Polymerase chain reaction (PCR) is a technique that has revolutionized the world of molecular biology and beyond. In this article, we will discuss a brief history of PCR and its principles, highlighting the different types of PCR and the specific purposes to which they are being applied.

pcrprinciples and history

In 1983, American biochemistKary Mulliswas driving home late at night when a flash of inspiration struck him. He wrote on the back of a receipt the idea that would eventually grant him the诺贝尔奖for Chemistry in 1993. The concept was straightforward: reproducing in a laboratory tube the DNA replication process that takes place in cells. The outcome is the same: the generation of new complementary DNA (cDNA) strands based upon the existing ones.

Mullis used the basis of桑格的DNA测序as a starting point for his new technique. He realized that the repeated use of DNA polymerase triggered a chain reaction resulting in a specific DNA segment's amplification.

1976年的发现，他的想法基础是一项可热稳定DNA聚合酶的发现，即Taq，，，，isolated from the bacteriumThermus aquaticus在温泉中发现。¹TaqDNA聚合酶的温度最佳72°C和幸存下来长期暴露于高达96的温度°C, meaning that it can tolerate several denaturation cycles.

Before the discovery ofTaqpolymerase, molecular biologists were already trying to optimize cyclic DNA amplification protocols, but they needed to add fresh polymerase at each cycle because the enzyme could not withstand the high temperatures needed for DNA denaturation. Having a thermostable enzyme meant that they could repeat the amplification process many times over without the need for fresh polymerase at every cycle, making the whole process scalable, more efficient and less time-consuming.

使用该聚合酶链反应（PCR）的第一个描述Taqpolymerase was published in Science in 1985.²

In 1993, the first FDA-approved PCR kit came to market. Since then, PCR has been steadily and systematically improved. It has become agame-changer从法医证据分析和诊断到疾病监测和基因工程，从事的一切。毫无疑问，这是20个最重要的科学进步之一^Th世纪。

标准PCR实验概述

PCR用于从起始材料的复杂混合物中扩增特定的DNA片段，称为template DNA。The sample preparation and purification protocols depend on the starting material, including the sample matrix and accessibility of target DNA. Often, minimal DNA purification is needed. However, PCR does require knowledge of the DNA sequence information that flanks the DNA fragment to be amplified (calledtarget DNA).

From a practical point of view, apcrexperimentis relatively straightforward and can be completed in a few hours. In general, a PCR reaction needs five key reagents:

DNA要扩增：also called PCR template or template DNA.This DNA can be of any source,such as genomic DNA (gDNA), cDNA, and plasmid DNA.

DNA聚合酶：所有PCR反应都需要一个可以在高温下起作用的DNA聚合酶。Taq聚合酶是一种常用的聚合酶，哪个行在70°C下以60碱基/秒的速率掺入核苷酸，并可以扩增高达5 kb的模板，使其适用于无需特殊要求的标准PCR。新一代的聚合酶正在设计以改善反应性能。例如，有些设计仅在高温下激活，以减少反应开始时的非特异性扩增。例如，重要的是，重要的是，重要的是，当放大序列与模板序列完全匹配（例如克隆期间）至关重要时。

底漆：DNA聚合酶需要短序列的核苷酸，以指示它们需要在何处进行扩增。在PCR中，这些序列称为底漆，是单链DNA的短片（约15-30个碱基）。在设计PCR实验时，研究人员确定了DNA的区域要放大并设计了一对引物，一个在正向链上，一个在反向上，特别是侧面靶区域。底漆设计是PCR实验的关键组成部分，应仔细进行。必须选择底漆序列以靶向感兴趣的唯一DNA，以避免结合与类似序列的可能性。它们应该具有相似的熔融温度，因为两个链的退火步骤同时发生。与腺嘌呤（a）或胸腺素（T）相比，底漆的熔化温度可能会受到鸟嘌呤（G）或胞嘧啶（C）的百分比的影响，而GC含量较高会增加熔融温度。调整引物长度可以帮助匹配底漆对。避免趋于形成二级结构或底漆二聚体的序列也很重要，因为这将降低PCR效率。许多免费的在线工具可用来帮助启动设计。

脱氧核苷酸三磷酸（DNTPS）：这些用作合成DNA的新链的构建块，并包括四个基本的DNA核苷酸（DATP，DCTP，DGTP和DTTP）。通常将DNTP添加到pCR反应中，以等摩尔量以进行最佳基础掺入。

pcrbuffer:PCR缓冲液确保在整个PCR反应过程中保持最佳条件。PCR缓冲液的主要成分包括氯化镁（MGCL₂), tris-HCl and potassium chloride (KCl). MgCl₂serves as a cofactor for the DNA polymerase, while tris-HCl and KCl maintain a stable pH during the reaction.

PCR反应是在单个管中通过混合上述试剂并将管放入热循环仪中的。

PCR扩增由三个定义的时间和所谓的温度组成steps：denaturation，，，，退火，，，，and延期(Figure 1).

图1： 单个PCR周期的步骤。

这些步骤中的每一个都称为cycles，重复30-40次，，，，，doubling the amount of DNA at each cycle and obtaining amplification (Figure 2).

图2： DNA分子扩增的不同阶段和周期通过PCR。

让我们仔细看看每个步骤。

1。Denaturation

The first step of PCR, called denaturation, heats the template DNA up to 95°C for a few seconds, separating the two DNA strands as the hydrogen bonds between them are rapidly broken.

2。Annealing

The reaction mixture is then cooled for 30 seconds to 1 minute. Annealing temperatures are usually 50 - 65°c然而，确切的最佳温度取决于引物的长度和序列。必须使用每组新的底漆仔细优化它。

两个DNA链couldrejoin at this temperature, but most do not because the mixture contains a large excess of primers that bind, or anneal, to the template DNA at specific, complementary positions. Once the annealing step is completed, hydrogen bonds will form between the template DNA and the primers. At this point, the polymerase is ready to extend the DNA sequence.

3。延期

The temperature is then raised to the ideal working temperature for the DNA polymerase present in the mixture, typically around 72°C, 74°C in the case ofTaq。

DNA聚合酶连接到每个底漆的一端，并合成与模板DNA互补的新的DNA链。现在，我们有四链DNA，而不是两个开始的DNA。

温度升至94°C和双链DNA分子 - “原始”分子和新合成的分子 - 再次变成单链。这开始了通道延伸的第二个周期。在第二个周期结束时，有八个单链DNA分子。通过重复30次循环，开始时存在的双链DNA分子被转换为超过1.3亿新的双链分子，每个分子都是由两个引物的退火位点描绘的起始分子区域的副本。

To determine if amplification has been successful, PCR products may be visualized using gel electrophoresis, indicating amplicon presence/absence, size and approximate abundance. Depending on the application and the research question, this may be the endpoint of an experiment, for example, if determining whether a gene is present or not. Otherwise, the PCR product may just be the starting point for more complex downstream investigations such as sequencing and cloning.

pcrvariations

由于它们的多功能性，PCR技术在近年来不断发展，导致开发或几种不同类型的PCR技术。

一些最广泛使用的是：

Quantitative real-time PCR (qPCR)

One of the most useful developments has been quantitative real-time PCR or qPCR. As the name suggests, qPCR is a quantitative technique that allows real-time monitoring of the amplification process and detection of PCR products as they are made.²It can be used to determine the starting concentration of the target DNA, negating the need for gel electrophoresis in many cases.This is achieved thanks to the inclusion of non-specific fluorescent intercalating dyes, such asSYBR®绿色，，，，That fluoresce when bound to double-stranded DNA, or DNA oligonucleotide sequence-specific fluorescent probes, such as水解（Taqman）探针andmolecular beacons。探针特异性与扩增子内的DNA靶序列结合，并使用förster共振能量转移（FRET）的原理通过一端的荧光分子的偶联来产生荧光，另一端是淬火器。对于荧光染料和探针，随着目标DNA的副本数量的增加，荧光水平会成比例地增加，从而可以参考包含已知拷贝数的标准来实时对扩增进行实时定量（图3）。

qpcruses specialized thermal cyclers equipped with fluorescent detection systems that monitor the fluorescent signal as the amplification occurs.

Figure 3:示例QPCR扩增图和标准曲线用于启用未知样品中拷贝数的量化。

Reverse transcription-PCR (RT-PCR)

逆转录（RT）-PCR和RT -QPCR是两个常用的PCR变体，可在临床和研究环境中对病毒RNA进行基因转录分析和定量。

RTis the process of making cDNA from single-stranded template RNA³and is consequently also called first-strand cDNA synthesis. The first step of RT-PCR is to synthesize a DNA/RNA hybrid between the RNA template and a DNA oligonucleotide primer. The reverse transcriptase enzyme that catalyzes this reaction has RNase activity that then degrades the RNA portion of the hybrid. Subsequently, a single-stranded DNA molecule is synthesized by the DNA polymerase activity of the reverse transcriptase. High purity and quality starting RNA are essential for a successful RT-PCR.

RT-pcrcan be performed following two approaches: one-step RT-PCR and two-step RT-PCR. In the first case, the RT reaction and the PCR reaction occur in the same tube, while in the two-step RT-PCR, the two reactions are separate and performed sequentially.

Reverse transcription-quantitative PCR (RT-qPCR)

The reverse transcription described above often serves as the first step in qPCR too, quantifying RNA in biological samples (either RNA transcripts or derived from viral RNA genomes).

与RT-PCR一样，RT-QPCR量化RNA的方法有两种方法：一步RT-QPCR和两步RT-QPCR。在这两种情况下，首先将RNA反向转录到cDNA中，该cDNA被用作qPCR扩增的模板。在两步方法中，逆转录和QPCR扩增依次作为两个独立的实验出现。在一步方法中，RT和QPCR在同一管中进行。

Digital PCR (dPCR) and digital droplet PCR (ddPCR)

数字PCR（DPCR）是原始PCR协议的另一种改编。⁴Like qPCR, dPCR technology uses DNA polymerase to amplify target DNA from a complex sample using a primer set and probes. The main difference, though, lies in the partitioning of the PCR reactions and data acquisition at the end.

DPCR和DDPCRare based on the concept of limiting dilutions. The PCR reaction is split into large numbers of nanoliter-sized sub-reactions (partitions). The PCR amplification is carried out within each droplet. Following PCR, each droplet is analyzed with Poisson statistics to determine the percentage of PCR-positive droplets in the original sample. Some partitions may contain one or more copies of the target, while others may contain no target sequences. Therefore, partitions classify either as positive (target detected) or negative (target not detected), providing the basis for a digital output format.

DDPCR是最近在2011年获得的技术。⁵ddPCR utilizes a water-oil emulsion to form the partitions that separate the template DNA molecules. The droplets essentially serve as individual test tubes in which the PCR reaction takes place.

Microfluidic PCR

The recent development of microfluidic handling systems with microchannels and microchambers has paved the way for a range of practical applications, including the amplification of DNA via PCR on microfluidic chips.

PCR以芯片的益处，敏感性和低消耗量的优势获得了芯片益处。这些功能使微流体PCR特别吸引了用于护理点测试的诊断应用。从实际的角度来看，样品流过微流体通道，反复通过三个温度区域，反映了PCR的不同步骤。10μL样品仅需90秒即可执行20个PCR周期。⁶The subsequent analysis can then be easily carried out off-chip.

PCR故障排除

The different PCR approaches all have advantages and disadvantages that impact the applications to which they are suited⁷。These are summarized in Table 1.

Approach	Advantages	Limitations
pcr	·Easiest PCR to perform ·设备和试剂的低成本 ·Several downstream applications (e.g., cloning)	·结果仅是定性的 ·Requires post-amplification analyses that increase time and risk of error ·可能需要通过测序确认产品
qpcr	·Produces quantitative results ·Probe use can ensure high specificity ·高分析灵敏度 ·较低的周转时间 ·消除放大后分析的要求	·Requires more expensive reagents and equipment ·底漆和探针选择的灵活性较小 ·Less amenable to other downstream product confirmation analyses (such as sequencing) due to the small length of the amplicon ·不适合某些下游应用，例如克隆
RT-pcrand RT-qPCR	·Can be used with all RNA types	·RNA is prone to degradation ·The RT step may increase the time and potential for contamination
DPCR和DDPCR	·快速地 ·无DNA纯化步骤 ·Provides absolute quantification ·在有限的临床样品中检测目标的灵敏度提高 ·Highly scalable	·昂贵 ·Based on several statistical assumptions
Microfluidic PCR	·Accelerated PCR process ·Reduced reagent consumption ·Can be adapted for high throughput ·Portable device for point-of-care applications ·允许单细胞分析	·仍然非常新的技术 ·需要大量样品准备以清除碎屑和不需要的化合物 ·Restricted choice of materials for the microfluidic device due to high temperatures

Table 1:不同PCR方法的关键优势和缺点。

pcroutput applications

pcrhas become an indispensable tool in modern molecular biology and has completely transformed scientific research. The technique has also opened up the investigation of cellular and molecular processes to those outside the field of molecular biology and consequently also finds utility by scientists in many disciplines.

Whilst PCR is itself a powerful standalone technique, it has also been incorporated into wider techniques, such as cloning and sequencing, as one small but important part of these workflows.

PCR的研究应用包括：

Gene transcription-pcrcan examine variations in gene transcription among cell types, tissues and organisms at a specific time point. In this process, RNA is isolated from samples of interest, and reverse-transcribed into cDNA. The original levels of RNA for a specific gene can then be quantified from the amount of cDNA amplified in PCR.

Genotyping-PCR可以检测特定细胞或生物的等位基因的序列变化。一个常见的例子是转基因生物的基因分型，例如敲除和敲除小鼠。在此应用中，底漆旨在扩增转基因部分（在转基因动物中）或突变（在突变动物中）。

克隆和诱变-PCR克隆是一种广泛使用的技术，其中将通过PCR扩增的双链DNA片段插入矢量（例如GDNA，cDNA，质粒DNA）。例如，这可以从中删除或插入遗传物质的细菌菌株。定点诱变也可用于通过克隆引入点突变。这通常采用一种称为recombinant PCR，，，，in which overlapping primers are specifically designed to incorporate base substitutions (Figure 4). This technique can also be used to create novel gene fusions.

Figure 4:Diagram depicting an example of recombinant PCR.

Sequencing - PCR可用于富集模板DNA进行测序。建议制备测序模板的PCR类型称为高保真PCR，能够维持DNA序列精度。在Sanger测序中，然后将PCR放大片段纯化并以测序反应运行。在下一代测序（NGS）中，在库制备阶段使用PCR，其中DNA样品富含PCR以增加起始数量并用测序适配器标记以允许多路复用。桥PCR也是第二代NGS测序过程的重要组成部分。

Both as an independent technique and as a workhorse within other methods, PCR has transformed a range of disciplines. These include:

Genetic research-PCR在全球大多数实验室中使用。最常见的应用之一是基因转录分析⁹旨在评估特定基因转录本的存在或丰度。它是通过克隆操纵生物（动物，植物和微生物）的遗传序列的强大技术。这使得在遗传标记中插入，删除或突变的基因或突变基因或突变的基因或部分可以改变表型，阐明基因功能并开发疫苗以命名为几种。在基因分型中，PCR可用于检测特定细胞或生物体中等位基因的序列变化。它的使用也不限于人类。Genotyping plants in agricultureassists plant breeders in selecting, refining, and improving their breeding stock. PCR is also the first step to enrich sequencing samples, as discussed above. For example, most mapping techniques in the人类基因组项目（HGP）relied on PCR.

Medicine and biomedical research-PCR用于许多医学应用中，从诊断与疾病相关的基因突变的诊断到鉴定感染剂。医学领域中PCR使用的另一个很好的例子是产前基因检测。Prenatal genetic testing通过PCR可以鉴定胎儿中的染色体异常和遗传突变，从而为父母提供有关婴儿是否患有某些遗传疾病的重要信息。PCR也可以用作植入前遗传诊断工具，用于筛选胚胎的胚胎体外fertilization (IVF) procedures.

Forensic science-Our unique genetic fingerprints mean that PCR can be instrumental in both paternity testing and法医调查查明样品的来源。例如，可以将从犯罪现场分离的小型DNA样品与DNA数据库或嫌疑人的DNA进行比较。这些程序确实改变了警察调查的方式。真实性测试还利用PCR遗传标记来确定衍生出肉类的物种。分子考古学也利用PCR从考古遗骸中扩增DNA。

环境微生物学和食品安全- PCR检测病原体，不仅在患者的样本中，而且在食物或水等基质中，对于诊断和预防传染病至关重要。

pcris the benchmark technology for detecting nucleic acids in every area, from biomedical research to forensic applications. Kary Mullis's idea, written on the back of a receipt on the side of the road, turned out to be a revolutionary one.

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