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

The goal of PCR, Polymerase Chain Reaction is to amplify a genetic sequence. In this example, a gene of interest will be amplified from purified DNA. In order to perform the amplification, a polymerase is required to synthesize the new DNA.

A primer, a short piece of single stranded DNA that shares homology to the gene of interest is also needed. Finally, single nucleotides called Deoxynucleoside triphosphates or dNTPs are used to make the new strand. The first step in PCR is to heat the mixture which denatures the DNA.

Next, the reaction is cooled and the primers will anneal to their homologous region. Once bound the reaction is heated to an optimal temperature for the polymerase. The polymerase then recognizes the primer DNA complex and begins synthesis of the new strand using the dNTPs in the solution.

The reaction is then heated and proceeds as described across additional cycles. Following the third cycle, there are eight total copies of the gene. After the fourth cycle, 16 copies.

And fifth cycle, 32 copies. The number of cycles will continue to grow exponentially. After 30 cycles, there are just over one billion copies.

Garibyan, Lilit i Nidhi Avashia. "Proste techniki badawcze: reakcja łańcuchowa polimerazy (PCR)". The Journal of Investigative Dermatology 133, nr 3 (marzec 2013): e6. [<a href="https://doi.org/10.1038/jid.2013.1" target="__blank">Źródło</a>]

Módl się, Leslie A. "Rewolucja biotechnologiczna: PCR i zastosowanie odwrotnej transkryptazy do klonowania genów wyrażonych przez geny." Edukacja przyrodnicza 1, nr 1 (2008): 94. [<a href="https://www.nature.com/scitable/topicpage/the-biotechnology-revolution-pcr-and-the-use-553" target="__blank">Źródło</a>]

<h4>Przegl&#x105;d</h4>

Reakcja &#x142;a&#x144;cuchowa polimerazy, czyli PCR, jest szeroko stosowan&#x105; technik&#x105; kopiowania segment&#xF3;w DNA. Ze wzgl&#x119;du na wyk&#x142;adnicz&#x105; amplifikacj&#x119;, PCR mo&#x17C;e wytworzy&#x107; miliony lub miliardy kopii DNA w ci&#x105;gu zaledwie kilku godzin. W reakcji PCR odporny na ciep&#x142;o enzym polimeraza DNA amplifikuje oryginalne DNA poprzez seri&#x119; zmian temperatury w zautomatyzowanej maszynie zwanej termocyklerem.

<h4>PCR to wszechstronna metoda, kt&#xF3;ra zrewolucjonizowa&#x142;a biologi&#x119; molekularn&#x105;</h4>

Kary Mullis opracowa&#x142; metod&#x119; PCR w 1983 r., za co w 1993 r. otrzyma&#x142; Nagrod&#x119; Nobla w dziedzinie chemii. B&#x119;d&#x105;c stosunkowo szybk&#x105;, niedrog&#x105; i precyzyjn&#x105; metod&#x105; kopiowania sekwencji DNA, PCR sta&#x142;a si&#x119; nieocenionym narz&#x119;dziem do wielu zastosowa&#x144;, w tym do klonowania molekularnego, mutagenezy gen&#xF3;w, wykrywania patogen&#xF3;w, analizy ekspresji gen&#xF3;w, oznaczania ilo&#x15B;ciowego i sekwencjonowania DNA oraz diagnostyki chor&#xF3;b genetycznych.

<h4>Cykl reakcji PCR</h4>

PCR na&#x15B;laduje naturalny proces replikacji DNA zachodz&#x105;cy w kom&#xF3;rkach. Mieszanina reakcyjna zawiera matrycow&#x105; sekwencj&#x119; DNA do skopiowania, par&#x119; kr&#xF3;tkich cz&#x105;steczek DNA zwanych starterami, wolne elementy DNA zwane trifosforanami deoksynukleotyd&#xF3;w (dNTP) i wyspecjalizowany enzym polimeraz&#x119; DNA.

PCR obejmuje szereg etap&#xF3;w prowadzonych w wysokich temperaturach, wymagaj&#x105;cych enzymu polimerazy DNA dzia&#x142;aj&#x105;cego w takich temperaturach. Najcz&#x119;&#x15B;ciej stosowan&#x105; polimeraz&#x105; DNA jest polimeraza Taq, nazwana na cze&#x15B;&#x107; bakterii Thermus aquaticus, z kt&#xF3;rej pocz&#x105;tkowo wyizolowano polimeraz&#x119;. Polimeraza DNA nie jest w stanie zsyntetyzowa&#x107; cz&#x105;steczki DNA od podstaw ani de novo. Zamiast tego polimeraza DNA dodaje kr&#xF3;tkie cz&#x105;steczki DNA, zwane starterami, kt&#xF3;re wi&#x105;&#x17C;&#x105; si&#x119; z matryc&#x105; DNA poprzez komplementarne parowanie zasad. Podk&#x142;ady zapewniaj&#x105; wolne 3&#x2019; grup&#x119; hydroksylow&#x105;, do kt&#xF3;rej polimeraza DNA mo&#x17C;e przy&#x142;&#x105;cza&#x107; nowe dNTP. W PCR wyst&#x119;puj&#x105; cztery typy dNTP, po jednym na ka&#x17C;dy nukleotyd w cz&#x105;steczce DNA: dATP, dCTP, dGTP i dTTP.

Ka&#x17C;dy cykl PCR sk&#x142;ada si&#x119; z trzech etap&#xF3;w: denaturacji, przy&#x142;&#x105;czania i syntezy DNA.

<ol>
	<li>Denaturacja. Cykl PCR inicjowany jest poprzez podgrzanie mieszaniny reakcyjnej do wysokiej temperatury, co powoduje rozdzielenie podw&#xF3;jnej helisy DNA na dwie nici. To &#x201E;topnienie&#x201D; proces zwykle zachodzi w temperaturze 90&#xB0;C&#xAD;&#xAD;&#x2013;100&#xB0;C.</li>
	<li>Wy&#x17C;arzanie. Mieszanin&#x119; reakcyjn&#x105; szybko sch&#x142;adza si&#x119; (zwykle do 50&#xB0;C&#x2013;65&#xB0;C)), umo&#x17C;liwiaj&#x105;c zwi&#x105;zanie dw&#xF3;ch starter&#xF3;w z ich komplementarnymi sekwencjami na niciach matrycowego DNA.</li>
	<li>Synteza DNA (przed&#x142;u&#x17C;anie startera). Mieszanin&#x119; reakcyjn&#x105; ponownie ogrzewa si&#x119;, tym razem do temperatury (zwykle 60&#xB0;C&#x2013;75&#xB0;C), kt&#xF3;ra pozwala polimerazie DNA na wyd&#x142;u&#x17C;enie starter&#xF3;w poprzez dodanie dNTP, kt&#xF3;re &#x142;&#x105;cz&#x105; si&#x119; z zasadami w nici matrycowej.</li>
</ol>

Typowy PCR obejmuje 20&#x2013;40 powtarzanych cykli tych trzech etap&#xF3;w zachodz&#x105;cych w termocyklerze. Poniewa&#x17C; liczba cz&#x105;steczek DNA podwaja si&#x119; w ka&#x17C;dym cyklu, DNA ulega wyk&#x142;adniczej amplifikacji.

<h4>Ograniczenia PCR</h4>

Je&#x15B;li naukowiec chce amplifikowa&#x107; okre&#x15B;lony odcinek genomu, musi zna&#x107; przynajmniej cz&#x119;&#x15B;&#x107; docelowej sekwencji DNA, aby zaprojektowa&#x107; odpowiednie startery. Innym potencjalnym problemem jest niespecyficzne przy&#x142;&#x105;czanie starter&#xF3;w do cz&#x119;&#x15B;ciowo podobnych sekwencji DNA, prowadz&#x105;ce do amplifikacji DNA innego ni&#x17C; docelowy. Problem ten mo&#x17C;na kontrolowa&#x107; optymalizuj&#x105;c warunki reakcji. PCR, b&#x119;d&#x105;c bardzo czu&#x142;&#x105; metod&#x105; wykrywania, jest r&#xF3;wnie&#x17C; podatna na zanieczyszczenia i nawet &#x15B;ladowe ilo&#x15B;ci zanieczyszczaj&#x105;cego DNA mog&#x105; powodowa&#x107; myl&#x105;ce wyniki. Polimerazy DNA stosowane w PCR mog&#x105; by&#x107; podatne na b&#x142;&#x119;dy. Je&#x15B;li mutacja nast&#x105;pi w ci&#x105;gu pierwszych kilku cykli, wi&#x119;kszo&#x15B;&#x107; amplifikowanego DNA b&#x119;dzie nosicielem mutacji.

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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