PCR中英对照自己总结背诵用.doc

Chapter 9Quantitative Analysis of Periodontal Pathogens by ELISA and Real-Time Polymerase Chain Reaction通过ELISA和实时-聚合酶联反应对牙周病病原菌进行定量分析Of the plethora of methodologies reported in the literature, the enzyme-linked immunosorbent assay (ELISA), which combines the specificity of antibody with the sensitivity of simple enzyme assays 结合了抗体的特异性和单独酶试验的敏感性and the polymerase chain reaction (PCR), has been widely utilized in both laboratory and clinical applications.Although conventional PCR does not allow quantitation of the target organism, real-time PCR (rtPCR) has the ability to detect amplicons扩增子as they accumulate in “real time” allowing subsequent quantitation.These methods enable the accurate quantitation of as few as 102 (using rtPCR) to 104 (using ELISA) periodontopathogens in dental plaque samples.Key words: Polymerase chain reaction (PCR), real-time PCR (rtPCR), enzyme-linked immunosorbent assay (ELISA), periodontitis, periodontal disease, oral periodontopathogens.What is PCR (polymerase chain reaction)?PCR (polymerase chain reaction) is a method to analyze a short sequence of DNA (or RNA) even in samples containing only minute quantities of DNA or RNA. PCR is used to reproduce (amplify) selected sections of DNA or RNA. Previously, amplification扩增of DNA involved cloning the segments of interest into vectors for expression in bacteria, and took weeks. But now, with PCR done in test tubes, it takes only a few hours. PCR is highly efficient in that untold numbers of copies can be made of the DNA. Moreover, PCR uses the same molecules that nature uses for copying DNA:Definition:Polymerase chain reaction (PCR) is a method of detecting specific sequences of DNA or RNA (types of genetic material) even when only a tiny amount is available. Thats because PCR targets sections of the DNA or RNA, and then reproduces and amplifies them. The process is performed in a test tube and takes only a few hours.Its common to come across this term in chronic fatigue syndrome research that involves testing for pathogens, such as viruses or bacteria. When used in this way, PCR allows scientists to identify the source of the genetic material and therefore identify which pathogens are present.Two primers, short single-stranded DNA（单链DNA）sequences that are synthesized to correspond to the beginning and ending of the DNA stretch to be copied;An enzyme called polymerase that moves along the segment of DNA, reading its code and assembling a copy; andA pile of DNA building blocks that the polymerase聚合酶needs to make that copy.How is PCR (polymerase chain reaction) done?As illustrated in the animated picture of PCR, three major steps are involved in a PCR. These three steps are repeated for 30 or 40 cycles. The cycles are done on an automated cycler, a device which rapidly heats and cools the test tubes containing the reaction mixture. Each step - denatauration变性(alteration of structure), annealing退火(joining), and extension延伸- takes place at a different temperature:Denaturation: At 94 C, the double-stranded双链DNA melts and opens into two pieces of single-stranded单链DNA.Annealing退火: At medium temperatures, around 54 C, the primers引物pair up (anneal) with the single-stranded单链template模板 (The template引物is the sequence of DNA to be copied.) On the small length of double-stranded DNA (the joined primer and template), the polymerase聚合酶attaches and starts copying the template.Extension: At 72 C, the polymerase聚合酶works best, and DNA building blocks complementary to the template are coupled to the primer, making a double stranded DNA molecule.With one cycle, a single segment of double-stranded DNA template is amplified into two separate pieces of double-stranded DNA. These two pieces are then available for amplification in the next cycle. As the cycles are repeated, more and more copies are generated and the number of copies of the template is increased exponentially.What is the purpose of doing a PCR (polymerase chain reaction)?To do PCR, the original DNA that one wishes to copy need not be pure or abundant. It can be pure but it also can be a minute part of a mixture of materials. So, PCR has found widespread and innumerable uses - to diagnose genetic diseases, do DNA fingerprinting, find bacteria and viruses, study human evolution, clone the DNA of an Egyptian mummy, establish paternity or biological relationships, etc. Accordingly, PCR has become an essential tool for biologists, DNA forensics labs, and many other laboratories that study genetic material.How was PCR (polymerase chain reaction) discovered?PCR was invented by Kary Mullis. At the time he thought up PCR in 1983, Mullis was working in Emeryville, California for Cetus, one of the first biotechnology companies. There, he was charged with making short chains of DNA for other scientists. Mullis has written that he conceived of PCR while cruising along the Pacific Coast Highway 128 one night on his motorcycle. He was playing in his mind with a new way of analyzing changes (mutations) in DNA when he realized that he had instead invented a method of amplifying any DNA region. Mullis has said that before his motorcycle trip was over, he was already savoring the prospects of a Nobel Prize. He shared the Nobel Prize in chemistry with Michael Smith in 1993.As Mullis has written in the Scientific American: Beginning with a single molecule of the genetic material DNA, the PCR can generate 100 billion similar molecules in an afternoon. The reaction is easy to execute执行，完成. It requires no more than a test tube, a few simple reagents, and a source of heat.What is RT PCR?RT-PCR (Reverse transcriptase-polymerase chain reaction) is a highly sensitive technique for the detection and quantitation of mRNA (messenger RNA). 逆转录PCR是一种高度敏感的用于信使RNA检测和定量的高度敏感的技术。The technique consists of two parts:这种技术包含两部分：The synthesis of cDNA (complementary DNA) from RNA by reverse transcription (RT) andThe amplification of a specific cDNA by the polymerase chain reaction (PCR).RNA通过逆转录合成互补DNA(cDNA)，通过PCR扩增特异性cDNA.Polymerase is an enzyme that helps replicate or repair DNA, and in the lab it can be used to very quickly make millions of copies.聚合酶是一种酶，可有助于复制或修复DNA,在实验室聚合酶可用于快速复制DNA。Real-Time PCRDefinition:RT-PCR is a somewhat confusing term because it can refer to two different methodologies used in biotechnology and genetic research. One of the definitions is real-time PCR. RT-PCR是一个令人困惑的名词，因为它指的是两种应用于生物技术和遗传研究的生物学方法。其中一种定义是实时PCR.This is a PCR process that requires highly sensitive equipment for measuring fluorescence throughout the PCR process.PCR过程需要高度敏感的用来通过PCR过程、测量荧光性的仪器。In real-time PCR, the PCR mixture contains a reagent that causes a fluorescent signal in proportion to the number of newly-formed DNA strands. 在实时PCR中，PCR混合物包含一种试剂，其可以引起一种与新形成的DNA链数量相符的荧光信号。The PCR reaction is run in a 96-well plate and the thermocycler热周期计is equipped with a very sensitive camera that can measure fluorescent signals and quantify the amount of DNA formed. PCR反应通过96孔板来进行，热周期计配备有敏感照相机，该照相机能够测量荧光信号及对已形成的DNA进行定量。Different techniques exist, based on the use of different fluorogenic reagents. 不同种技术的存在是基于不同荧光试剂的应用。Two of these are the probe-based and intercalator-based methods.这两种技术是急于探针的方法和急于嵌入剂的方法。An advantage to RT-PCR, over conventional PCR, is that quantification can take place much sooner, without waiting until completion of the entire reaction and using gel electrophoresis. RT-PCR相对于传统PCR的一个优势在于，定量可以发生地更快，不需要等到整个反应完成和使用凝胶电泳后再定量。The sensitivity for detection is much greater than agarose gels琼脂糖凝胶, and can differentiate between as little as a 2-fold change in DNA concentrations, versus 5-fold-plus changes detected by electrophoresis methods. 检测的敏感性比琼脂糖凝胶更大，能够区分DNA浓度2倍变化与电泳疗法检测的5倍变化之间的不同。Another advantage is that quantification takes place before the PCR products begin to degrade, or the reaction rates plateau进入停滞期.另一个优势在于，定量发生在PCR产物开始降解或者反应速率进入停滞期之前。Also Known As: RT-PCR, quantitative PCR, kinetic PCR, qPCR, qRT-PCR, RT-qPCR又叫做：RT-PCR, 定量PCR, 动力学PCR, qPCR, qRT-PCR, RT-qPCR.What is ELISA?什么是ELISA?ELISA is an abbreviation for enzyme-linked immunosorbent assay.ELISA是“酶联免疫吸附试验”的简称。What is an ELISA test?An ELISA test uses components成分，组分of the immune system免疫系统and chemicals化学物质to detect immune responses免疫反应in the body (for example, to infectious microbes有传染性的细菌). ELISA实验使用免疫系统和化学物质成分，检测体内的免疫反应。The ELISA test involves an enzyme (a protein that catalyzes催化a biochemical reaction). ELISA实验包含一种酶（一种催化生物化学反应的酶）。It also involves an antibody or antigen (immunologic molecules).ELISA实验也包含一种抗体或抗原（免疫学分子）。What is the use of an ELISA test?ELISA tests are widely utilized to detect substances that have antigenic抗原的properties, primarily proteins (as opposed to small molecules and ions such as glucose葡萄糖and potassium钾离子). ELISA实验被广泛应用于检测具有抗原性的物质，主要是蛋白质（而不是小分子和离子，比如说葡萄糖和钾离子）。The substances detected by ELISA tests include hormones激素, bacterial antigens细菌抗原and antibodies抗体.ELISA实验检测的物质包含激素、细菌抗原和抗体。How does an ELISA test work?There are variations of the ELISA test, but the most basic type consists of an antibody attached to a solid surface. 存在多种ELISA实验，但是最基本的类型包含一种附着于固体表面的抗体。The substance on which the enzyme acts is then added and the amount of product is measured in some way, such as a change in color of the solution.与酶反应的底物被加入，产物的量可通过某种方式得以测定，比如说：溶液颜色的一种变化。What are the advantages of ELISA?ELISA的优势在哪儿？ELISA tests are generally accurate tests. ELISA实验通常是很准确的。They are considered highly sensitive and specific and compare favorably with other methods used to detect substances in the body, such as radioimmune assay (RIA) tests. ELISA实验被认为是高度敏感和特异性的，优于其他用于检测机体内物质的方法。PCR英文课件：What is PCR?PCR is the abbreviation for Polymerase Chain Reaction. plimreis tein rikn PCR stands for代表“Polymerase Chain Reaction”, a process to replicate复制 DNA.PCR importance:PCR is a technique used to make numerous copies of (or amplify) a specific segment of DNA. PCR makes it possible to quickly and accurately obtain large quantities of DNA needed in carrying out research in molecular biology and in clinical diagnosis. PCR is a fast and inexpensive technique used to amplify, or make many copies of, small specific segments of DNA. The objective of PCR is to amplify DNA segment whose base sequence located its two ends that have been cleared. PCR的目的在于扩增位于两段已知序列之间的DNA区段。PCR consists of three steps:PCR cycle:(1)Denaturation phase di:netren 变性(2)Annealing phase ni:l 退火(3)Extension phase延伸The PCR stages are repeated for many cycles.Double stranded DNA 双链DNADNA is the template模板of PCR. DNA是PCR的模板。Step 1: denaturation 变性 92-96The template DNA is separated into single strands by heating to 94 for 1 minute.Step 2: annealing 52-54When the temperature is reduced to around 54(45 seconds), the primers引物will bind to their complementary sequences互补序列. The primers binding is annealing.Step 3: extension 72The temperature is increased to 72(1 minute) for the polymerase to extend the primers into new complementary strands, which uses up dNTPs and required Mg2+.When one PCR cycle is over, the target DNA will increase two times. Repeated denaturation, annealing and extension cycles周期 multiply the target DNA exponentially指数.PCR principle原理 can be described as follows:PCR amplification of a template requires two oligonucleotide primers引物, the four dNTPs, magnesium ions, and a thermostable DNA polymerase to perform DNA synthesis合成.oligonucleotide lgnju:kltad 寡核苷酸magnesium mgni:zim 镁离子Thermostable :mstebl 耐热的PCR is a process based on the ability of DNA polymerase enzyme that can synthesize a complementary strand互补链to a targeted segment of DNA in a test tube mixture of the four DNA bases.enzyme enzam 酶（1） The mixture is first heated to denature (separate) the sides of the double-stranded DNA and then cooled to allow: (2) the primers to find and bind to their complementary sequences on the separated strands; (3) the polymerase to extend the primers into new complementary strands.Repeated heating and cooling cycles multiply the target DNA exponentially, since each new double strand separtes to become two templates for further sythesis.In about 1 hour, 20 PCR cycles can amplify the target by a million fold. In 32 cycles at 100% efficiency, 1.07 billion copies of targeted DNA region are created.Primary components of PCR mixture:Template 模板Primers 引物DNA polymerase DNA聚合酶10乘以PCR buffer PCR缓冲液dNTPs 脱氧核苷三磷酸Magnesium ions Mg2+ ion anSterile deionized water 灭菌去离子水deionized di:anazd 去离子的TemplateThe DNA or RNA tested in cellsWhen your vials(瓶) of cells arrive at the lab for testing, they are first mixed with a detergent去垢剂which causes the cells to burst open and release their DNA along with other cell contents.The mixture is then washed with a phosphate磷酸盐缓冲液containing buffer (mild salt) solution to dilute稀释dalu:t cellular debris残渣. With minimal preparation, the sample is ready and the DNA on the targeted area of a chromosome染色体 krmsm can be amplified.What are primers?PCR mixture must also contain two DNA fragments片段, each about 20 bases long, called primers引物, that have sequences序列complementary to areas adjacent to邻近each sides of the target sequence目标序列. To do PCR, you need to know the DNA sequence around the region you want to amplify.These primers can be constucted in the lab, or purchased from commercial suppliers.If chosen well, the 20-25 base pair sequence will be unique独一无二的in the entire template so will match only the place specifically特异性地chosen thus limiting and defining the area to be copied.Why important are primers ?Though DNA polymerase can elongate a polynucleotide plnju:kljtad多聚核苷酸strand by adding new nucleotidesnju:kltad核苷酸, it cannot start a strand from scratch从头开始because it can only bond new nucleotides to a free sugar (3) end (-OH, hydroxy hadrks ) of a nucleotide chain核苷酸链.DNA polymerase requires the assistance帮助of a primer, a previously existing short strand of DNA (or RNA) that is complementary to the first part of the DNA segment being copied.DNA聚合酶需要引物的帮助，一种之前存在的DNA或者RNA的短链（与已被复制的DNA片段的第一部分互补）。（看不懂）This small strand of nucleotides anneals (binds) by complementary base互补碱基 pairing to the beginning of the area being copied.通过与已复制的区域的开始相配对的互补碱基，核苷酸的这部分短链退火（绑定）。With the primer in place, DNA polymerase is then able to continue adding the rest of the pairs of the segment until a new double strand of DNA is completed.只要引物在对的位置，DNA聚合酶就能够继续添加片段的剩余那一对儿，知道一个新的双链DNA完成。Primers are formed from free nucleotides游离核苷酸in the cell by enzymes called RNA primases引物酶.引物的形成是在RNA引物酶的作用下，由游离核苷酸形成的。Primase 引物酶 pramez Through some quirk巧合 of evolution, DNA polymerase is only able to add nucleotides to the 3 (sugar) end of a growing chain of DNA.通过一些进化的巧合，DNA聚合酶仅仅能够将核苷酸添加到DNA正在增长的链的3糖末端。 Therefore, it is not able just to start right up building a complementary strand互补链for a length of DNA that has been separated from its partner.因此，不能够开始建造互补链。（不理解）To solve this problem in the cell, an enzyme called a primase pramez引物酶 produces a short string短线 of nucleotides (usually 15-30) that are complementary to the first part of the segment of DNA that is being copied.为了解决细胞中的这个问题，一种叫做引物酶的酶产生了一个核苷酸的短链（经常15-30），它与已被复制的DNA片段的第一部分互补。（还是不懂）With all the improvements in PCR, what is currently considered standard PCR? PCR amplification of a template requires two oligonucleotide primers寡核苷酸引物, the four dNTPs, magnesium ions镁离子, and a thermostable DNA polymerase耐热DNA聚合酶 to perform DNA synthesis. The target region to be amplified should be between 150 and 400 nucleotides. 需要扩增的目标区域应该再150-400个核苷酸之间。The quantities of oligonucleotide primers, dNTPs, and magnesium, and the final volume, may vary for each specific application.寡核苷酸引物，dNTP,镁离子的量因应用不同而有所不同。Controls对照Even with the best reagents试剂, primers, and supplies, proper controls must be performed to test for PCR performance and presence of DNA contamination. 即使有最好的试剂、引物和供应物，为了检测PCR的性能和DNA污染的存在，也是需要进行恰当的对照的。As a rule, there should be a panel of negative and positive control sam