Polymerase chain reaction: applications in environmental microbiology.

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Annu. Rev. Microbial. 1991.45:137-41 Copyright © 1991 by Annual Reviews 1nc. All rights reserved

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POLYMERASE CHAIN REACTION: Applications in Environmental Microbiology R. J. Steffan} Gesellschaft fUr Biotechnologische Forschung mbH, Braunschweig, Germany

R. M. Atlas Department of Biology, University of Louisville, Louisville, Kentucky 40292 KEY WORDS:

PCR, gene probes

CONTENTS INTRODUCTION ...................................... .. ............................... . . . .........

138

THE POLYMERASE CHAIN REACTION. . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . ........... . . . . . . .... PCR Reaction Mixture . . . . . . .......................................... ............... .. . ........ PCR Reaction Conditions ...................... ...................... . ....... . . . . . . . . ....... . . . . Multiplex PCR . . ............ . . . . . . . . . . . . . . . . . . . ... . . . . . . . . . . . . . . . . . . .. . . . . . . . .. . .. . .. . . . ...... . . . . . Enhancing PCR Efficiency . . . . ................................................... . .. . . . . . . . . . . . .

1 38 138 140 141 141

ISOLATION OF ENVIRONMENTAL NUCLEIC ACIDS FOR PCR . . . . . . . . . . . ....... . . . Isolation of Nucleic Acids from Aquatic Environments.. . . . . . . ... ........ . . ......... . . .... Isolation of Nucleic Acids from Soil and Sediment ........................ . . . . . . . ....... . .

142 142 143

DETECTION OF PCR PRODUCTS .... . . . . . . ..... . . . . . . .... . . . . ... . . . . . . . . . . . . . . . . . . . . ..... . . . . . Capture Probes-Reverse Hybridization.. ........... . . ... . . . . . . .. ................ . ... . . . . . . . .

144 146

USE OF PCR TO DETECT MICROORGANISMS IN ENVIRONMENTAL SAMPLES Detection of Genetically t;ngineered Microorganisms .... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Detection of Indicator Organisms and Pathogens... . . . . . . . . . ...... . . . . . . . . . . . . ....... . . . . .

147 147 148

QUANTITATION OF PCR-AMPLIFIED PRODUCT . .. . . . . . . ..... .... . . . . .... Quantification of DNA Target Sequence . . . . .. ... . . . . . . . . . . . . . . . ...... . . . . . . . . . . ...... . . . . . Quantification of mRNA .... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

149 149 1 50

USE OF PCR TO ISOLATE AND CLONE SPECIFIC DNA SEQUENCES ... . . . ...... Use of PCR to Analyze Ribosomal RNA Sequences ....... . . . ............ . . . . . . ........ . . . Cloning DNA and Creating Gene Probes Using Degenerate Primers . .. . ....... . . . ...

1 52 152 153

CONCLUSION . . . ................. . . . . . . . . ................ . . . ........................ . . ... . .........

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'Current address: Envirogen Inc., Lawrenceville, New Jersey 08648. 137

0066-4227/91/1001-0137$02.00


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INTRODUCTION The development of the polymerase chain reaction (PCR) in 1983 (71-73a) was a major methodological breakthrough in molecular biology. The method has found its way into nearly every type of laboratory interested in molecular biology from forensics to ecology and diagnostics to pure research (22, 42). PCR permits the in vitro replication of defined sequences of DNA whereby gene segments can be amplified. Perhaps the most obvious application of this technique is to enhance gene probe detection of specific gene sequences. By exponentially amplifying a target sequence, PCR significantly enhances the probability of detecting rare sequences in heterologous mixtures of DNA. In this review, we describe some of the recent work in environmental microbiol ogy that has been done with the aid of PCR as well as some uses of PCR that may be applicable to answering future questions in molecular microbial ecology.

THE POLYMERASE CHAIN REACTION PCR involves three stages: 1. the DNA is melted to convert double-stranded DNA to single-stranded DNA; 2. primers are annealed to the target DNA; and 3. the DNA is extended by nucleotide addition from the primers by the action of DNA polymerase. The oligonucleotide primers are designed to hybridize to regions of DNA flanking a desired target gene sequence. The primers are then extended across the target sequence using DNA polymerase (currently almost exclusively Taq DNA polymerase) in the presence of free deoxynucleotide triphosphates, resulting in a duplication of the starting target material. Melt ing the product DNA duplexes and repeating the process many times result in an exponential increase in the amount of target DNA (Figure 1).

peR Reaction Mixture The essential components of the PCR reaction mixture are Taq DNA polymerase, oligomer primers, deoxynuc1eotides (dNTPs), template DNA, and magnesium ions. The recommended concentration range for Taq DNA polymerase is 1.0-2.5 units per 100 pJ (58). If the enzyme concentration is too high, nonspecific background products often form. If the enzyme concen tration is too low, the amount of desired product made is insufficient. The Taq DNA polymerase has an optimum activity around 70°C and is not inactivated by short incubations at temperatures at which PCR-generated fragments will denature (usually 90-95°C). Primer concentrations of 0.1-0.5 pM are recommended. Higher primer concentrations may promote nonspecific product formation and in particular may increase the generation of a primer-dimer. Nonspecific products and


POLYMERASE CHAIN REACTION Target DNA

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PCR C}des Figure

1

The stages of peR and the resultant amplification of DNA copies of the target region.

primer-dimer artifacts are substrates for PCR and result in a lower yield of the desired product. Typical primers are 18 to 28 nucleotides in length with 50--60% G + C composition. The primers must have similar melting tempera tures (Tms). One must avoid complementarity at the 3' ends of primer pairs as this arrangement favors the formation of primer-dimer and reduces the yield of the desired product. Also, three or more Cs or Gs at the 3' ends of primers may promote mispriming at G + C-rich sequences and should be avoided. Avoiding primers with 3' overlaps reduces the incidence of primer-dimer formation. Palindromic sequences within primers likewise must be avoided. Where possible, primers should have a G + C content of around 50% and a random base distribution. Secondary structure in the target template DNA and in the primers likewise should be minimized. dNTP concentrations should be 20--200 fl-M to give optimal specificity and fidelity. The four dNTPs should be used at equivalent concentrations to minimize misincorporation errors. The lowest dNTP concentration appropri ate for the length and composition of the target sequence should be used to minimize mispriming at nontarget sites and reduce the likelihood of extending misincorporated nucleotides (44). Twenty fl-M of each dNTP in a 100 fl-l reaction is theoretically sufficient to synthesize 2.6 fl-g of a 400-bp sequence.


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PCRs should contain 0.5 to 2.5 mM magnesium over the total dNTP concentration. The presence of EDTA or other chelators in the primer stocks or template DNA may disturb the apparent magnesium optimum. The magne sium ion concentration affects primer annealing, DNA melting temperatures, and enzyme activity. The recommended buffer for PCR is 10- to 50-mM Tris-HCl (pH 8.3-8.8). Up to 50-mM KCl can be included in the reaction mixture to facilitate primer annealing. KCI >50 mM inhibits Taq DNA polymerase activity (44). Gelatin or bovine serum albumin (100 j.tg/ml) and nonionic detergents such as Tween 20 can be used to stabilize the Taq DNA polymerase. PCR Reaction Conditions PCR involves repetitive cycling between a high temperature to melt the DNA, a relatively low temperature to allow the primers to hybridize (anneal) with the complementary region of the target DNA, and an intermediate tempera ture for primer extension. The temperature cycling can be achieved using an automated thermal cycler. The temperature and length of time required for primer annealing depend upon thc base composition, length, and concentration of the amplification primers (43). The annealing temperature generally is 5°C below the true Tm of the primers. Annealing temperatures in the range of 55 to noc generally yield the best results. At typical primer concentrations of 0. 2 pM, annealing requires only a few seconds. Increasing the annealing temperature enhances discrimination against incorrectly annealed primers and reduces addition of incorrect nucleotides at the 3' end of the primers. Stringent annealing temper atures, especially during the first several cycles, help increase specificity. If the temperature is lower than the optimum, the reaction products frequently include additional DNA fragments that can be visualized using agarose gel electrophoresis and ethidium bromide staining. The range of enzyme activity varies by two orders of magnitude between 20 and 85°C (26). Extension time depends upon the length and concentration of the target sequence and upon temperature. Primer extensions typically are performed at noc, which is optimal for Taq DNA polymerase. Low exten sion temperature together with high dNTP concentrations favors extension of misincorporated nuc1eotides. Using longer primers and only two tempera tures, e.g. 55 to 75°C for annealing and extension and 94°C for DNA melting, can yield better results (52). Typical denaturation conditions are 94-95°C for 30-60 s (43). Lower temperatures may result in incomplete denaturation of the target template and/or the PCR product and failure of the PCR. In contrast, denaturation steps that are too high and/or too long lead to unnecessary loss of enzyme activity.


POLYMERASE CHAIN REACTION

141

The half-life of Taq DNA polymerase activity is 40 min at 95°C. Too many cycles can increase the amount and complexity of nonspecific background products. Too few cycles give low product yield.

Multiplex PCR One can amplify several DNA segments simultaneously using multiple pairs of primers. Chamberlain et al (11) developed this procedure, called multiplex PCR, to detect human genes. Bej et al (7) modified the approach of simulta neous PCR amplification to detect gene sequences associated with different groups of bacteria in environmental samples. Multiplex PCR amplification of two different Legionella genes, one specific for Legionella pneumophila (mip) and the other for the genus Legionella (5S rRNA), was achieved by staggered additions of primers. Multiplex PCR amplification using differing amounts of primers specific for lacZ and lamB genes permitted the detection of coliform bacteria and those associated with human fecal contamination, including the indicator bacterial species Escherichia coli and enteric pathogens Salmonella and Shigella.

Enhancing PCR Efficiency One way of enhancing the probability of successful and specific amplification of environmental DNA is to dilute the sample (1 : 10 to 1 : 200) after the first few cycles of PCR (73, 89) or after a completed reaction (81), and then perform an additional round of PCR. This process may effectively dilute potential inhibitors to an acceptable level and allow for successful amplifica tion. The diluted DNA will also contain relatively high ratios of target sequences to total, nontarget, background DNA. The second round of PCR may be performed with the same primer set, or with nested primers designed to recognize regions within the initial amplified region (35, 73). Using nested primers provides an additional level of specific ity and increases amplification efficiency by minimizing nonspecific primer annealing (35), which is particularly useful in detecting organisms in environ mental samples in which the exact specificity or uniqueness of the primers is not known. Ruano et al (83) described a diphasic amplification strategy, termed booster peR, to improve the efficiency of amplifying target sequences present in low numbers. Initial PCR cycles are performed with a low concentration (2. 5-8.3 pM) of primers; after 20 PCR cycles, primer concentrations are increased to 0.1 /LM and up to 50 more PCR cycles are performed. This methodology minimizes the formation of primer dimers that may result from primer excess (86) in the early PCR cycles and compete for polymerase molecules. Using this method, one can amplify and detect as few as 5 to 10 copies of a specific human DNA target.


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ISOLATION OF ENVIRONMENTAL NUCLEIC ACIDS FOR peR For PCR analysis of DNA from environmental samples, the DNA must be isolated and may have to undergo extensive purification. Purification pro cedures can be any combination of CsCl-EtBr ultracentrifugation (40, 81, 1 02), hydroxylapatite or affinity chromatography (76, 8 1 , 102, 108), phenol! chloroform extractions, ethanol precipitations (24), dialysis, or repeated poly vinylpolypyrrolidone (PVPP) treatments (40, 81,102,117). For the isolation of RNA, humic-contaminated pellets may be resuspended in a solution of guanidine-hydrochloride and then subjected to ethanol precipitation and phe nol extraction (33, 34). Unfortunately, standard purification protocols will probably not work with every environmental sample, and the required con ditions must be ascertained on an individual basis. Isolation of Nucleic Acids from Aquatic Environments In general, water samples are the easiest environmental matrix from which to isolate nucleic acids. In many cases, water samples need only be filtered to collect microorganisms that may then be lysed to isolatc their nucleic acids (8, 24, 30, 95). in some cases, filter-collected cells can be analyzed directly. The cells may be lysed directly on specific filters, e.g. by freeze-thaw cycling, and the PCR can be run without removing the filters (A. K. Bej, personal communication). Alternately, filtered cells may first be subjected to enzymat ic lysis, andlor phenol-chloroform extraction procedures, to yield adequate DNA for peR analysis. Sommerville et al (95) demonstrated a simple method for isolating nucleic acids from aquatic samples that allows filtering relatively large volumes of water. Cell collection and lysis are performed in a single filter cartridge, and chromosomal DNA, plasmid DNA, and speciated RNA can be selectively recovered. Dissolved and particulate DNA may also be isolated from aquatic environments for di;:ect PCR analysis (19, 81). In aquatic studies, microbial nucleic acids of sufficient purity for enzymatic analysis have also been isolated from algal mats (115, 117) and filtered algal (81) and cyanobacterial cells (125). Paul et al (81) reported that with certain algal DNA preparations, and all preparations of environmental DNA, purification by CsCI-EtBr ultracentrifugation was required for successful amplification of target genes. Multiple PVPP treatment was required to recover purified rRNA from microbial mats (117). In other studies, multiple phenol or phenol:chloroform extractions havc produced sufficiently pure DNA from cyanobacteria (125) or planktonic microorganisms (24, 61). We (lOO-102) observed that adding solid ammonium acetate to cell lysates from aquatic sediments to a final concentration of 2.5 M, followed by high



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speed centrifugation and then ethanol precipitation of the DNA in the super natant, also removed great amounts of organic impurities from the recovered DNA. This treatment was necessary to obtain pure DNA even if additional purification treatments, such as ultracentrifugation or hydroxyapatite chromatography, were used.

Isolation of Nucleic Acids from Soil and Sediment Two approaches have been used to recover DNA from soils and sediments: isolation of microbial cells followed by cell lysis and nucleic acid purification (cell extraction) and direct lysis of microbial cells in the environmental matrix followed by nucleic acid purification (direct extraction). Balkwill et al (4) and GoksYlyr and colleagues (23, 107) first demonstrated cell extraction methods, and several groups have since used cell extraction, with some modifications, to isolate DNA from soil (3,40, 45, 102, 106, 108) or sediment (99, 102). Hahn et a1 (33) used a similar approach to recover rRNA from soils to detect Frankia sp. One simple but significant improve ment of this approach is the inclusion of a PVPP treatment to decrease sample humic content prior to cell lysis (40, 102), thereby simplifying DNA purifica tion . Ogram et al (76) were first to describe direct extraction. In this method, cells are lysed while still within the soil matrix by incubation with SDS followed by physical disruption with a bead beater. The DNA is then ex tracted using alkaline phosphate buffer. This method has successfully recov ered DNA from sediments (76, 102) and soil (102). B. Olson (personal communication) has developed an alternate direct DNA extraction procedure in which DNA is released through treatment with lysozyme and freeze-thaw disruption to lyse bacterial cells followed by phenol-chloroform extraction. The extracted DNA is then purified, e.g. by centrifugation or chromatogra phy. One may also separate DNA from impurities by using gel electrophoresis (J. Armstrong, personal communication; B. Olson, personal communication). Pvpp treatments may also be incorporated into this procedure to reduce the great amounts of humic materials inherently co-extracted during the treatment of soil or sediment with hot SDS (102). Significantly higher yields of DNA are recovered with the direct extraction method, but the DNA may contain impurities that can inhibit enzymatic manipulation (102). Because the yield is so great, however, more purification steps (e.g. CsCL-EtBr centrifugation, hydroxyapatite chromatography, or affinity chromatography) may be incorporated with a lower percentage of DNA loss. Also, both eukaryotic and prokaryotic DNA can be recovered. With the cell extraction method, extracted bacterial cells can be separated and washed to remove impurities before cell lysis, but one might recover only DNA from easily extractable cells.


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DETECTION OF PCR PRODUCTS Gibbs (27) has estimated that, if peR was perfonned at 100% of its theoreti cal maximum efficiency, one could generate 100 J,Lg of a l -kb unique human DNA fragment from 100 ng of total human DNA in only 25 peR cycles. Generally, however, only a few J,Lg of target are produced, even under the most controlled conditions. Paul et al (81) commented that they have never been able to generate enough amplified target from environmental DNA to visualize it on an EtBr-stained agarose gel, but the product was readily detectable following Southern blotting and probe hybridization. We (99) also made this observation during our studies with sediment DNA in which the amplified product was sufficient for detection with dot blot analysis but not with direct gel visualization. To explain this inability to directly visualize amplified DNA, let us pose the following hypothetical scenario. First we will assume all cells in a sediment or soil have the same genome size (5 X 106 bp) and extractability by the cell-extraction procedure. Let us also assume that the sediment or soil sample has 1010 ceUs/g as detennined by direct counting, and that we have added 1 target cell/g, each of which contains a single copy of a 500-bp target frag ment. Our target cell and its genome thus represent 10-10 of the total cells and genomes present in the sample. Now we extract the cells and DNA from 100 g of this sample and recover 100 J,Lg of the highly purified DNA. The target genome represents 10-10 of the DNA, or 10-8 t-tg of DNA. Because the total genome size of the target cell is 5 x 106 bp and the target fragment is 500 bp (10-4 of the total genome), the total target in the extracted DNA is 10-12 t-tg. Now, if we perfonn peR with 1 JLg (10-2 of the total) of the extracted DNA, the sample will contain 10-14 JLg (0.01 ag) of target DNA. If we assume that 0.1 ng of a 500 bp fragment can be detected in an EtBr-stained agarose gel, we would require a IOIO-fold (approximately 233) amplification to see the fragment (assuming the entire sample is run on the gel). Gene probes, however, can detect target DNA in the 0.1 pg range (40), thus requiring only a 107-fold amplification (approximately 224). The gene probe detection limit observed without amplification is generally in the range of 103 to 104 cells/g of soil or sediment (40,98,99). With peR amplification and approximately 30 cycles, however, single target cells can be detected using gene probes (2). Because of the great amounts of target DNA generated by peR amplifica tion, detection methods no longer need rely on the ability to produce strong signals using radiolabeled probes for the identification of minimal copies of target (62). This breakthrough has allowed the development of several nonra-



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dioactive detection methods that have many advantages over radioactive

methods (9). PeR-based nonradioactive detection methods can be grouped into three categories: 1. use of nonradioactively labeled probes to detect amplified product (77); 2. the direct incorporation of label into the amplified product for use as a probe (18); and 3. the direct incorporation of label into the peR product for probe or capture-mediated detection of amplified product (18, 51, 87, 104). In the first case, analysis can be performed using standard dot-blot (68) or Southern blot procedures (96), and the methodology does not differ greatly from previous methods . Increased detection sensitivity results from the pro duction of great quantities of target, and also the increase in target to nontarget sequence ratio. An additional advantage of peR methodology is that it provides two levels of stringency; primer annealing and probe hybridization. The latter cases have allowed for the development of several new tech niques with great potential for the rapid screening of large numbers of

(89, 104), horseradish peroxidase or alkaline peroxidase (62), or fluorescent dyes (13), or they can include sequences specifically recognized by DNA binding pro teins (109). Following amplification, each copy of the amplified product has the specific label. Similarly, one primer can contain a sequence to facilitate capture (i.e. biotin or DNA binding protein attachment site) while the op posite primer contains a label to facilitate detection (89). Label can also be incorporated into the amplified product by performing peR in the presence of labeled deoxyribonucleotides such as biotin-l l-dUTP (2, 8, 18, 64) or digoxigenin-Iabeled nucleotides (56). Day et al (18) per formed extensive studies to evaluate the parameters necessary to successfully

environmental samples. Primers can be labeled with either biotin

incorporate biotinylated nucleotides (biotinylated-dNTP, where

n

is the num

PCR products, and they evaluated how these incorporated biotinylated-dNTPs affect the efficien cy of hybridization to these amplified products. Initial concentrations of biotin-l l-dUTP from 1. 5 to 200 J.LM were incorporated into a 123-bp product through 30 cycles of peR. Greatest biotin incorporation occurred when the reaction was performed in the presence of 200 J.LM biotin- l l-dUTP (total replacement of dTTP), but this level of biotin incorporation significantly

ber of carbons in the biotin-nucleotide spacer arm) into

affected both mobility on an agarose gel, and hybridization to a radiolabeled

probe. Hybridization inhibition was also observed with 100-JLM biotin- l l dUTP, but it decreased below 50 J.LM. Replacement of dTTP with biotin-7dUTP or biotin-16-dUTP at concentrations greater than 180 J.LM inhibited primer extension, suggesting that the e ll spacer arm may be optimal for successful amplification.


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Capture Probes-Reverse Hybridization Potentially, any number of target sequences may be amplified in a single reaction mixture, or a number of samples could be screened for the presence of a specific sequence in a single hybridization assay and the resulting products could be screened against a bank of immobilized capture probes. Saiki et al (87) described a reverse hybridization procedure utilizing im mobilized capture probes for detection of several specific allelic mutations in human HLA-DQA DNA types . One could achieve high incorporation of biotin by incorporating biotin-labeled nucleotides rather than using 5' -end labeled biotinylated primers during PCR amplifications, thereby reducing IO-fold the amount of biotin-labeled DNA that could be detected using the immobilized capture probes. Abbott et al (1) reported similar findings. The biotin-labeled amplified product is captured by an immobilized nonlabeled probe-the reverse of the typical reaction in which the target is immobilized and the probe is labeled. One can use several methods to immobilize hybridization probes . In a relatively simple approach, long homomeric tails are added to the 3' end of oligomeric probes by the use of terminal deoxyribonucleotydltransferase and the tailed probes are then attached to nylon membranes through UV irradia tion (7, 87). Because the tails are much longer than the probe, they are preferentially bound to the membrane and the probes function normally. Single-base-pair differences can still be detected (87). In general, long poly (dT) tails of several hundred bases produce the most efficient immobilization because of the efficiency at which light-activated thymidine reacts with primary amines present in nylon. However, when the intensity of the UV light source cannot be carefully controlled, less-reactive poly (dC) tail may be more desirable for preventing excessive binding of the tail to the membranes. Bej et al (7) used hybridization to immobilize poly-dT-tailed capture probes with a dot- or slot-blot approach to detect Legionella and coliform bacteria in environmental waters. Hybridization of biotin-labeled amplified DNA, in which the biotin was incorporated during PCR amplification from biotiny lated-dUTP, to immobilized 400-dT-tailed capture probes permitted specific and sensitive detection of target gene sequences. The sensitivity of col orimetric detection achieved by peR amplification of target DNA was equiv alent to 1-2 bacterial cells, which is the same level of sensitivity obtained with radioactive detection. Running & Urdea (84) linked oligomeric probes to polystyrene plates by first coating the plates with the polypeptide poly (Lys-HBr, Phe) and then coupling an oligomeric probe synthesized with an alkylamine linker to the coated plate. Immobilizing probes in this fashion made them available for direct hybridization. Using a different approach that doesn't require a probe, Kemp et al (51) generated a peptide fusion between glutathione S-transferase



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(GST) and the GNC4 DNA, binding the protein from Saccharomyces cere and coated it onto microtiter wells. PCR was performed with one primer containing the consensus sequence recognized by GNC4 and another containing a biotin molecule. After PCR amplification, the amplified product was introduced into the GST -GNC4-coated wells where GNC4 bound amplified product containing the incorporated consensus binding sequence. The biotin of the second primer was then detected using a standard colorimet ric detection system. Because these methods utilize microtiter plates and colorimetric detection methods, robotic techniques can be used to dispense reagents, wash the wells, and perform the color-generating reaction (91). Positive reactions can be rapidly determined and the data analyzed using an automated spectrophoto metric plate reader. Large numbers of environmental samples (e.g. standard water quality analysis) could be processed rapidly with a great degree of visiae (GST-GNC4),

reproducibility.

USE OF PCR TO DETECT MICROORGANISMS IN ENVIRONMENTAL SAMPLES

Detection of Genetically Engineere d Microorganisms (98) used the polymerase chain reaction (PCR) to amplify a 1.0-kilobase (kb) probe-specific region of DNA from the herbicide-degrading bacterium Pseudomonas cepacia AC1100 to increase the sensitivity of dot-blot detection of the organism. The 1.0-kb region was an integral portion of a larger 1.3-kb repeat sequence present as 15 to 20 copies on the P. cepacia AC l lOO genome. PCR was performed by melting the target DNA, annealing 24-base oligonucleotide primers to unique sequences flanking the 1.0-kb region, and performing extension reactions with DNA polymerase. After extension, the DNA was again melted, and the procedure was repeated for a total of 25 to 30 cycles. After amplification, the reaction mixture was transferred to nylon filters and hybridized against radiolabeled 1.0-kb fragment probe DNA. Amplified target DNA was detectable in samples initially containing as little as 0.3 pg of target. The addition of 20 /-Lg of nonspecific DNA isolated from sediment samples did not hinder amplification or detection of the target DNA. The detection of 0.3 pg of target DNA showed that the sensitivity of gene sequence detection had increased at least 103-fold compared with dot-blot analysis of nonamplified samples. PCR performed after bacterial DNA was isolated from sediment samples permitted the detection of as few as 100 cells of P. cepacia AC1100 per 100 g of sediment sample against a background of 1011 diverse nontarget organisms; that is, P. cepacia AC1100 was positively detected at a concentration of 1 cell per gram of sediment. This observation We


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represented a 103-fold increase in sensitivity compared with nonamplified samples. Chaudhry et al (12) also used PCR for detecting genetically engineered microorganisms (GEMs). They cloned O.3-kb napier grass (Pennisetum pur pureum) genomic DNA that did not hybridize to DNAs isolated from various microorganisms, soil sediments, and aquatic environments into a derivative of a 2A-dichlorophenoxyacetic acid-degradative plasmid, pRClO, and trans ferred the construct into E. coli. This genetically altered microorganism was seeded into filter-sterilized lake and sewage-water samples (l04/ml). The PCR method amplified and detected the O.3-kb DNA marker of the GEM even after 10 to 14 days of incubation. The PCR method required only picogram amounts of DNA and had an advantage over the plate count technique, which can detect only culturable microorganisms. They concluded that the method may be useful for monitoring GEMS in complex environ ments, where discrimination between GEMs and indigenous microorganisms is either difficult or requires time-consuming tests.

Detection of Indicator Organisms and Pathogens PCR is useful for the identification of clinically important pathogens (10, 36, 55) and can be similarly applied for environmental surveillance (2). Bej et al (8) used PCR amplification and gene probe detection of regions of two genes, lacZ and lamB, to detect coliform bacteria in environmental waters. Amplification of a segment of the coding region of E. coli lacZ using a PCR-primer annealing temperature of 50°C detected E. coli and other coli form bacteria (including Shigella spp.) but not Salmonella spp. and noncoli form bacteria. Amplification of a region of E. coli lamB using a primer annealing temperature of 50°C selectively detected E. coli and Salmonella and Shigella spp. PCR amplification and radiolabeled gene probes detected as little as 1 to 10 fg of genomic E. coli DNA and as few as 1 to 5 viable E. coli cells in 100 rn1 of water. Thus, they demonstrated the potential use of PCR amplification of lacZ and lamB as a method to detect indicators of fecal contamination of water. They showed that amplification of lamB in particular permits detection of E. coli and enteric pathogens (Salmonella and Shigella spp.) with the necessary specificity and sensitivity for monitoring the bacteri ological quality of water so as to ensure the safety of water supplies. They also developed a method for the detection of the fecal coliform bacterium E. coli using PCR and gene probes based upon amplifying regions of the uid gene that code for j3-glucuronidase-expression of which forms the basis for fecal coliform detection by the commercially available Colilert method (A. K. Bej, personal communication). Amplification and gene probe detection of four different regions of uid specifically detected E. coli and Shigella species including f3-glucuronidase-negative strains of E. coli; no amplification was observed for other coliform and nonenteric bacteria.



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Starnbach et al (97) reported the detection of Legionella pneumophila using amplification of a fragment of DNA of unknown function from Legionella spp. using peR. The sensitivity of detection was equivalent to 35 colony forming units detected by viable plating. Mahbubani et al (67) developed a method for the detection of Legionella spp. in environmental water sources based upon peR and gene probes. All species of Legionella, including all 15 serogroups of L . pneumophila tested, were detected using peR amplification of a 104-bp DNA sequence that codes for a region of 58 rRNA followed by radiolabeled oligoprobe hybridization to an internal region of the amplified DNA. Strains of L. pneumophila (all serogroups) were specifically detected based upon amplification of a portion of the coding region of the macrophage infectivity potentiator (mip) gene. Pseudomonas spp. that exhibit antigenic cross-reactivity in serological detection methods did not produce positive signals in the PeR-gene probe method using Southern blot analyses. Single cell, single-gene Lesionella detection was achieved with the PeR-gene probe methods. M . H. Mahbubani (personal communication) investigated the ability of PeR-gene probe methods to detect viable L. pneumophila in water by exam ining bacterial cells exposed to biocide or elevated temperature for varying times. Both viable culturable and viable nonculturable cells of L. pneumophi la, formed during exposure to hypochlorite, showed positive peR amplifica tion, whereas nonviable cells did not. Viable cells of L. pneumophila were also specifically detected using mip mRNA as the target, reverse transcription to form eDNA, and peR to amplify the signal. When cells were killed by elevated temperature, only viable cuIturable cells were detected. and detec tion of these cells corresponded precisely with positive p eR amplification.

QUANTITATION OF PCR-AMPLIFIED PRODUCT

Quantification of DNA Target Sequence Quantitative estimates of a target population based on peR reaction products are hampered by the fact that the amount of peR products formed during the reaction increases exponentially; therefore, minute differences in any of the parameters that affect the efficiency of amplification can dramatically affect the outcome of the reaction. Gilliland et al (28, 29) developed a competitive peR scheme in which target DNA is quantified by co-amplifying target DNA in the presence of known quantities of a competitive DNA. In effect, they used an internal standard that competes for the same reagents as the target templates. In their work, competitive DNA (chromosomal) was amplified by the same primer set as the target DNA (eDNA), but it contained a small internal sequence and, thus, produced a larger reaction product. Because the primers and their sites on the two templates are identical, the two DNAs should be amplified with the same efficiency, provided the amplification of


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the competitive DNA is not affected by the presence of the intervening sequences. By titrating unknown amounts of target DNA against a dilution series of competitor, one can reliably and reproducibly quantify the amount of target DNA in the original sample. One quantifies target DNA by performing peR in the presence of one radiolabeled nucleotide, separating the products by electrophoresis, and quantifying the radioactivity in the resulting bands. Activity differences caused by the difference in the lengths of the two templates and, thus, the resulting peR product, are adjusted for by multiply ing the activity of the target band by the ratio of competitive DNA to target DNA length. Quantitation does not depend on cycle number or on concentra tion of primers or dNTPs. This approach could easily be applied to the quantitation of the number of target sequences in an environmental sample by first inserting a small transposon, such as those developed by Herrero et al (38), into a cloned copy of the target DNA. If enough amplified product is obtained, the sample can simply be resolved using electrophoresis and the intensity of the ethidium bromide-stained bands quantitated using videodensi tometry. Gilliland et al (28, 29) also demonstrated the possibility of using peR mediated site-directed mutagenesis (37, 39, 47) to insert or destroy a unique restriction site in the competitive DNA. The amplified competitive DNA could then be easily distinguished from target DNA following restriction enzyme digestion after competitive amplification. Again, this method could easily be adapted to environmental analysis by mutating a cloned copy of the target sequence. One limitation of this approach is that under conditions in which priming is rate limiting (i.e. late peR cycles), the sequence similarities between the target and competitive DNA may allow for the formation of heteroduplex molecules that will not be digested by the restriction endonucle ase (74).

Quantification of mRNA Several groups have also used peR to quantify mRNA (6, 28, 29, 93, 113). These mRNA levels may provide a valuable estimate of gene expression and/or cell viability under different environmental conditions (50). Singer Sam et al (93) first converted mRNA into cDNA by treatment with reverse transcriptase directly in a peR reaction mixture in the presence of specific primers, and then performed peR to amplify the desired target. They then determined relative amounts of initial mRNA by separating the products using electrophoresis and comparing the resulting bands by videodensitometry or by autoradiography after Southern transfer (96) and hybridization to a specific radioactive probe. A linear response in activity was observed over a lOOO-fold dilution range, from 0.02 to 2.00 jLg of total mouse-liver mRNA, with an



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average deviation of 40% from the best-fit line. The linear response to template concentration could only be maintained if primer, nucleotide, and polymerase concentrations remained in excess. Consequently,the appropriate number of PCR cycles for adequate quantitation must be empirically de termined for each sample because the number of cycles required to reach saturation depends on priming efficiency and the abundance of target tran script. Other reported mRNA quantitation methods (6,28,29,113) are essentially the same as the previously described method for DNA quantitation by com petitive PCR. Competitor RNA can be generated by cloning a mutant (either with a deletion or intervening sequence or with an inserted or deleted restric tion site) copy of the target DNA into a vector containing a RNA polymerase promoter (70, 99). Large amounts of RNA templates can then be easily produced by in vitro transcription with RNA polymerase for use as competitor in these reactions. Alternately, PCR primers can be designed that amplify the mutated copy of competitive DNA while at the same time insert a copy of a RNA polymerase promoter sequence (88, 116). PCR results in the production of large quantities of promoter-containing templates that can then be used for the production of RNA transcripts. This method eliminates the need for subcloning of the mutant template, for plasmid isolation, and for restriction digestion,and it allows for tailoring the length of the resulting RNA transcript without restriction enzyme digestion. To quantify the specific mRNA, both the target and the competitor must first be converted to cDNA by the use of reverse transcriptase. This process can be done in a separate reaction (6, 118), or directly in the PCR reaction mixture by adding 5 VI ILg RNA of AMV reverse transcriptase to the reaction mixture, incubating for an appropriate amount of time at 37-50°C, and then performing normal cycles of PCR (93). Alternately,Taq polymerase may also be used to reverse transcribe the RNA directly, circumventing the use of a separate reverse transcriptase (46, 110). The ultimate advantage of utilizing the reverse transcriptase activity of this enzyme is that the reaction can be performed at an elevated temperature (68°C) to overcome problems of transcribing RNAs with stable secondary structure. The amplified eDNA is then quantitated as previously described for competitive DNA amplification. To demonstrate the sensitivity of this method, Becker-Andre & Hahlbrock (6) mixed samples of 50 pg of target mRNA with 100 pg of mutant competitor mRNA, diluted it to 10-4, added 2.0 ILg of nonspecific carrier mRNA, and subjected the samples to reverse transcription and PCR. At the 10-4 dilution, approximately 100 copies of target mRNA were present, and quantitative detection was still accurate. Similar results were obtained in the absence of carrier RNA. The method was, therefore, quantitative, whether the target RNA was present as a small portion of a large amount of total RNA or as a


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large proportion of a small amount of RNA. These investigators estimated the accuracy of the method at ± 10% by analyzing portions of an identical titration mixture, but they observed considerable variation with fewer than 100 target molecules. In an interesting adaptation of this technique, Wang et al (113, 114) developed a synthetic competitive gene that could be amplified by any of a large set of primers. The synthetic gene was constructed in a RNA polymerase promoter vector by PeR-mediated oligonucleotide overlap extension (39) and contained target sites for at least 12 sets of peR primers that flanked an internal linker region and that were the same as those on the target DNA. This gene allowed them to use the same competitive RNA to quantitate many specific mRNA transcripts. This method could provide environmental micro biologists with a simple tool to study the relative expression of many environ mentally important genes without developing separate competitive genes for each target.

USE OF PCR TO ISOLATE AND CLONE SPECIFIC DNA SEQUENCES A potentially useful application of peR is the direct cloning of genomic DNA sequences (25, 90). Historically, genes have been cloned from organisms of interest by first generating a gene library of the organism in either a ,.\ phage or cosmid vector (68) and then screening the library (usually in E. coli) for expression of the desired phenotype by selective plating, the use of specific antibodies, or detection of the cloned sequence with gene probes. peR provides a relatively simple alternative to these procedures that may be utilized in many cases in which specific or related sequence information (e.g. highly conserved DNA/amino acid regions) is known. This feature makes peR particularly attractive for cloning and analyzing mutants of known genes (87), for cloning similar genes from different organisms (125), for subcloning genes or regions of genes where the nucleotide sequence is known (90), and even for isolating genes directly from natural environments (81). Several applications of peR provide mechanisms by which only limited (48, 82) or virtually no specific sequence information is known (103, 105, 119). Like wise, peR procedures have been described for directly cloning or analyzing genes that have been disrupted by the insertion of a transposon (21), and peR has even been used to construct new gene sequences (41, 124), add expres sion sequences (e.g. promoters) (65, 121), and to insert or delete sequences (39, 49) in cloned genes.

Use of peR to Analyze Ribosomal RNA Sequences The use of ribosomal RNA (rRNA) sequences for identification and phylogenetic characterization of microorganisms has been a major advance-



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ment in the study of microbial ecology (5,20,30,33,34,78,79, 115,117, 125). Generally, the methodology used to study these sequences has relied upon the successful isolation of rRNAs followed by direct sequencing with reverse transcriptase and chain terminators. To sequence both strands of the DNA coding for these transcripts, the genes must first be located within a gene library of the organism by using gene probes and then subcloned and sequenced. Alternately, double-stranded cDNA can be generated and sub cloned for sequencing (115, 117). PCR allows one to specifically amplify the region of DNA to be sequenced without developing gene libraries or performing extensive screening. The wealth of information presently available concerning highly conserved and variable regions within 5S and 16S rRNAs (17) allows for relatively simple selection of primer target sites for amplifying desired rRNA gene sequences (5). These specific amplified sequences may then be subcloned for con ventional sequencing (30) or directly sequenced using any of the many currently available methods (32, 54, 92, 122, 123). Ginovannoni et al (30) utilized these techniques to isolate and characterize rRNA sequences from Sargasso Sea bacterioplankton.

Cloning DNA and Creating Gene Probes Using Degenerate Primers Because of the degeneracy inherent the genetic triplet code (16), the exact nucleic acid sequence of a given piece of DNA cannot be deduced from the amino acid sequence alone. For PCR amplification, however, degenerate primers can be developed so that every possible combination of nucleic acid sequence that could code for a given amino acid sequence can be generated and used as a primer mixture to amplify the desired DNA fragment (15, 31, 60, 120, 125). Because of the speed of the Taq polymerase (approximately 150 nucleotides/s per enzyme molecule) (26, 43), only instantaneous anneal ing of the primer is needed to begin transcription,and an authentic copy of the intervening region is produced (60, 120). The thermal stability of Taq polymerase also allows the polymerase reaction to be performed at an ele vated temperature that may prevent priming by highly mismatched primers, thus increasing the probability of producing authentic transcripts (60). Several parameters are important for amplifying DNA using degenerate primers (14,59,60,66,80,94,120). Generally,short oligomeric primers (:5 20 nucleotides) with a limited degeneracy « 64-fold) appear to provide the most satisfactory amplification results, but longer primers (> 30 nucleotides) with greater degeneracies (to 535-fold) may also provide adequate amplifica tion in some instances (14, 94, 120). Primers with degeneracies as high as 516-fold have been successfully used for peR (14). With primers of 17-20 nucleotides,great degrees of degeneracy may be acceptable provided the first three nucleotides on the 3' end are a perfect match (94). The degeneracy of


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primers may be compensated for by substituting deoxynucleotide analogs such as de0xyinosine at ambiguous positions (53, 80). When using DNA polymerase I Klenow fragment to perform peR reac tions, temperatures must remain low, and yields of authentic clones as low as 2% can be expected (59). The Taq polymerase, however, allows the use of much higher primer annealing temperatures that potentially limit priming by highly mismatched primers (31). The first few cycles of- peR can be per formed by using low annealing temperatures to ensure priming, and later cycles can be performed at a higher annealing temperature. Alternately, the ramping rate from annealing temperature to extension temperature can be decreased to allow for adequate priming (14). When short degenerate primers are used, annealing temperatures greater than the calculated Tm of the primers may produce sufficient amplification (66). Degenerate primer methods may be useful for creating DNA probes based on N-terminal amino acid sequences. In many instances, native proteins may be easier to isolate and sequence than their respective genes, and the resulting amino acid sequence can be translated into its original nucleic acid sequence for use as a probe to screen a gene library (57). When little is known about an environmental isolate, however, nothing may be known about the organism's preferred codon usage, and probes must be developed with a very high degree of degeneracy. The short length and great degeneracy of these sequences may prevent their use as probes because of thermodynamic instability of the resulting mismatched duplexes (69, 85) . The use of peR circumvents this problem by allowing one to generate authentic transcripts of the sequenced region (59, 60, 120). By generating the amplified product from a cloned library with one degenerate primer and one primer targeted towards a known region flanking the cloning site in the vector, even regions with very little known sequence can be amplified. The nonprobe (vector) DNA can be trimmed away by digesting the amplified product with the restriction enzyme used to generate the library. Degenerate primer peR procedures have been applied to environmental samples, and several methods performed with nonenvironmental samples may also be directly applicable to this type of analysis. Zehr & McReynolds (125) used degenerate oligonucleotide primers to amplify nitrogen fixation (nif) genes from the marine cyanobacterium Trichodesmium thiebautii, an organ ism that has never been maintained in pure culture. By analyzing known amino acid sequence information for nif gene products of other nitrogen fixing organisms, including the heterocystous cyanobacteria Anabaena and several other nitrogen-fixing eubacteria, they identified regions of conserved amino acid sequence, and subsequently translated the amino acid sequence into a potential DNA sequence. They selected regions with less than 200-fold degeneracy and designed mixed peR primers ( l7-mers) to account for all



1 55

possible nucleotide combinations for the conserved amino acid region. T. thiebautii bundles were isolated from the west Caribbean Sea by using plankton tows and were then washed in buffer and frozen for later analysis. DNA was isolated from the bundles using a phenol/chloroform extraction protocol, and then subjected to PCR amplification with a degenerate primer mixture of 1 26 and 96 oligomers for the up- and downstream primers, respectively. The resulting amplified product was resolved by elec trophoresis,cloned into a M13 cloning/sequencing vector,and later subjected to sequence analysis . Although this method allows for sequencing of only a portion of the total gene or operon (i.e. the area between the conserved-sequence-directed prim ers) , several chromosomal walking techniques (63, 75) will allow amplifica tion and sequencing of regions up- and downsteam of the initially sequenced region. Tung et al ( 111) used such a method to clone an exocrine protein gene from the salivary gland of a South American bat by first constructing a cDNA library in A gt22. One PCR primer was then targeted against the SP6 promoter of the phage DNA, and a set of degenerate primers (containing 256 de generacies) was targeted against a region of DNA coding for a known amino acid sequence. PCR resulted in the production of a 450-base pair region that was subsequently sequenced. Once this nucleic acid sequence was known, new PCR primers targeted towards the now known sequence were developed. Subsequent PCR reactions were performed with these primers and primers targeted towards the SP6 and T7 promoters flanking the cloning sites. Se quence information derived from this amplified DNA could then be used to walk along the DNA in either direction to clone or sequence DNA flanking the target DNA. This method should be applicable to microorganisms of environmental significance, such as cloning catabolic genes from isolated organisms or from DNA isolated from environments shown to exhibit a specific catabolic activity.

CONCLUSION The polymerase chain reaction permits the amplification of specified DNA sequences. The reaction involves melting DNA (e.g. at 94°C), annealing oligomeric primers (approximately 20-mers at 50-70°C) to sites flanking the region to be amplified, and extending from the primers by deoxynucleotide addition using Taq DNA polymerase. The PCR cycle is repeated to increase exponentially the amplified DNA product. The process requires relatively purified DNA that can be obtained from environmental samples by cell extraction followed by cell lysis and DNA purification or by cell rupture within the environmental matrix followed by DNA extraction and purifica tion. Removal of humic material is critical in the purification process. PCR


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amplifi cati on permits the detection of as few as 1 00 cells per 100-g sample and is useful for tracking genetically engineered microorganisms and monitor ing indicator popUlations and pathogens in waters, soils, and sediments. The peR product can be quantified , permitting estimates of organisms and specif ic mRNAs in environmental s ample s . peR is useful for measuring gene expression by viable microorganisms as well as detecting specific popUlations based upon diagnostic gene sequences. peR is also useful for cloning genes, permitting sequencing of genes,even from environmentally important micro organisms that cannot yet be cultured. Thus, peR promises to have a wide range of applications in microbial ecology and environmental molecular microbial analyses . Literature Cited I . Abbott, M. A . , Poiesz, B . J . , Byrne, B . c., Kwok, S . , Sninsky , J. J., Ehrlich, O. D. 1 988. Enzymatic gene amplifi cation: qualitative and quantitative methods for detecting proviral DNA am plified in vitro. J. Infect. Dis. 1 5 8 : 1 1 5869 2. Atlas, R. M . , Bej, A. K. 1 990. Detect ing bacterial pathogens in environmental water samples by using PCR and gene probes. See Ref. 42, pp. 399-407 3. Bakken, L. R . 1 985. Separation and purification of bacteria from soil. Appl. Environ . Microbiol. 49: 1 482-87 4. Balkwill, D. L . , Labeda, D. P . , Casida, L. E. Jr. 1975. Simplified procedure for releasing and concentrating microorgan isms from soil for transmission electron microscopy viewing as thin-section and frozen-etched preparations. Can. J. Mi crobial. 2 1 :252-62 5 . Barry, T. , Powell, R . , Gannon , F. 1 990. A general method to generate DNA probes for microorganisms. Bioi Technology 8:233-36 6. Becker-Andre , M . , Halbrock, K. 1989. Absolute mRNA quantification using the polymerase chain reaction (PCR). Novel approach by a PCR-aided transcript titra tion assay (PATrY). Nucleic Acids Res.

1 7 :9437-46 7. Bej , A. K. , Mahbubani, M. H . , Miller, R . , DiCesare, J. L . , Haff, L . , Atlas, R . M . 1 990. Multiplex PCR amplification and immobilized capture probes for de tection of bacterial pathogens and in dicators in water. Mol. Cell. Probes 4:353-65 8. Bej , A. K . , Steffan, R. J. , DiCesare, J . , Haff, L . , Atlas, R . M . 1990. Detection of coliform bacteria in water by polymerase chain reaction and gene probes. Appl. Environ. Microbial. 56: 307- 1 4

9. Bugawan, T. L . , Saiki, R . K . , Leven son, C. H . , Watson, R. M . , Erlich, H . A . 1 988. The use o f non-radioactive oli gonucleotide probes to analyze enzymat ically amplified DNA for prenatal di agnosis and forensic HLA typing. Bioi Technology 6:943-47 1 0 . Byrne, B. c . , Li, J. 1. , Sninsky, J., Poiesz, B . 1. 1 988. Detection of HIV- 1 RNA sequences by in vitro DNA amplification. Nucleic Acids Res. 1 6: 4 1 65 I I . Chamberlain, J S . , Gibbs, R. A . , Ranier, J . E . , Nguyen , P . N . , Cashey, C . T. 1 988. Deletion screening of the Duchenne muscular dystrophy locus via multiplex DNA amplification. Nucleic Acids Res. 1 6: 1 1 1 4 1-56 1 2 . Chaudhry, G. R . , Toranzos, O . A . , Bhatti, A . R. 1 989. Novel method for monitoring genetically engineered mi croorganisms in the environment. Appl. Environ. Microbial. 55: 1 30 1 -4 1 3 . Chehab, F. F . , Kan Y. W. 1 989. Detec tion of specific DNA sequences by fluorescence amplification: A color complementation assay. Proc. Natl. Acad. Sci. USA 86:9178-82 1 4 . Compton, T. 1 990. Degenerate primers for DNA amplification. See Ref. 42, pp. 39-45 1 5 . Cooper, D. L. , Isola, N. 1 990. Full length eDNA cloning utilizing the polymerase chain reaction, a degenerate oligonucleotide sequence and a universal mRNA primer. BioTechniques 9:60-64 1 6. Crick, F. H. C. 1 965. The origin of the genetic code . J. Mol. BioI. 38: 367-79 1 7 . Dams, E . , Hemdricks, L . , Van der Peer, Y . , Neifs, J. B . , Smits G . , et al. 1988 . Compilation of small ribosomal subunit RNA sequences. Nucleic Acids Res. 1 6(Suppl . ):r87-r 1 73 1 8. Day, P. J. R . , Bevan, I. S . , Gurney, S . ,



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Applications of the polymerase chain reaction to genome analysis.

The polymerase chain reaction. History, methods, and applications.

Quantitative polymerase chain reaction.

Inverse polymerase chain reaction.

Polymerase chain reaction techniques.

Polymerase chain reaction strategy.

The polymerase chain reaction.

Polymerase chain reaction.

Recent advances in the polymerase chain reaction.

Heteroduplex formation in polymerase chain reaction.

DNA polymerase fidelity and the polymerase chain reaction.

Deletion mutagenesis during polymerase chain reaction: dependence on DNA polymerase.

Targeted gene walking polymerase chain reaction.

Polymerase Chain Reaction on a Viral Nanoparticle.

Genetic analysis using the polymerase chain reaction.

DNA amplification by the polymerase chain reaction.

Red light-controlled polymerase chain reaction.

[Gene amplification by the polymerase chain reaction].

Error Rate Comparison during Polymerase Chain Reaction by DNA Polymerase.

The polymerase chain reaction (PCR): general methods.

Polymerase chain reaction: trenches to benches.

Genotyping errors with the polymerase chain reaction.

Amplification of nucleic acids by polymerase chain reaction (PCR) and other methods and their applications.

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