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  • 聚合酶链反应 - 高级

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    How do you make a scientific process easier?
    ::你如何使科学过程更容易?

    Use a machine. The polymerase chain reaction has revolutionized biological and biomedical research and applications. Luckily many machines have been developed that allow this process to be performed rapidly and with precision.
    ::使用机器。聚合酶链反应使生物和生物医学研究和应用发生了革命性的变化。幸运的是,已经开发了许多机器,使得这一过程能够迅速和精确地进行。

    The Polymerase Chain Reaction
    ::聚合酶链反应

    The Polymerase Chain Reaction (PCR) is used to amplify specific regions of a strand millions of times. A region may be a number of loci , a single gene , a part of a gene, or a non-coding sequence. This technique produces a useful quantity of DNA for analysis, be it medical, forensic or some other form of analysis. Amplification of DNA from as little as a single is possible. Whole genome amplification is also possible.
    ::聚合酶链反应(PCR)用于扩大几百万倍的特定区域,一个区域可以是若干地缘、单一基因、基因的一部分或非编码序列。这一技术产生有用的DNA,供分析使用,无论是医学、法医学还是其他形式的分析。从一个小到一个小可能放大DNA。整个基因组的扩大也是可能的。

    PCR utilizes a heat stable DNA polymerase, Taq polymerase (or Taq DNA polymerase), named after the thermophilic Thermus aquaticus, from which it was originally isolated. T. aquaticus is a bacterium that lives in hot springs and hydrothermal vents, and Taq polymerase is able to withstand the high temperatures required to denature DNA during PCR (discussed below). Taq polymerase’s optimum temperature for activity is between 75°C and 80°C. Recently other DNA polymerases have also been used for PCR.
    ::T. 水生生物是一种细菌,生活在温泉和热液喷口中,Taq聚合酶能够承受在PCR(下文讨论)期间使DNA变硬所需的高温。 Taq聚合酶的活动最佳温度在75°C至80°C之间。 最近,其他DNA聚合酶也被用于PCR。

    A basic PCR involves a series of repeating cycles involving three main steps (see Figure ):
    ::基本的PCR涉及一系列涉及三个主要步骤的重复周期(见图):

    1. Denaturation of the double stranded DNA.
      ::双层悬浮DNA的衰减
    2. Annealing of specific oligonucleotide primers .
      ::特定核糖核酸核糖核酸基质的安纳林
    3. Extension of the primers to amplify the region of DNA of interest.
      ::扩大入门器以扩大感兴趣的DNA区域。

    These steps will be discussed in additional detail below.
    ::下文将进一步详细讨论这些步骤。

    The oligonucleotide primers are single stranded pieces of DNA that correspond to the 5’ and 3’ ends of the DNA region to be amplified. These primers will anneal to the corresponding segment of denatured DNA. Taq Polymerase, in the presence of free deoxynucleotide triphosphates (dNTPs), will extend the primers to create double stranded DNA. After many cycles of denaturation, annealing and extension, the region between the two primers will be greatly amplified.
    ::核糖核酸核酸二酸基质是单一的DNA碎片,它们与需要放大的DNA区域5和3端相对应。 这些基质将与相应的减肥DNA相匹配。 Taq 聚氨酯酶在自由脱氧核糖核酸三磷酸酯(dNTPs)的出现下,将扩大基质以生成双倍的被搁置的DNA。 经过许多衰减、消化和扩展周期后,两个基质之间的区域将大大扩大。

    The PCR is commonly carried out in a thermal cycler, a machine that automatically allows heating and cooling of the reactions to control the temperature required at each reaction step (see below). The PCR usually consists of a series of about 30 to 35 cycles. Most commonly, PCR is carried out in three repeating steps, with some modifications for the first and last step.
    ::PCR通常在热循环器中进行,这种机器自动允许对反应进行供暖和冷却,以控制每一反应步骤所需的温度(见下文),PCR通常由大约30至35个周期的系列组成,最常见的是,PCR分三个重复步骤进行,对第一步和最后一步作了一些修改。

    PCR is usually performed in small tubes or wells in a tray, each often beginning with the complete genome of the being studied. As only a specific sequence from that genome is of interest, the sequence specific primers are targeted to that sequence. PCR is done with all the building blocks necessary to create DNA: template DNA, primers, dNTPs, and a DNA polymerase.
    ::PCR通常在小管或水井中用托盘进行,每个托盘通常从所研究的整个基因组开始。由于只对基因组的某个具体序列感兴趣,因此具体序列的开源器是针对该序列的。PCR使用创建DNA所需的所有构件:模板DNA、开源器、dNTP和DNA聚合酶。

    The three basic steps of PCR ( Figure ) are:
    ::PCR的三个基本步骤(图)是:

    • Denaturation step : This step is the first regular cycling event and consists of heating the reaction to 94 - 98°C for 30 to 60 seconds. It disrupts the hydrogen bonds between complementary bases of the DNA strands, yielding single strands of DNA.
      ::拒绝饱和步骤:这是第一次定期自行车事件,包括将反应加热至94-98°C30至60秒,破坏DNA线互补基础之间的氢联结,产生单一的DNA线。
    • Annealing step : The reaction temperature is lowered to 50-65°C for 30 to 60 seconds, allowing annealing of the primers to the single-stranded DNA template. Stable hydrogen bonds form between the DNA strand (the template) and the primers when the primer sequence very closely matches the complementary template sequence. Primers are usually 17 - 22 nucleotides long and are carefully designed to bind to only one site in the genome. The polymerase binds to the primer-template hybrid and begins DNA synthesis.
      ::Annaling 步骤:反应温度降低到50-65°C,30至60秒,允许单弦DNA模版的基质喷射。当基质序列与补充性模板序列非常接近时,DNA线(模板)和基质之间形成稳定的氢联结。初级元素通常长17至22核糖核酸,经过仔细设计,只粘合到基因组的一个地点。聚合酶与基质板混合并开始DNA合成。
    • Extension step : A temperature of around 72°C is used for this step, which is close to the optimum temperature of Taq polymerase. At this step the Taq polymerase extends the primer by adding dNTPs, using one DNA strand as a template to create a the other (new) DNA strand. The extension time depends on the length of the DNA fragment to be amplified. As a standard, at its optimum temperature, the DNA polymerase will polymerize a thousand bases in one minute.
      ::扩展步骤:该步骤使用的温度约为72°C左右,接近塔克聚合酶的最佳温度。在这一步骤,塔克聚合酶通过添加dNTP来扩展开源器,使用一个DNA线作为模板来创建另一个(新的)DNA线。延长时间取决于需要放大的DNA碎片的长度。作为一个标准,在最佳温度下,DNA聚合酶将在一分钟内聚合一千个基数。

    lesson content

    The Polymerase Chain Reaction. The polymerase chain reaction involves three steps. High temperatures are needed for the process to work. The enzyme Taq polymerase is used in step 3 because it can withstand high temperatures.
    ::聚合酶链反应:聚合酶链反应涉及三个步骤。该过程工作需要高温。酶Taq聚合酶用于第3步,因为它能承受高温。

    Utilizing PCR, DNA can be amplified millions of times to generate quantities of DNA that can be used for a number of purposes. These include the use of DNA for prenatal or genetic testing, such as testing for a specific . PCR has revolutionized the fields of , human genetics , and a number of other sciences. Many of the applications will be discussed in the additional concepts. PCR was developed in 1983 by Kary Mullis. Due to the importance of this process and the significance it has had on scientific research, Dr. Mullis was awarded the in Chemistry in 1993, just 10 years after his discovery. 
    ::利用PCR,DNA可以被放大数百万次,以产生可用于若干目的的DNA数量,其中包括将DNA用于产前或基因测试,如具体测试。PCR已经使人体遗传学领域和其他一些科学领域发生革命性变化。许多应用将在其他概念中讨论。PCR是Kary Mullis1983年开发的。由于这一过程的重要性及其对科学研究的重要性,Mullis博士在发现后10年才于1993年获得化学奖。

    To say that PCR, molecular and the has revolutionized biology and medicine would be an understatement. These efforts have led to numerous accolades, including Nobel prizes, and more may follow. Some of the ways that these discoveries have shaped our lives are the focus of the Concept Biotechnology (Advanced) concepts.
    ::说PCR、分子和医学革命化的生物学和医学是低估了这一点。 这些努力带来了无数的赞誉,包括诺贝尔奖,并可能随之而来。 这些发现塑造了我们生活的一些方式是“生物技术概念(先进)”概念的焦点。

    Summary
    ::摘要

    • The Polymerase Chain Reaction (PCR) is used to amplify millions of times specific regions of a DNA strand. This can be a number of loci, a single gene, a part of a gene, or a non-coding sequence.
      ::聚合酶链反应(PCR)用于放大DNA链的成百上千万倍的具体区域。 这可能是一些地缘、单一基因、基因的一部分或非编码序列。
    • PCR usually involves the following steps:
      • Denaturation
        ::失饱和度
      • Annealing
        ::安妮
      • Extension
        ::延长

      ::PCR通常涉及以下步骤:

    Review
    ::回顾

    1. What is PCR?
      ::什么是PCR? (PCR)? (PCR)? (PCR) (PCR)? (PCR)
    2. What allows PCR to be done at high temperatures?
      ::是什么允许在高温下进行PCR?
    3. Describe the PCR process.
      ::描述PCR过程。
    4. Illustrate the PCR process.
      ::印证PCR程序