Successivamente la
reazione viene raffreddata,

e i primer si ricopriranno
nella loro regione omologa.

Una volta legati, la
reazione viene riscaldata

a una temperatura ottimale
per la polimerasi.

La polimerasi quindi riconosce

il complesso del DNA dell'innesco,

e inizia la sintesi del nuovo filone,

utilizzando i dNTP nella soluzione.

La reazione viene quindi riscaldata

e procede come descritto,

attraverso ulteriori cicli.

Dopo il terzo ciclo,

ci sono otto copie totali del gene.

Dopo il quarto ciclo, 16 copie.

E il quinto ciclo, 32 copie.

Il numero di copie continua a
crescere in modo esponenziale.

Dopo 30 cicli, ci sono poco
più di 1 miliardo di copie.

Garibyan, Lilit e Nidhi Avashia. "Tecniche di ricerca rese semplici: reazione a catena della polimerasi (PCR)." The Journal of Investigative Dermatology 133, n. 3 (marzo 2013): e6. [<a href="https://doi.org/10.1038/jid.2013.1" target="__blank">Sorgente</a>]

Pray, Leslie A. "La rivoluzione biotecnologica: PCR e l'uso della trascrittasi inversa per clonare geni espressi". Educazione alla natura 1, n. 1 (2008): 94. [<a href="https://www.nature.com/scitable/topicpage/the-biotechnology-revolution-pcr-and-the-use-553" target="__blank">Sorgente</a>]

pcr - polymerase chain reaction

<h4>Panoramica</h4>

La reazione a catena della polimerasi, o PCR, è una tecnica ampiamente utilizzata per copiare segmenti di DNA. Grazie all'amplificazione esponenziale, la PCR può produrre milioni o miliardi di copie di DNA in poche ore. In una reazione PCR, un enzima di polimerasi del DNA resistente al calore amplifica il DNA originale attraverso una serie di variazioni di temperatura all'interno di una macchina automatizzata chiamata termociclo.

<h4>PCR è un metodo versatile che ha rivoluzionato la biologia molecolare</h4>

Kary Mullis ha sviluppato la PCR nel 1983, per la quale è stato insignito del Premio Nobel per la chimica nel 1993. Essendo un modo relativamente veloce, economico e preciso di copiare una sequenza di DNA, la PCR è diventata uno strumento inestimabile per numerose applicazioni, tra cui la clonazione molecolare, la mutagenesi genica, il rilevamento di patogeni, l'analisi dell'espressione genica, la quantificazione del DNA e il sequenziamento e la diagnosi delle malattie genetiche.

<h4>Il ciclo di reazione PCR</h4>

La PCR imita il processo di replicazione del DNA naturale che si verifica nelle cellule. La miscela di reazione include una sequenza di DNA modello da copiare, una coppia di molecole di DNA corto chiamate primer, blocchi di costruzione di DNA libero chiamati triplofati deossinucleotidi (dNTP) e un enzima specializzato di polimerasi del DNA.

La PCR comporta una serie di passaggi ad alte temperature, che richiedono un enzima di polimerasi del DNA che è funzionale a tali temperature. La polimerasi del DNA più comunemente utilizzata è la polimerasi Taq, dal nome della Thermus aquaticus, il batterio da cui la polimerasi è stata inizialmente isolata. La polimerasi del DNA non è in grado di sintetizzare una molecola di DNA da zero, o de novo. Invece, la polimerasi del DNA si aggiunge alle molecole di DNA corto, chiamate primer, che si legano al modello di DNA attraverso l'accoppiamento di base complementare. I primer forniscono un gruppo di idrossile gratuito da 3' a cui la polimerasi del DNA può collegare nuovi dNTP. Ci sono quattro tipi di dNTP in una PCR, uno per ogni nucleotide nella molecola di DNA: dATP, dCTP, dGTP e dTTP.

Ogni ciclo di PCR è costituito da tre fasi: Denaturazione, Annealing e Sintesi del DNA.

<ol><li>Denaturazione. Il ciclo PCR viene avviato riscaldando la miscela di reazione ad una temperatura elevata, causando la separazione del DNA doppia elica in due fili. Questo processo di "fusione" di solito si verifica ad una temperatura di 90-100 gradi centigradi.</li>
	<li>Ricottura. La miscela di reazione viene raffreddata rapidamente (di solito a 50oC–65oC), permettendo ai due primer di legarsi alle loro sequenze complementari sui filamenti di DNA del modello.</li>
	<li>Sintesi del DNA (Estensione Primer). La miscela di reazione viene riscaldata di nuovo, questa volta ad una temperatura (di solito 60-75gradi) che consente alla polimerasi del DNA di estendere le primer aggiungendo dNTP che si accoppiano con le basi nel filo modello.</li>
</ol>Una PCR tipica coinvolge 20-40 cicli ripetuti di questi tre passaggi, che si verificano nel termociclore. Poiché il numero di molecole di DNA è raddoppiato in ogni ciclo, il DNA viene amplificato in modo esponenziale.

<h4>Limitazioni della PCR</h4>

Se lo scienziato vuole amplificare un tratto specifico del genoma, lo scienziato deve conoscere almeno una parte della sequenza del DNA bersaglio per progettare i primer appropriati. Un altro potenziale problema è l'annessione non specifica dei primer a sequenze di DNA parzialmente simili, portando all'amplificazione del DNA non bersaglio. Questo problema può essere controllato ottimizzando le condizioni di reazione. Essendo un metodo di rilevamento altamente sensibile, la PCR è anche vulnerabile alla contaminazione e anche tracce di DNA contaminato possono causare risultati fuorvianti. Le polimerasi del DNA utilizzate nella PCR possono essere soggette a errori. Se una mutazione avviene entro i primi cicli, la maggior parte del DNA amplificato porterà la mutazione.

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

Didattica

JoVE Core

Molecular Biology

Biologia Molecolare

Unit 2

Unità 2

Studying DNA and RNA

Studiare il DNA e l'RNA

KEY TERMS AND CONCEPTS

TERMINI E CONCETTI CHIAVE

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 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

Le video animazioni di JoVE Core Molecular Biology, organizzate in video lezioni, illustrano i processi molecolari cellulari in maniera chiara e concisa. Inoltre, nei video di "scienziati in azione", i concetti scientifici vengono applicati e dimostrati da ricercatori attraverso esperimenti in laboratorio.

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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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.
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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