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» ポリメラーゼ連鎖反応(PCR)

ポリメラーゼ連鎖反応(PCR)

1983
  • Kary Mullis
分子生物学実験室でDNAを増幅するサーマルサイクラー。.

(画像はイメージです)

The Polymerase Chain Reaction (PCR) is a laboratory technique used to amplify a specific segment of DNA, generating millions to billions of copies from a small initial sample. The 方法 relies on thermal cycling, consisting of repeated cycles of heating and cooling for DNA melting, primer annealing, and enzymatic replication of the DNA using a thermostable DNA polymerase, resulting in exponential amplification.

The Polymerase Chain Reaction (PCR) is a revolutionary in vitro method for exponentially amplifying a specific target DNA sequence. Its power lies in its ability to generate billions of copies from a minute starting quantity of DNA, making it possible to analyze DNA from samples as small as a single cell. The process is driven by a series of temperature changes, or thermal cycles, managed by a machine called a thermal cycler. Each cycle consists of three core steps. First, Denaturation: the reaction mixture is heated to 94–98 °C for a short period, causing the double-stranded DNA template to separate into single strands. Second, Annealing: the temperature is lowered to 50–65 °C, allowing short, single-stranded DNA primers to bind (anneal) to their complementary sequences on the now single-stranded template DNA. These primers flank the target region to be amplified. Third, Extension: the temperature is raised to 72 °C, the optimal temperature for the key enzyme, a thermostable DNA polymerase, to work. The most commonly used is Taq polymerase, isolated from the thermophilic bacterium *Thermus aquaticus*. The polymerase binds to the primer-template complex and begins synthesizing a new complementary DNA strand, extending from the primer. This three-step cycle is repeated 20-40 times. With each cycle, the number of copies of the target DNA sequence doubles, leading to an exponential amplification, mathematically described as [latex]2^n[/latex], where n is the number of cycles. The result is a solution containing a vast quantity of the specific DNA fragment, which can then be easily visualized by gel electrophoresis or used in subsequent molecular biology applications like sequencing or cloning.

UNESCO Nomenclature: 2406
分子生物学

タイプ

化学プロセス

混乱

革命的

使用法

広く普及している

前駆物質

  • discovery of DNA structure
  • understanding of DNA replication principles
  • isolation of DNA polymerase enzymes
  • discovery of thermostable organisms and their enzymes (e.g., Taq polymerase)
  • development of oligonucleotide synthesis for primers

アプリケーション

  • DNA fingerprinting for forensics
  • medical diagnostics for infectious diseases (e.g., COVID-19)
  • genetic testing for hereditary diseases
  • cloning genes
  • sequencing genomes
  • phylogenetic analysis

特許:

  • US4683195
  • US4683202
  • US4965188

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Related to: PCR, polymerase chain reaction, DNA amplification, kary mullis, taq polymerase, thermal cycling, forensics, diagnostics, molecular cloning, genetic testing.

歴史的背景

ポリメラーゼ連鎖反応(PCR)

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