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

- [Eğitici] Polimeraz Zincir Reaksiyonu PCR'nin amacı, genetik bir diziyi çoğaltmaktır.
 Bu örnekte, ilgilenilen bir gen saflaştırılmış DNA'dan çoğaltılacaktır.
 Çoğaltmayı gerçekleştirmek için, yeni DNA'yı sentezlemek üzere bir polimeraz gereklidir.
İlgilenilen gen ile aynı homolojiyi paylaşan kısa bir tek zincirli DNA parçası yani bir primer de gereklidir.
 Son olarak, yeni iplikçik yapmak için Deoksinükleotit Trifosfat veya dNTP adı verilen tekli nükleotidler kullanılır.
 PCR'nin ilk adımı karışımı ısıtmaktır ve bu DNA'yı denatüre eder.
Daha sonra reaksiyon soğutulur ve primerler kendi homolog bölgelerine tutturulur.
 Bağlanmadan sonra, reaksiyon polimeraz için en uygun sıcaklığa ısıtılır.
 Polimeraz daha sonra primer DNA kompleksini tanır ve çözeltideki dNTP'leri kullanarak yeni zincirin sentezine başlar.
Reaksiyon daha sonra ısıtılır ve ilave döngüler boyunca belirtildiği şekilde ilerler.
 Üçüncü döngüyü takiben, genin toplam sekiz kopyası oluşur.
 Dördüncü döngüden sonra 16 kopya olur.
 Ve beşinci döngüden sonra 32 kopya.
 Kopya sayısı katlanarak artmaya devam eder.
 30 döngüden sonra 1 milyardan fazla kopya vardır.

Garibyan, Lilit ve Nidhi Avashia. "Araştırma Teknikleri Basitleştirildi: Polimeraz Zincir Reaksiyonu (PCR)." Araştırmacı Dermatoloji Dergisi 133, no. 3 (Mart 2013): e6. [<a href="https://doi.org/10.1038/jid.2013.1" target="__blank">Kaynak</a>]

Dua edin, Leslie A. "Biyoteknoloji Devrimi: PCR ve İfade Edilen Genleri Klonlamak için Ters Transkriptaz Kullanımı." Doğa Eğitimi 1, hayır. 1 (2008): 94. [<a href="https://www.nature.com/scitable/topicpage/the-biotechnology-revolution-pcr-and-the-use-553" target="__blank">Kaynak</a>]

pcr - polimeraz zincir reaksiyonu

<h4>Genel bakış</h4>

Polimeraz zincir reaksiyonu veya PCR, DNA segmentlerini kopyalamak için yaygın olarak kullanılan bir tekniktir. Üstel amplifikasyon nedeniyle, PCR sadece birkaç saat içinde milyonlarca veya milyarlarca DNA kopyası üretebilir. Bir PCR reaksiyonunda, ısıya dayanıklı bir DNA polimeraz enzimi, termocycler adı verilen otomatik bir makinenin içindeki bir dizi sıcaklık değişimi ile orijinal DNA'yı çoğaltır.

<h4>PCR Moleküler Biyolojide Devrim Yapan Çok Yönlü Bir Yöntemdir</h4>

Kary Mullis 1983 yılında PCR'yi geliştirdi ve 1993 yılında Nobel Kimya Ödülü'ne layık görüldü. Bir DNA dizisini kopyalamanın nispeten hızlı, ucuz ve hassas bir yolu olan PCR, moleküler klonlama, gen mutagenezi, patojen tespiti, gen ekspresyonu analizi, DNA kantitasyonu ve dizilemesi ve genetik hastalık tanısı gibi çok sayıda uygulama için paha biçilmez bir araç haline gelmiştir.

<h4>PCR Reaksiyon Döngüsü</h4>

PCR hücrelerde oluşan doğal DNA çoğaltma işlemini taklit eder. Reaksiyon karışımı kopyalanacak bir şablon DNA dizisi, primer adı verilen bir çift kısa DNA molekülü, deoksinükleotid trifosfat (dNTP) adı verilen serbest DNA yapı taşları ve özel bir DNA polimeraz enzimi içerir.

PCR yüksek sıcaklıklarda bir dizi adım içerir ve bu tür sıcaklıklarda işlevsel bir DNA polimeraz enzimi gerektiririr. En sık kullanılan DNA polimeraz Taq polimeraz’dır ve adını ilk olarak izole edildiği Thermus aquaticus bakterisinden alır. DNA polimeraz yöntemi ile bir DNA molekülünü sıfırdan yani de novo şekilde sentezlenemez. Bunun yerine, DNA polimeraz kısa DNA moleküllerini oluşturur, primer denilen tamamlayıcı baz eşleştirme yoluyla DNA şablonuna bağlar. Primerler, DNA polimerazın yeni dNTP'ler takabileceği 3' hidroksil grubu sağlarlar. Bir PCR'de dört tip dNTP vardır ve DNA molekülündeki her nükleotit için bir tanedir: dATP, dCTP, dGTP ve dTTP.

Her PCR döngüsü üç adımdan oluşur: Denatürasyon, Tavlama ve DNA Sentezi.

<ol>
<li>Denatürasyon. PCR döngüsü, reaksiyon karışımının yüksek bir sıcaklığa ısıtılmasıyla başlatılır ve DNA’nın iki iplikçik halinde ayrılmasına neden olur. Bu "erime" işlemi genellikle 90°C-100°C sıcaklıkta gerçekleşir.</li>
<li>Tavlama. Reaksiyon karışımı hızlı bir şekilde soğutularak (genellikle 50°C-65°C'ye kadar), iki primerin şablon DNA iplikçiklerindeki tamamlayıcı dizilerine bağlanmasını sağlanır.</li>
<li>DNA Sentezi (Primer Uzantısı). Reaksiyon karışımı bu kez tekrar ısıtılır, fakat bu kez DNA polimerazın şablon iplikçikteki bazlarla eşleşen dNTP'ler ekleyerek astarları genişletmesini sağlayan bir sıcaklığa (genellikle 60°C-75°C) kadar ısıtma gerçekleştirilir.</li>
</ol>Tipik bir PCR sürecinde termocycler içinde meydana gelen bu üç adım, 20-40 kez tekrarlanır. DNA moleküllerinin sayısı her döngüde iki katına çıktığı için, DNA katlanarak yükseltilir.

<h4>PCR’ın Sınırlı Yönleri</h4>

Eğer bilim insanı genomun belirli bir bölümünü çoğaltmak istiyorsa, uygun primeri tasarlamak için hedef DNA dizisinin en azından bir kısmını bilmelidir. Bir diğer potansiyel sorun da primerin kısmen benzer DNA dizilerine non-spesifik şekilde tavlanmasıdır. Bu sorun, reaksiyon koşulları optimize edilerek kontrol edilebilir. Son derece hassas bir algılama yöntemi olan PCR, kontaminasyona karşı da savunmasızdır ve DNA'nın eser miktarda kontaminasyonu bile yanıltıcı sonuçlara neden olabilir. PCR'de kullanılan DNA polimerazlar hatalara yatkın olabilir. Eğer ilk birkaç döngü içinde bir mutasyon gerçekleşirse, güçlendirilmiş DNA'nın çoğu bu mutasyonu taşıyacaktır.

PCR - Polimeraz Zincir Reaksiyonu

Genel bakış

Polimeraz zincir reaksiyonu veya PCR, DNA segmentlerini kopyalamak için yaygın olarak kullanılan bir tekniktir. Üstel amplifikasyon nedeniyle, PCR üretebilir ...

Education

JoVE Core

Molecular Biology

Moleküler Biyoloji

Unit 2

Ünite 2

Studying DNA and RNA

DNA ve RNA Çalışmaları

KEY TERMS AND CONCEPTS

TEMEL TERİMLER VE KAVRAMLAR

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 Moleküler Biyoloji, hücrenin moleküler süreçlerini açık ve özlü animasyonlu video dersleriyle sergiler. Buna ek olarak, bilim insanı-eylem videolarında, bu kavramların uygulaması modern laboratuvarlarda gerçek deneylerle gösterilmektedir.

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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Investigate means by which karyotypes are utilized in diagnostic medicine.
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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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