什么是聚合酶链反应（PCR）？

如果没有发现PCR，将无法实现生命科学的有效研究和进步。PCR has contributed greatlyto innovations in biopharmaceuticals, biotech crops, and工业生物技术. The development of hundreds of technologies in the aforementioned areas, such as genetic cloning, forensics, and genetic analysis, is the result of PCR.

他的tory of PCR

Theidea to reproduce or make copiesof a DNA segment (or DNA replication) in a laboratory tube (which normally takes place in a cell) was introduced by Kary Mullis in 1983. The invention eventually granted the scientist the 1993 Nobel prize in chemistry (shared with the Cetus Corporation) and brought on a revolution in生物科学研究领域.

尽管Taq DNA聚合酶当时已经发现了，在没有引入PCR的情况下，它没有充分认识到其对分子生物学的影响。PCR使科学家能够在仅使用自动化设备的几小时内放大任何DNA序列，而不是手动DNA扩增的方法。

What is PCR?

Polymerase chain reaction (PCR)is a rapid and inexpensivein vitro用于扩增DNA小段拷贝的技术。此过程可以快速有效地产生数十亿份。它有时也称为“分子复印”。

PCR has several applications in the area of medical and molecular biology. Some of them are given below:

• Selective DNA Isolation:PCR用于实验室to isolate specific DNA fragments by amplifying only a selected region of DNA. It can synthesize large amounts of pure DNA by using a small DNA sample. PCR also assists in generating hybridization probes required for southern or northern blots and DNA cloning.
• Medical and Diagnostic uses:PCR用于诊断疾病相关的遗传突变，鉴定传染性药物和产前遗传检测。它可以识别胎儿中的任何染色体异常或遗传突变，测试父母是任何疾病的遗传载体，并用作筛选胚胎胚胎胚胎的体外施肥（IVF）程序。

PCR assays are also used in the detection,诊断分析和病毒基因组的测序。例如，一个变体PCR (RT-PCR) was extensively usedto testCOVID-19 patients用SARS-COV-2病毒基因组感染。

传统上，这些测试依赖于存在血液中的抗体存在。RT-PCR（实时PCR）的发现促进了甚至在宿主中的最小病毒基因组的检测只是几个小时。

在RT-PCR中，酶逆转录酶使用RNA作为模板，并将RNA逆转录成DNA分子，然后使用PCR扩增。除了RT-PCR，其他一些类型的PCR包括：

• qPCR
• RT-qPCR
• DPCR.
• DDPCR.
• 热态启动聚合酶链反应
• High fidelity PCR
• Multiplex PCR
• 微流体PCR

• 遗传研究：PCR is used to study gene expression, evaluate the presence or absence of gene transcripts, manipulate genetic sequences, enrich sequencing samples, and genotyping. It was also extensively involved in the人类基因组项目(HGP) for most of the mapping procedures.
• DNA指纹识别：PCR is a benchmark technology in paternity testing and forensic investigations to find the source of DNA samples.
• 食品安全：PCR技术可用于检测食物和水中的病原体，并确保食物的安全性。

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聚合酶链式反应如何工作？

A thermal cycler (orthermocycler）用于运行PCR反应并扩增DNA序列in vitro. It mimics the process of DNA replication that occurs inside a cell. A tube containing a reaction mixture of PCR reagents is put into the machine, which changes the temperature to suit each stage of the process. An essential requirement before performing PCR is the knowledge of sequence information of the target DNA.

PCR反应在几个小时内完成并需要五个主要试剂：

1. DNA模板：The sample DNA that contains the target sequence that needs to be amplified. The source of a DNA template can either be genomic DNA (gDNA), cDNA, or plasmid DNA.
2. DNA Polymerase:PCR需要DNA polymerases这可以在高温下工作。Taq聚合酶是PCR中最常用的聚合酶。它是一种从细菌中分离的热稳定DNA聚合酶Thermus Aquaticus.found in hot springs.
3. 引物：引物是单链DNA的短片（约15-30个碱基对），称为寡核苷酸。它引发了新的DNA股线的形成。
4. Deoxynucleotide triphosphate (dNTPs):These are the four DNA nucleotides that serve as building blocks to synthesize new DNA strands.
5. PCR Buffer:The main components for a PCR buffer are magnesium chloride (MgCl2), tris-HCl, and potassium chloride (KCl). They are required to maintain the optimal conditions throughout the PCR reaction.

After an initial denaturation step, where the DNA is heated to 95 for a few minutes (up to 10 depending on complexity), the three steps below are repeated cyclically.

变性阶段

At this stage, the templated DNA is heated up to 95°C for up to a minute. It produces two single strands of DNA by breaking the hydrogen bonds between DNA helices. This process is called nucleic acid denaturation.

退火阶段

在该阶段，使反应混合物冷却30秒至1分钟。然后，引物对每个单链DNA结合或退火。退火温度落在50-65°C之间。精确的温度低于引物的熔融温度5°C，取决于PCR反应中使用的引物的长度和序列。

延伸/伸长阶段

在该阶段，温度升高至约72℃-74℃，为Taq聚合酶的最佳温度约1-2分钟。聚合酶结合引物的末端并使用DNTP开始DNA合成。这导致形成新的DNA链。

在一个循环结束时，PCR管中存在四种单链DNA分子。重复超过20-30次的循环导致数十亿拷贝的靶DNA序列。

One can then analyze the PCR product using agarose gel electrophoresis and check for the successful amplification of the target sequences.

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PCR的发现彻底改变了生物技术，制药，生物医学和其他生命科学领域的研究。今天，它是一种使用的常用工具research labs在检测病原体，法医研究，遗传异常和食品安全方面的应用。

热循环仪使用PCR原理在百万和数十亿份中扩增DNA样品。它调整每个扩增阶段的最佳温度，涉及DNA的变性，退火和伸长率。

高科技PCR Thermocycler.can cost as much as $25,000. Additionally, its high maintenance and cost of repairs make buying PCR equipment outright impractical for small biotech labs.

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