Name: pcr cycle for dna amplification
Uploaded: 2026-08-26T08:19:52.163Z
Duration: 2 min 7 s
Channel: JoVE

- [E&#x11F;itici] Polimeraz Zincir Reaksiyonu PCR&#x27;nin amac&#x131;, genetik bir diziyi &#xE7;o&#x11F;altmakt&#x131;r.
 Bu &#xF6;rnekte, ilgilenilen bir gen safla&#x15F;t&#x131;r&#x131;lm&#x131;&#x15F; DNA&#x27;dan &#xE7;o&#x11F;alt&#x131;lacakt&#x131;r.
 &#xC7;o&#x11F;altmay&#x131; ger&#xE7;ekle&#x15F;tirmek i&#xE7;in, yeni DNA&#x27;y&#x131; sentezlemek &#xFC;zere bir polimeraz gereklidir.
&#x130;lgilenilen gen ile ayn&#x131; homolojiyi payla&#x15F;an k&#x131;sa bir tek zincirli DNA par&#xE7;as&#x131; yani bir primer de gereklidir.
 Son olarak, yeni iplik&#xE7;ik yapmak i&#xE7;in Deoksin&#xFC;kleotit Trifosfat veya dNTP ad&#x131; verilen tekli n&#xFC;kleotidler kullan&#x131;l&#x131;r.
 PCR&#x27;nin ilk ad&#x131;m&#x131; kar&#x131;&#x15F;&#x131;m&#x131; &#x131;s&#x131;tmakt&#x131;r ve bu DNA&#x27;y&#x131; denat&#xFC;re eder.
Daha sonra reaksiyon so&#x11F;utulur ve primerler kendi homolog b&#xF6;lgelerine tutturulur.
 Ba&#x11F;lanmadan sonra, reaksiyon polimeraz i&#xE7;in en uygun s&#x131;cakl&#x131;&#x11F;a &#x131;s&#x131;t&#x131;l&#x131;r.
 Polimeraz daha sonra primer DNA kompleksini tan&#x131;r ve &#xE7;&#xF6;zeltideki dNTP&#x27;leri kullanarak yeni zincirin sentezine ba&#x15F;lar.
Reaksiyon daha sonra &#x131;s&#x131;t&#x131;l&#x131;r ve ilave d&#xF6;ng&#xFC;ler boyunca belirtildi&#x11F;i &#x15F;ekilde ilerler.
 &#xDC;&#xE7;&#xFC;nc&#xFC; d&#xF6;ng&#xFC;y&#xFC; takiben, genin toplam sekiz kopyas&#x131; olu&#x15F;ur.
 D&#xF6;rd&#xFC;nc&#xFC; d&#xF6;ng&#xFC;den sonra 16 kopya olur.
 Ve be&#x15F;inci d&#xF6;ng&#xFC;den sonra 32 kopya.
 Kopya say&#x131;s&#x131; katlanarak artmaya devam eder.
 30 d&#xF6;ng&#xFC;den sonra 1 milyardan fazla kopya vard&#x131;r.

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>]

<h4>Genel bak&#x131;&#x15F;</h4>

Polimeraz zincir reaksiyonu veya PCR, DNA segmentlerini kopyalamak i&#xE7;in yayg&#x131;n olarak kullan&#x131;lan bir tekniktir. &#xDC;stel amplifikasyon nedeniyle, PCR sadece birka&#xE7; saat i&#xE7;inde milyonlarca veya milyarlarca DNA kopyas&#x131; &#xFC;retebilir. Bir PCR reaksiyonunda, &#x131;s&#x131;ya dayan&#x131;kl&#x131; bir DNA polimeraz enzimi, termocycler ad&#x131; verilen otomatik bir makinenin i&#xE7;indeki bir dizi s&#x131;cakl&#x131;k de&#x11F;i&#x15F;imi ile orijinal DNA&#x27;y&#x131; &#xE7;o&#x11F;alt&#x131;r.

<h4>PCR Molek&#xFC;ler Biyolojide Devrim Yapan &#xC7;ok Y&#xF6;nl&#xFC; Bir Y&#xF6;ntemdir</h4>

Kary Mullis 1983 y&#x131;l&#x131;nda PCR&#x27;yi geli&#x15F;tirdi ve 1993 y&#x131;l&#x131;nda Nobel Kimya &#xD6;d&#xFC;l&#xFC;&#x27;ne lay&#x131;k g&#xF6;r&#xFC;ld&#xFC;. Bir DNA dizisini kopyalaman&#x131;n nispeten h&#x131;zl&#x131;, ucuz ve hassas bir yolu olan PCR, molek&#xFC;ler klonlama, gen mutagenezi, patojen tespiti, gen ekspresyonu analizi, DNA kantitasyonu ve dizilemesi ve genetik hastal&#x131;k tan&#x131;s&#x131; gibi &#xE7;ok say&#x131;da uygulama i&#xE7;in paha bi&#xE7;ilmez bir ara&#xE7; haline gelmi&#x15F;tir.

<h4>PCR Reaksiyon D&#xF6;ng&#xFC;s&#xFC;</h4>

PCR h&#xFC;crelerde olu&#x15F;an do&#x11F;al DNA &#xE7;o&#x11F;altma i&#x15F;lemini taklit eder. Reaksiyon kar&#x131;&#x15F;&#x131;m&#x131; kopyalanacak bir &#x15F;ablon DNA dizisi, primer ad&#x131; verilen bir &#xE7;ift k&#x131;sa DNA molek&#xFC;l&#xFC;, deoksin&#xFC;kleotid trifosfat (dNTP) ad&#x131; verilen serbest DNA yap&#x131; ta&#x15F;lar&#x131; ve &#xF6;zel bir DNA polimeraz enzimi i&#xE7;erir.

PCR y&#xFC;ksek s&#x131;cakl&#x131;klarda bir dizi ad&#x131;m i&#xE7;erir ve bu t&#xFC;r s&#x131;cakl&#x131;klarda i&#x15F;levsel bir DNA polimeraz enzimi gerektiririr. En s&#x131;k kullan&#x131;lan DNA polimeraz Taq polimeraz&#x2019;d&#x131;r ve ad&#x131;n&#x131; ilk olarak izole edildi&#x11F;i Thermus aquaticus bakterisinden al&#x131;r. DNA polimeraz y&#xF6;ntemi ile bir DNA molek&#xFC;l&#xFC;n&#xFC; s&#x131;f&#x131;rdan yani de novo &#x15F;ekilde sentezlenemez. Bunun yerine, DNA polimeraz k&#x131;sa DNA molek&#xFC;llerini olu&#x15F;turur, primer denilen tamamlay&#x131;c&#x131; baz e&#x15F;le&#x15F;tirme yoluyla DNA &#x15F;ablonuna ba&#x11F;lar. Primerler, DNA polimeraz&#x131;n yeni dNTP&#x27;ler takabilece&#x11F;i 3&#x27; hidroksil grubu sa&#x11F;larlar. Bir PCR&#x27;de d&#xF6;rt tip dNTP vard&#x131;r ve DNA molek&#xFC;l&#xFC;ndeki her n&#xFC;kleotit i&#xE7;in bir tanedir: dATP, dCTP, dGTP ve dTTP.

Her PCR d&#xF6;ng&#xFC;s&#xFC; &#xFC;&#xE7; ad&#x131;mdan olu&#x15F;ur: Denat&#xFC;rasyon, Tavlama ve DNA Sentezi.

<ol>
<li>Denat&#xFC;rasyon. PCR d&#xF6;ng&#xFC;s&#xFC;, reaksiyon kar&#x131;&#x15F;&#x131;m&#x131;n&#x131;n y&#xFC;ksek bir s&#x131;cakl&#x131;&#x11F;a &#x131;s&#x131;t&#x131;lmas&#x131;yla ba&#x15F;lat&#x131;l&#x131;r ve DNA&#x2019;n&#x131;n iki iplik&#xE7;ik halinde ayr&#x131;lmas&#x131;na neden olur. Bu &#x22;erime&#x22; i&#x15F;lemi genellikle 90&#xB0;C-100&#xB0;C s&#x131;cakl&#x131;kta ger&#xE7;ekle&#x15F;ir.</li>
<li>Tavlama. Reaksiyon kar&#x131;&#x15F;&#x131;m&#x131; h&#x131;zl&#x131; bir &#x15F;ekilde so&#x11F;utularak (genellikle 50&#xB0;C-65&#xB0;C&#x27;ye kadar), iki primerin &#x15F;ablon DNA iplik&#xE7;iklerindeki tamamlay&#x131;c&#x131; dizilerine ba&#x11F;lanmas&#x131;n&#x131; sa&#x11F;lan&#x131;r.</li>
<li>DNA Sentezi (Primer Uzant&#x131;s&#x131;). Reaksiyon kar&#x131;&#x15F;&#x131;m&#x131; bu kez tekrar &#x131;s&#x131;t&#x131;l&#x131;r, fakat bu kez DNA polimeraz&#x131;n &#x15F;ablon iplik&#xE7;ikteki bazlarla e&#x15F;le&#x15F;en dNTP&#x27;ler ekleyerek astarlar&#x131; geni&#x15F;letmesini sa&#x11F;layan bir s&#x131;cakl&#x131;&#x11F;a (genellikle 60&#xB0;C-75&#xB0;C) kadar &#x131;s&#x131;tma ger&#xE7;ekle&#x15F;tirilir.</li>
</ol>Tipik bir PCR s&#xFC;recinde termocycler i&#xE7;inde meydana gelen bu &#xFC;&#xE7; ad&#x131;m, 20-40 kez tekrarlan&#x131;r. DNA molek&#xFC;llerinin say&#x131;s&#x131; her d&#xF6;ng&#xFC;de iki kat&#x131;na &#xE7;&#x131;kt&#x131;&#x11F;&#x131; i&#xE7;in, DNA katlanarak y&#xFC;kseltilir.

<h4>PCR&#x2019;&#x131;n S&#x131;n&#x131;rl&#x131; Y&#xF6;nleri</h4>

E&#x11F;er bilim insan&#x131; genomun belirli bir b&#xF6;l&#xFC;m&#xFC;n&#xFC; &#xE7;o&#x11F;altmak istiyorsa, uygun primeri tasarlamak i&#xE7;in hedef DNA dizisinin en az&#x131;ndan bir k&#x131;sm&#x131;n&#x131; bilmelidir. Bir di&#x11F;er potansiyel sorun da primerin k&#x131;smen benzer DNA dizilerine non-spesifik &#x15F;ekilde tavlanmas&#x131;d&#x131;r. Bu sorun, reaksiyon ko&#x15F;ullar&#x131; optimize edilerek kontrol edilebilir. Son derece hassas bir alg&#x131;lama y&#xF6;ntemi olan PCR, kontaminasyona kar&#x15F;&#x131; da savunmas&#x131;zd&#x131;r ve DNA&#x27;n&#x131;n eser miktarda kontaminasyonu bile yan&#x131;lt&#x131;c&#x131; sonu&#xE7;lara neden olabilir. PCR&#x27;de kullan&#x131;lan DNA polimerazlar hatalara yatk&#x131;n olabilir. E&#x11F;er ilk birka&#xE7; d&#xF6;ng&#xFC; i&#xE7;inde bir mutasyon ger&#xE7;ekle&#x15F;irse, g&#xFC;&#xE7;lendirilmi&#x15F; DNA&#x27;n&#x131;n &#xE7;o&#x11F;u bu mutasyonu ta&#x15F;&#x131;yacakt&#x131;r.

Overview

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

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pcr cycle for dna amplification

PCR is a laboratory method for making many copies of a DNA segment. It can turn a small amount of DNA into millions or even billions of copies in just a few hours. The process uses a heat-resistant DNA polymerase inside a machine called a thermocycler.

PCR was developed by Kary Mullis in 1983, and he received the 1993 Nobel Prize in Chemistry for it. The method is fast, relatively inexpensive, and precise. It has become useful for molecular cloning, gene mutagenesis, pathogen detection, gene expression analysis, DNA quantitation, sequencing, and genetic disease diagnosis.

PCR copies DNA in a way that resembles natural DNA replication. The reaction mixture includes template DNA, short DNA primers, free DNA building blocks called deoxynucleotide triphosphates, or dNTPs, and a special DNA polymerase. The most common enzyme is Taq polymerase, which comes from the bacterium Thermus aquaticus.

DNA polymerase cannot start a DNA strand from scratch. It needs primers, which are short DNA molecules that bind to the template by complementary base pairing. The primers provide a free 3' hydroxyl group so the polymerase can add new dNTPs. The four dNTPs used in PCR are dATP, dCTP, dGTP, and dTTP.

Each PCR cycle has three steps: denaturation, annealing, and DNA synthesis. During denaturation, the mixture is heated to about 90°C to 100°C, which separates the double helix into two strands. During annealing, the mixture cools to about 50°C to 65°C so the primers can bind to their matching sequences.

During DNA synthesis, also called primer extension, the mixture is heated again to about 60°C to 75°C. At this stage, DNA polymerase extends the primers by adding dNTPs that match the template strand. A typical PCR uses 20 to 40 cycles in the thermocycler, and the DNA amount doubles each cycle, so amplification is exponential.

PCR has limits that scientists must watch for. To copy a specific genome region, the target sequence must be known well enough to design the right primers. Primers can also bind to similar non-target DNA, which can create unwanted products, but reaction conditions can be adjusted to reduce this problem.

Because PCR is very sensitive, contamination can cause misleading results even when only trace amounts of outside DNA are present. The DNA polymerases used in PCR can also make mistakes. If a mutation happens early in the cycle, most of the amplified DNA will carry that mutation.

PCR Cycle for DNA Amplification

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key terms and concepts

Learn how PCR amplifies DNA through denaturation, annealing, and extension, plus the role of primers, Taq polymerase, and common PCR limits.

PCR Cycle for DNA Amplification | JoVE K12

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PCR Cycle for DNA Amplification

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