聚合酵素鏈鎖反應(PCR)是一個簡便而有效的方法,它可使DNA在微量試管中擴增至106X以上。這個方法的原理十分簡單,在要擴增的DNA片段兩端分別設計一個前置引子(forward primer)和反置引子(reverse primer)使之與已變性的單股目標DNA緩冷配對(annealing)後,利用DNA聚合酵素(DNA polymerase)以目標DNA的兩股分別做為模板(template)來合成新的DNA股。像這樣經由(1)變性反應 (denaturation),使DNA的兩股分離。(2)緩冷配對反應(annealing)，使引子與目標DNA配對。(3)延長反應 (extension)，合成新的DNA股。的循環操作每次可使DNA的量添加一倍，若重複操作多次，以數學公式計算，DNA增加的量將會是2n，n是代表重複操作的次數。在理想的聚合酵素鏈鎖反應條件下，DNA是以幾何級數增加。理論上 ，一個DNA分子若重複操作 PCR 25次，那麼DNA的分子數將會擴增到225 = 106 個分子。這個DNA的量已足夠在Agarose凝膠電泳中觀察到。
PCR操作過程主要分成三大部份:(一)以高溫(92℃-95℃)使雙股模板DNA分離(denature)，(二)使引子與單股模板DNA做緩冷配對(40℃-52℃)，(三)再將溫度調整到DNA聚合酵素作用的有效溫度而合成新的DNA股。一般使用的DNA聚合酵素的有效作用溫度是37℃，因此在高溫分離雙股時會破壞DNA聚合酵素的活性，然而在耐高溫的細菌(Thermus aquaticus)中分離出來的DNA聚合酵素(Taq DNA polymerase)在95℃中其活性的半衰期(half life)長達40分鐘，故可供PCR操作使用。Taq聚合酵素的有效作用溫度為72℃，在這溫度下，每分鐘可合成2000-4000個核甘酸(nucleotides)。由於Taq聚合酵素的發現，使PCR之操作得以自動化。
Taq聚合酵素缺乏3'至5'端外切酵素(exonuclease)的特性，因而在DNA合成時沒有校對(proofreading)的功能，因此核甘酸在PCR合成時的濃度是影響錯誤配對的一個十分重要的因素，所以核甘酸的量應控制在20-200贡M之間，過多或過少都會影響PCR之精確性。一般而言，Taq聚合酵素合成DNA時，在每一個循環中錯誤配對的頻率可高達1/6000個核甘酸。此外，影響PCR DNA合成時的精確性的因素尚有，所欲合成的DNA的長度(以1kb以下為宜);循環數(越多時精確度越低);聚合酵素的種類(有校對能力者為佳)和Mg2+(0.5-2.5mM)的量等。
目前，PCR的技術已廣泛地應用在學術，工業和醫學上的研究，例如DNA序列的直接分析，DNA及細胞種類的分析，基因定位突變，遺傳病及病源之診斷，基因表現與選殖，水質及食品檢驗等。 詳細說明如下:
Isolation of genomic DNA
PCR allows isolation of DNA fragments from genomic DNA by selective amplification of a specific region of DNA. This use of PCR augments many methods, such as generating hybridization probes for Southern or northern hybridization and DNA cloning, which require larger amounts of DNA, representing a specific DNA region. PCR supplies these techniques with high amounts of pure DNA, enabling analysis of DNA samples even from very small amounts of starting material.
Other applications of PCR include DNA sequencing to determine unknown PCR-amplified sequences in which one of the amplification primers may be used in Sanger sequencing, isolation of a DNA sequence to expedite recombinant DNA technologies involving the insertion of a DNA sequence into a plasmid or the genetic material of another organism. Bacterial colonies (E.coli) can be rapidly screened by PCR for correct DNA vector constructs. PCR may also be used for genetic fingerprinting; a forensic technique used to identify a person or organism by comparing experimental DNAs through different PCR-based methods.
Some PCR 'fingerprints' methods have high discriminative power and can be used to identify genetic relationships between individuals, such as parent-child or between siblings, and are used in paternity testing. This technique may also be used to determine evolutionary relationships among organisms.
Amplification and quantitation of DNA
Because PCR amplifies the regions of DNA that it targets, PCR can be used to analyze extremely small amounts of sample. This is often critical for forensic analysis, when only a trace amount of DNA is available as evidence. PCR may also be used in the analysis of ancient DNA that is tens of thousands of years old. These PCR-based techniques have been successfully used on animals, such as a forty-thousand-year-old mammoth, and also on human DNA, in applications ranging from the analysis of Egyptian mummies to the identification of a Russian Tsar.
Quantitative PCR methods allow the estimation of the amount of a given sequence present in a sample – a technique often applied to quantitatively determine levels of gene expression. Real-time PCR is an established tool for DNA quantification that measures the accumulation of DNA product after each round of PCR amplification.
PCR in diagnosis of diseases
PCR allows early diagnosis of malignant diseases such as leukemia and lymphomas, which is currently the highest developed in cancer research and is already being used routinely. PCR assays can be performed directly on genomic DNA samples to detect translocation-specific malignant cells at a sensitivity which is at least 10,000 fold higher than other methods.
PCR also permits identification of non-cultivatable or slow-growing microorganisms such as mycobacteria, anaerobic bacteria, or viruses from tissue culture assays and animal models. The basis for PCR diagnostic applications in microbiology is the detection of infectious agents and the discrimination of non-pathogenic from pathogenic strains by virtue of specific genes.
Viral DNA can likewise be detected by PCR. The primers used need to be specific to the targeted sequences in the DNA of a virus, and the PCR can be used for diagnostic analyses or DNA sequencing of the viral genome. The high sensitivity of PCR permits virus detection soon after infection and even before the onset of disease. Such early detection may give physicians a significant lead in treatment. The amount of virus ("viral load") in a patient can also be quantified by PCR-based DNA quantitation techniques.
*Viral load is a measure of the severity of a viral infection, and can be calculated by estimating the amount of virus in an involved body fluid. For example, it can be given in RNA copies per milliliter of blood plasma. Determination of viral load is part of the therapy monitoring during chronic viral infections, and in immunocompromised patients such as those recovering from bone marrow or solid organ transplantation. Currently, routine testing is available for HIV-1, cytomegalovirus, hepatitis B virus, and hepatitis C virus.
Variations on the basic PCR technique
* Allele-specific PCR: This diagnostic or cloning technique is used to identify or utilize single-nucleotide polymorphisms (SNPs) (single base differences in DNA). It requires prior knowledge of a DNA sequence, including differences between alleles, and uses primers whose 3' ends encompass the SNP. PCR amplification under stringent conditions is much less efficient in the presence of a mismatch between template and primer, so successful amplification with an SNP-specific primer signals presence of the specific SNP in a sequence.
* Assembly PCR or Polymerase Cycling Assembly (PCA): Assembly PCR is the artificial synthesis of long DNA sequences by performing PCR on a pool of long oligonucleotides with short overlapping segments. The oligonucleotides alternate between sense and antisense directions, and the overlapping segments determine the order of the PCR fragments thereby selectively producing the final long DNA product.
* Asymmetric PCR: Asymmetric PCR is used to preferentially amplify one strand of the original DNA more than the other. It finds use in some types of sequencing and hybridization probing where having only one of the two complementary stands is required. PCR is carried out as usual, but with a great excess of the primers for the chosen strand. Due to the slow (arithmetic) amplification later in the reaction after the limiting primer has been used up, extra cycles of PCR are required. A recent modification on this process, known as Linear-After-The-Exponential-PCR (LATE-PCR), uses a limiting primer with a higher melting temperature (Melting temperature|Tm) than the excess primer to maintain reaction efficiency as the limiting primer concentration decreases mid-reaction.
* Helicase-dependent amplification: This technique is similar to traditional PCR, but uses a constant temperature rather than cycling through denaturation and annealing/extension cycles. DNA Helicase, an enzyme that unwinds DNA, is used in place of thermal denaturation.
* Hot-start PCR: This is a technique that reduces non-specific amplification during the initial set up stages of the PCR. The technique may be performed manually by heating the reaction components to the melting temperature (e.g., 95˚C) before adding the polymerase. Specialized enzyme systems have been developed that inhibit the polymerase's activity at ambient temperature, either by the binding of an antibody or by the presence of covalently bound inhibitors that only dissociate after a high-temperature activation step. Hot-start/cold-finish PCR is achieved with new hybrid polymerases that are inactive at ambient temperature and are instantly activated at elongation temperature.
* Intersequence-specific PCR (ISSR): a PCR method for DNA fingerprinting that amplifies regions between some simple sequence repeats to produce a unique fingerprint of amplified fragment lengths.
* Inverse PCR: a method used to allow PCR when only one internal sequence is known. This is especially useful in identifying flanking sequences to various genomic inserts. This involves a series of DNA digestions and self ligation, resulting in known sequences at either end of the unknown sequence.
* Ligation-mediated PCR: This method uses small DNA linkers ligated to the DNA of interest and multiple primers annealing to the DNA linkers; it has been used for DNA sequencing, genome walking, and DNA footprinting.
* Methylation-specific PCR (MSP): The MSP method was developed by Stephen Baylin and Jim Herman at the Johns Hopkins School of Medicine, and is used to detect methylation of CpG islands in genomic DNA. DNA is first treated with sodium bisulfite, which converts unmethylated cytosine bases to uracil, which is recognized by PCR primers as thymine. Two PCRs are then carried out on the modified DNA, using primer sets identical except at any CpG islands within the primer sequences. At these points, one primer set recognizes DNA with cytosines to amplify methylated DNA, and one set recognizes DNA with uracil or thymine to amplify unmethylated DNA. MSP using qPCR can also be performed to obtain quantitative rather than qualitative information about methylation.
* Miniprimer PCR: Miniprimer PCR uses a novel thermostable polymerase (S-Tbr) that can extend from short primers ("smalligos") as short as 9 or 10 nucleotides, instead of the approximately 20 nucleotides required by Taq. This method permits PCR targeting smaller primer binding regions, and is particularly useful to amplify unknown, but conserved, DNA sequences, such as the 16S (or eukaryotic 18S) rRNA gene. 16S rRNA miniprimer PCR was used to characterize a microbial mat community growing in an extreme environment, a hypersaline pond in Puerto Rico. In that study, deeply divergent sequences were discovered with high frequency and included representatives that defined two new division-level taxa, suggesting that miniprimer PCR may reveal new dimensions of microbial diversity. By enlarging the "sequence space" that may be queried by PCR primers, this technique may enable novel PCR strategies that are not possible within the limits of primer design imposed by Taq and other commonly used enzymes.
* Multiplex Ligation-dependent Probe Amplification (MLPA): permits multiple targets to be amplified with only a single primer pair, thus avoiding the resolution limitations of multiplex PCR.
* Multiplex-PCR: The use of multiple, unique primer sets within a single PCR mixture to produce amplicons of varying sizes specific to different DNA sequences. By targeting multiple genes at once, additional information may be gained from a single test run that otherwise would require several times the reagents and more time to perform. Annealing temperatures for each of the primer sets must be optimized to work correctly within a single reaction, and amplicon sizes, i.e., their base pair length, should be different enough to form distinct bands when visualized by gel electrophoresis.
* Nested PCR: increases the specificity of DNA amplification, by reducing background due to non-specific amplification of DNA. Two sets of primers are being used in two successive PCRs. In the first reaction, one pair of primers is used to generate DNA products, which besides the intended target, may still consist of non-specifically amplified DNA fragments. The product(s) are then used in a second PCR with a set of primers whose binding sites are completely or partially different from and located 3' of each of the primers used in the first reaction. Nested PCR is often more successful in specifically amplifying long DNA fragments than conventional PCR, but it requires more detailed knowledge of the target sequences.
* Overlap-extension PCR: is a genetic engineering technique allowing the construction of a DNA sequence with an alteration inserted beyond the limit of the longest practical primer length.
* Quantitative PCR (Q-PCR): is used to measure the quantity of a PCR product (preferably real-time). It is the method of choice to quantitatively measure starting amounts of DNA, cDNA or RNA. Q-PCR is commonly used to determine whether a DNA sequence is present in a sample and the number of its copies in the sample. The method with currently the highest level of accuracy is Quantitative real-time PCR. It is often confusingly known as RT-PCR (Real Time PCR) or RQ-PCR. QRT-PCR or RTQ-PCR are more appropriate contractions. RT-PCR commonly refers to reverse transcription PCR, which is often used in conjunction with Q-PCR. QRT-PCR methods use fluorescent dyes, such as Sybr Green, or fluorophore-containing DNA probes, such as TaqMan, to measure the amount of amplified product in real time.
* RT-PCR: (Reverse Transcription PCR) is a method used to amplify, isolate or identify a known sequence from a cellular or tissue RNA. The PCR is preceded by a reaction using reverse transcriptase to convert RNA to cDNA. RT-PCR is widely used in expression profiling, to determine the expression of a gene or to identify the sequence of an RNA transcript, including transcription start and termination sites and, if the genomic DNA sequence of a gene is known, to map the location of exons and introns in the gene. The 5' end of a gene (corresponding to the transcription start site) is typically identified by an RT-PCR method, named RACE-PCR, short for Rapid Amplification of cDNA Ends.
* Solid Phase PCR: encompasses multiple meanings, including Polony Amplification (where PCR colonies are derived in a gel matrix, for example), 'Bridge PCR' (the only primers present are covalently linked to solid support surface), conventional Solid Phase PCR (where Asymmetric PCR is applied in the presence of solid support bearing primer with sequence matching one of the aqueous primers) and Enhanced Solid Phase PCR.
* TAIL-PCR: Thermal asymmetric interlaced PCR is used to isolate unknown sequence flanking a known sequence. Within the known sequence TAIL-PCR uses a nested pair of primers with differing annealing temperatures; a degenerate primer is used to amplify in the other direction from the unknown sequence.
* Touchdown PCR: a variant of PCR that aims to reduce nonspecific background by gradually lowering the annealing temperature as PCR cycling progresses. The annealing temperature at the initial cycles is usually a few degrees (3-5˚C) above the Tm of the primers used, while at the later cycles, it is a few degrees (3-5˚C) below the primer Tm. The higher temperatures give greater specificity for primer binding, and the lower temperatures permit more efficient amplification from the specific products formed during the initial cycles.
* PAN-AC: This method uses isothermal conditions for amplification, and may be used in living cells.
* Universal Fast Walking: this method allows genome walking and genetic fingerprinting using a more specific 'two-sided' PCR than conventional 'one-sided' approaches (using only one gene-specific primer and one general primer - which can lead to artefactual 'noise') by virtue of a mechanism involving lariat structure formation. Streamlined derivatives of UFW are LaNe RAGE (lariat-dependent nested PCR for rapid amplification of genomic DNA ends), 5'RACE LaNe and 3'RACE LaNe.
應用例證:
一.
JOURNAL OF CLINICAL MICROBIOLOGY, Oct. 1996, p. 2552–2558
A single-round PCR method with primers specific for the 3* noncoding region (NCR) of hepatitis C virus (HCV) has been developed. Using a double RNAzol-B extraction, a high-temperature reverse-transcription step with SuperScript II reverse transcriptase, and a 40-cycle two-temperature PCR with a TaqStart antibody hot-start procedure, we were able to detect a 92-nucleotide fragment of the recently discovered 98-nucleotide highly conserved sequence at the 3* terminus of the HCV genome. Direct sequencing of the PCR products confirmed the specificity of the PCR and demonstrated conservation in this region. Only one nucleotide change in 14 specimens was found. End point dilution titration of sera with known viral RNA titers showed the sensitivity of the single-round 3* NCR PCR to be comparable to those of the established nested 5* NCR assays (fewer than 25 HCV genome equivalents). To evaluate specificity and sensitivity, a panel of 116 serum samples characterized by nested 5*-end PCR, genotyping, and quantitative assays was tested. A high degree of concordance (96%) between the 3* NCR and 5* NCR PCR results was found. The sequence conservation at the 3* end of the HCV genome among common genotypes and the savings in time, labor, and reagents from a single-round PCR make this assay a useful addition to the detection systems available to identify and monitor HCV infection.
二.
Acta Med Okayama. 2000 Dec;54(6):253-7.
We have developed a reliable internally controlled RT-nested PCR method for the detection of hepatitis C virus (HCV) RNA using in vitro synthesized Renilla luciferase (Rluc) RNA as an internal control. Using this method, the 5'-noncoding region of HCV RNA (144 nucleotides) and Rluc RNA (276 nucleotides) were efficiently amplified in a single tube, and the sensitivity and specificity of this method were comparable to standard RT-nested PCR. This method was successfully performed on RNA specimens obtained from in vitro HCV-infected human hepatocyte PH5CH8 cells, which support HCV replication. In addition, we demonstrated that this method was useful for the evaluation of antiviral reagents by confirming the anti-HCV activity of bovine lactoferrin, which we previously found to be a new inhibitor of HCV infection. Therefore, this method may be useful for the studies of not only HCV but also of other viruses.
三.
JOURNAL OF CLINICAL MICROBIOLOGY, June 2002, p. 2051–2056
The degrees of effectiveness of reverse transcription (RT)-PCR, virus isolation, and antigen enzyme-linked immunosorbent assay (ELISA) for the detection of influenza A virus were evaluated with nasopharyngeal swabs from 150 patients (1 week to 86 years old) with influenza A virus infection. RT-PCR had a sensitivity for influenza A virus in stock virus preparations 103 times higher than virus isolation and 106 to 107 times higher than ELISA. The detection rate achieved by RT-PCR in clinical samples was clearly higher (93%) than that by virus isolation (80%) and ELISA (62%). Despite low overall detection rates achieved by antigen ELISA, samples from patients younger than 5 years old yielded higher-than-average rates in this rapid assay (88%). The likelihood of negative results in the ELISA increased significantly with increasing age of the patient (P <0.01). The degrees of effectiveness of RT-PCR and virus isolation were not influenced by the age of the patient. Neither influenza immunizations nor the interval between onset of symptoms and laboratory investigation (mean, 4.7 days; standard deviation, 3.3 days) affected results obtained by the three test systems. Our results demonstrate that the ELISA is reliable for rapid laboratory diagnosis of influenza in infants and young children, but for older patients application of RT-PCR or virus isolation is necessary to avoid false negative results.
四.
中國獸醫科技 2003年03期
將豬流行性腹瀉病毒分離株HLJ02,經過胰酶處理後,在Vero細胞上增殖.利用Gen-Bank中的基因序列設計合成了2對M基因引物,應用RT-PCR和RT-nested PCR分別擴增出HLJ02的854 bp的M全基因片段和412 bp的M基因部分片段,而在豬傳染性胃腸炎病毒中和Vero培養液中未擴增出上述產物.結果表明,RT-nested PCR可用於檢測豬流行性腹瀉病毒(PEDV).
五.
林美娟 Mei-Chuan Lin 陳福旗 Fure-Chyi Chen
蘭花是台灣目前重要的園藝作物之一，但近年來蘭花產業卻飽受蕙蘭嵌紋病毒(Cymbidium mosaic virus；CyMV)感染之苦，造成蘭科植物組織出現明顯的嵌紋及壞疽病斑，降低商品價值。因此，降低此病毒對蘭花的影響並提高蘭株的品質，是目前最重要的工作之一。本研究採用蝴蝶蘭Phalaenopsis Sogo Diana ‘F1184’ 不同組織及文心蘭試管苗Oncidium Gower Ramsey之組織RNA，以RT-PCR及real-time RT-PCR法來進行CyMV RNA replicase之檢測，結果顯示real-time RT-PCR之靈敏度較RT-PCR高，在植物組織總量RNA濃度為1ag/μl時仍可偵測到病毒。利用real-time RT-PCR還可偵測出不同組織中病毒之表現量，其中以文心蘭試管苗表現量最高，蝴蝶蘭花瓣次之，而以蝴蝶蘭葉片之表現量最低。此外，從蝴蝶蘭及文心蘭中選殖出的CyMV各基因片段之相似度都在89%以上，顯示感染兩種蘭花之CyMV應為同一病毒不同系統。而利用RNA干擾技術構築CyMV RNA replicase干擾載體，以農桿菌轉殖系統轉殖入菸草中，經分子檢測後，可觀察到CyMV RNA replicase及GUS linker之條帶，初步確認構築之載體已成功地殖入菸草基因組中。
六.
JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 2004, p. 5189–5198
A panel of 23 real-time PCR assays based on TaqMan technology has been developed for the detection and monitoring of 16 different viruses and virus families including human polyomaviruses BK virus and JC virus, human herpesviruses 6, 7, and 8, human adenoviruses, herpes simplex viruses 1 and 2, varicella-zoster virus, cytomegalovirus, Epstein-Barr virus, parvovirus B19, influenza A and B viruses, parainfluenza viruses 1 to 3, enteroviruses, and respiratory syncytial virus. The test systems presented have a broad dynamic range and display high sensitivity, reproducibility, and specificity. Moreover, the assays allow precise quantification of viral load in a variety of clinical specimens. The ability to use uniform PCR conditions for all assays permits simultaneous processing and detection of many different viruses, thus economizing the diagnostic work. Our observations based on more than 50,000 assays reveal the potential of the real-time PCR tests to facilitate early diagnosis of infection and to monitor the kinetics of viral proliferation and the response to treatment. We demonstrate that, in immunosuppressed patients with invasive virus infections, surveillance by the assays described may permit detection of increasing viral load several days to weeks prior to the onset of clinical symptoms. In virus infections for which specific treatment is available, the quantitative PCR assays presented provide reliable diagnostic tools for timely initiation of appropriate therapy and for rapid assessment of the efficacy of antiviral treatment strategies.
摘錄 MATERIALS AND METHODS
Sample preparation. (i) Nucleic acid extraction. For the isolation of DNA and RNA, commercially available kits were used, essentially as recommended by the manufacturer. DNA extraction from largely cell-free liquids, except urine, and from peripheral blood leukocytes for the detection of intracellular virus particles was performed by using the QIAamp DNA mini kit (Qiagen GmbH, Hilden, Germany). Isolation of virus DNA from urine was done by using the QIAamp viral RNA mini kit, and isolation of virus DNA from stool was done by using the QIAamp DNA stool mini kit (Qiagen). For the isolation of RNA from each of these sources, the QIAamp viral RNA mini kit was used. The only modifications performed included the adjustment of the input and the elution volumes. For nucleic acid extraction, the input volume for all samples was 200 l for DNA and 140 l for RNA and the elution volume was 240 l for DNA and 120 l for RNA.
(ii) Reverse transcription. For reverse transcription of purified viral RNA, a total of 30 l of viral RNA eluate and 5 l of nuclease-free water were mixed with 1 mM concentrations of each of the deoxynucleoside triphosphates and 25 M pd(N)6, and this mixture was incubated at 72。 for 5 min. The denatured RNA was placed on ice for 1 min before the addition of 12 l of reaction buffer (50 mM Tris-HCl [pH 8.3], 75 mM KCl, 5 mM MgCl2), 10 mM dithiothreitol, 1.5 l of RNasin (40 U/l; Promega, Mannheim, Germany), and 1.5 l of Moloney murine leukemia virus reverse transcriptase (200 U/l; Invitrogen, Carlsbad, Calif.). The reaction mixture was incubated at 37。 for 45 min, and finally, the enzymes were inactivated by heating at 98。 for 3 min.
Target sequence selection and primer and probe design. Specific primers and probes were selected and designed by using the Primer Express, version 2.0, software (Applied Biosystems [AB], Foster City, Calif). ...some of the primers reveal a degenerated code. This was a prerequisite for the detection of viral subspecies differing from each other by single nucleotides. For the experiments described below, hydrolysis probes labeled with 6-carboxyfluorescein reporter molecules at the 5 end and 6-carboxy-tetramethylrhodamine quencher molecules at the 3 end (AB) were used. The optimal concentration of primers was assessed by performing serial PCRs across a concentration range from 50 to 900 nM.
Real-time PCR. All reactions were set up as singleplex PCRs in a total volume of 25 l containing 12.5 l of Universal Master mix (2 concentration, includingROX reference dye and uracil N-glycosylase [UNG]; AB), 50 to 900 nM concentrations of primers, 200 nM TaqMan probe (Table 1), and 6 l of genomic DNA or cDNA template. The mixtures were prepared in 96-well optical microtiter plates (AB), centrifuged for 1 min at 272 g and amplified on the ABI 7700 or 7900 sequence detection system by using the following uniform cycling parameters: 2 min at 50。 (degradation of potentially present contaminating dUTP-containing amplicons by UNG), 10 min at 95。 (inactivation of UNG and activation of AmpliTaq Gold DNA polymerase), and 50 cycles of 15 s at 95。 and 60 s at 60。 (amplification of the specific target sequence).
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