Optimization of arbitrarily primed PCR for the identification of bacterial isolates

Journal of Microbiological Methods 24 (1995) 55-63

ELSEVIER

Journal ofMicrobiological Methods

Optimization of arbitrarily primed PCR for the identification of bacterial isolates Eileen Maura Jutras”, Raina M. Miller, Ian L. Pepper University of Arizona,

Dept. of Soil and Water Science, Shantz 38 Rm 429, University of Arizona,

Tucson, AZ 85721, USA

Received 12 September 1994; revised received15 February 1995 accepted 20 March 1995

Abstract Arbitrarily primed polymerase chain reaction (AP-PCR) has been used extensively for genetic mapping, and the identification of bacterial isolates. To ensure that the results will be reproducible and due to true genetic variations, the AP-PCR reaction conditions must be optimized. In this study, three cultured bacterial isolates were screened with 100 arbitrary primers. Of these, five were chosen for the optimization study. The parameters optimized included: the operating conditions of the thermal cycler, the agarose gel concentration, the annealing temperature, and the concentrations of Taq polymerase enzyme, magnesium chloride, primer, and template. The final optimized PCR reaction conditions were 1 x buffer (3.5 mM MgCl,, 10 mM Tris-HCl, 50 mM KC1 and 0.1 mg ml-’ gelatin), 200 PM dNTP, 0.4 PM primer, 2.5 U AmpliTaqQ (Perkin-Elmer Cetus) polymerase enzyme, and 5 ~1 of template (at least lo6 lysed bacterial cells). The Perkin-Elmer Gene-AmpTM 9600 PCR System was used with the following cycling conditions; a 94°C 15 s denaturation step, a 45°C 15 s annealing step, and 72°C 30 s extension step for a total of 35 cycles. Reproducible, unique fingerprints were generated for the three isolates using each of the five arbitrary primers. Keywords:

AP-PCR;

Bacterium;

Identification;

Optimization

1. Introduction

and species without prior sequence

The polymerase chain reaction (PCR) was originally developed using 2 primers of known sequence to amplify a specific region of target DNA. Arbitrarily primed PCR (AP-PCR) uses a single primer of arbitrary sequence that amplifies random regions of target DNA resulting in a unique banding pattern which can be species specific [l]. These patterns can be used to distinguish between different bacterial genera

AP-PCR is subject to artifactual variation that can interfere with interpretation of the amplification fragments generated [3]. This variation can result from inconsistencies in the reaction such as magnesium, primer, and template concentrations that are not due to true genetic variability of the DNA. Duplication of AP-PCR in any given laboratory and among different laboratories is crucial to the applicability of AP-PCR for the identification of bacterial species [4]. Therefore successful implementation of AP-PCR must include consideration of these important parame-

* Corresponding author. Tel: + 1 (602) 621-5988; fax: + 1 (602) 621-1647.

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ters including; annealing temperature and magnesium chloride (MgCl,), primer, template, and polymerase enzyme concentration. AP-PCR identification of bacterial species based on unique banding patterns or fingerprints is an emerging alternative to the standard methods presently used for identification. These methods have been developed for clinical isolates in pure culture with little recognition of potential application for environmental organisms. AP-PCR of mixed populations of environmental isolates would be a practical identification method because no prior sequence information is needed. This technique would require construction of a database to be used in fingerprint interpretation. As with any identification method, the results have to be reproducible to have functional value. In this study, the parameters of temperature, AP-PCR, MgCl, , annealing primer, and template concentration were optimized to give unique banding patterns for individual isolates of Escherichia coli, Pseudomonas aeruginosa, and Bacillus subtilis. These diverse bacteria were chosen as representative Gram negative and positive clinical and soil isolates.

2. Materials and methods 2.1. Preparation of target DNA from individual

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min in the Perkin-Elmer Gene-AmpTM 9600 PCR System (9600) or the Perkin-Elmer 480 DNA Thermal CyclerTM (480) (Perkin-Elmer Cetus) [2]. Total DNA in the 10’ aliquots was measured in the TKO 100 Mini-fluorometer (Hoeffer Scientific Instruments) using the manufacturer’s recommended protocol. 2.2. Arbitrary primer selection A set of 100 arbitrary primers were obtained from the Biotechnology Laboratory at the University of British Columbia. The initial screening of primers was performed using 25 ~1 reactions with 2.5 ~1 of 10 x buffer (15 mM MgCl,, 100 mM Tris-HCl, 500 mM KC1 and 1 mg ml-’ gelatin), 10 mM dNTP, 0.2 /_LMprimer, 1.25 U AmpliTaq@ (Perkin-Elmer Cetus) polymerase enzyme, and 2.5 ~1 of template (heat lysed cells). A Perkin-Elmer Cetus 480 thermal cycler was used with the following cycling conditions: A 94°C 1 min denaturation step; 36°C 1 min annealing step; and finally, a 72°C 2 min extension step, over a total of 40 cycles. Primers were selected for their ability to generate unique banding patterns with the bacterial isolates used in this study. Five oligonucleotides, 10 bases each, were selected for optimization in this study. Table 1 shows the sequence and percent GC content for each of the selected primers.

isolates

2.3. AP-PCR Escherichia coli (ATCC 15224)) Pseudomonas aeruginosa (ATCC 9027), and Bacillus subtilis

(ATCC 21332) were grown in nutrient broth and maintained on nutrient agar plates. A pure culture isolate was used to inoculate broth cultures, and cells were grown to late log phase at 37°C. The culture was then serially diluted in 0.1% peptone and spread plate counts were used to enumerate colony forming units (CFU). Ten ml of the late log phase culture were concentrated by centrifugation at 10000 rpm for 10 min at 25°C. The supernatant was discarded and the cells were resuspended in 100 or 1000 ~1 of 0.1% pegtone. One hundred ~1 aliquots of the lo”10 CFU ml-’ dilutions were lysed at 98°C for 10

conditions

The cycling conditions adopted for the 9600 thermal cycler utilized similar temperatures as the 480, but used shorter cycle times due to reduced ramp times. Denaturation and annealing

Table 1 Random primers selected for this study Number

Sequence 5’-3’

615 620 623 625 682

CGT TTG TGC CCG CTG

CGA CGC GGG CTG CGA

%GC content GCG CCG ACT GAG CGG

G G G C T

80 80 70 80 70

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was shortened to 15 s and the extension cycle was deceased to 310 s. The total number of amplification cycles needed was determined by using a PCR reaction containing primer 682 with E. coli as template. After every five cycles the thermal cycler was paused and 3 replicate samples removed. This ‘was repeated until a total of 40 cycles had been completed. When the replicate samples from increasing numbers of cycles were compared, no change in the banding patterns was detected beyond 35 cycles. The cycling conditions for the 9600 prior to optimization were a 94°C 15 s denaturation step, a 36°C 15 s annealing step, and a 72°C 30 s extension step for a total of 35 cycles. 2.4. Optimization

of AP-PCR

The initial PCR 50 ~1 master mix contained 1 X buffer (10 mM Tris-HCl, 50 mM KCl, 1.5 mM MgCl,, and 0.3. mg ml-’ gelatin), 200 PM dNTPs, 0.2 PM primer, 1.25 U AmpliTaq polymerase, and lo6 cells. The MgCl, concentration, annealing temperature, primer concentration, template concentration, and units of polymerase enzyme were optimized for the five primers using E. co&. As each parameter was tested, banding patterns of the five primers were compared, and the concentration that gave the best overall resolution for all five primers was used for the next optimization step. Table 2 shows the parameters that were optimized in the PCR reaction. 2.5. Agarose gel electrophoresis AP-PCR can generate up to 20 amplification products per reaction for bacterial DNA [6].

Table 2 Parameters

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Since the rationale of the protocol is to generate unique banding patterns, some consideration must be given to the method of visualizing the results. To test the influence of gel concentration on the resolution of the banding patterns, a 1.0% Fisher BioTech Low EEO agarose gel was cast and allowed to cool. A section of four lanes was removed from the gel and 1.2% agarose was cast in its place. The procedure was repeated using 1.4% agarose. Two DNA standards and a sample of a arbitrarily amplified E. cofi DNA were loaded into each gel concentration and electrophoresed. This allowed for simultaneous comparison of each gel concentration. The DNA standards were a 123 bp ladder (Gibco BRL Research Products Life Technologies Inc.), and Hind111 digested 1 phage. The gel was electrophoresed at 100 volts (2.7 volts cm-‘) for 2 h in 1 x TBE buffer. As the concentration of agarose increased, individual bands were easily distinguished. Concentrations below 1.4% did not resolve the larger fragments and overall the bands appeared diffuse (data not shown). Subsequent AP-PCR reactions were electrophoresed in 1.4% Fisher BioTech agarose gels at 100 volts (2.7 volts cm-‘) for 2.5 h in 1 x TBE buffer. Fifteen ~1 of the AP-PCR reaction was mixed with 3 ~1 of loading buffer which contained 20% Ficoll 400, 0.1 M Na,EDTA pH8, 1.0% SDS, and 0.25% bromophenol blue. Gels were stained in 1 pg ml-’ ethidium bromide for 15 min and destained for up to 1 h in distilled water. A UV transilluminator was used to visualize the banding patterns and photographs were taken with a Polaroid MP-L Land camera using Polaroid 667 black and white film.

optimized in randomly primed PCR reactions

MgCl, mM

Annealing “C

Primer FM

Template #cells

AmpliTaq U

1.5 2.0 2.5 3.0 3.5 4.0

36 40 45 50 55

0.2 0.4 0.8 1.6

lo9 lo* 10’ lo6 lo5 lo4

1.25 2.50 3.75

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3.1. AmpliTaq concentration

polymerase

enzyme

Varying the amount of polymerase enzyme resulted in no amplification below 2.5 U per 50 ~1 reaction (data not shown). AP-PCR reactions which used either 2.5 or 3.75 U resulted in banding patterns that remained the same. No other changes to the banding patterns were seen. Thus, there was no amplification below 2.5 U, and this amount of enzyme was used for the rest of the study. 3.2. MgCl, concentration The

MgCl,

concentration

governs

the

ef-

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Fig. 1. Increasing MgCl, concentration using E. coli DNA as a template. AP-PCR: 10 mM Tris-HCl, 50 mM KCl, 0.1 mg/ml gelatin, 200 PM dNTP, 0.2 pM primer, 2.5 U AmpliTaq polymerase, lo6 cells, and 1.5-4.0 mM MgC& concentration. Thermal cycler conditions: 94°C 15 s denaturation, 36°C 15 s annealing, and 72°C 30 s extension, for 35 cycles. Primer 625. Lanes: 1, 123 bp ladder; 2, negative control; 3, 1.5 mM; 4, 2.0 mM; 5, 2.5 mM; 6, 3.0 mM; 7, 3.5 mM; 8, 4.0 mM.

3. Results

An arbitrary primer should define a set of amplification products that are consistent from one reaction to the next. Although reproducible banding patterns were eventually generated from AP-PCR reactions, it was found that several factors in the reaction influenced the observed banding patterns. The MgCl, concentration, annealing temperature, primer, and template concentration had such influence and therefore were optimized using E. coli ATCC strain 15224. The AP-PCR with all five primers were optimized resulting in unique fingerprints for each primer. Figs. 1-4 illustrate the major results of the optimization.

Fig. 2. Increasing annealing temperature using E. coli DNA as a template. AP-PCR: 10 mM Tris-HCl, 50 mM KCl, 0.1 mg/ml gelatin, 3.5 mM MgCl,, 200 PM dNTP, 0.2 PM primer, 2.5 U AmpliTaq polymerase, and lo6 cells. Thermal cycler conditions: 94°C 15 s denaturation, 36”-55°C 15 s annealing, and 72°C 30 s extension, for 35 cycles. Primer 615. Lanes: 1, negative control; 2, 36°C; 3,4O”C; 4,45”C; 5, 50°C; 6, 55°C; 7. marker.

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ficiency of the Taq polymerase in the PCR reaction. Though it may be possible to vary this for PCR protocols that use specific primers without significant loss of the amplification product of interest, AP-PCR requires a narrower concentration range because incorrect concentrations can result iin poor efficiency of amplification or production of non-specific products [7]. This may be especially true for environmental samples that may contain increased concentrations of magnesium.. Increasing the concentration typically resulted in a banding pattern with more amplification fragments regardless of the primer used in the reaction. The results of optimizing Mg2+ concentration using primer 625 is shown in Fig. 1 for E. coli. When MgCl, was increased in the reaction, there was a shift in amplification from

12

3

4

5

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smaller fragment to larger fragment sizes. The intensity of the amplification products indicated that larger fragments were preferentially amplified when primers 61.5 and 620 were used with increasing Mg*+ concentrations. As shown in Fig. 1 for primer 625, this change in intensity of an ethidium stained fragment was a reflection of the efficiency of amplification during the PCR reaction. Amplification with primers 615 and 620 (data not shown) with increasing Mg*+ yielded results similar to primer 625. In contrast, primers 623 and 682 did not exhibit changes in their banding patterns over the range of concentrations used (data not shown). The Mg*+ concentration above 3.0 mM did not appear to affect the amplified products of any of the five primers. Thus, the 3.5 mM MgCl, was chosen as the optimal concentration to continue the study.

6

7

8

91071

Fig. 3. Increasing primer concentration using E. coli as a template. AP-PCR: 10 mM Tris-HCI, 50 mM KCl, 0.1 mgiml gelatin, 3.5 mM MgCl,, 200 PM dNTP, 0.2-1.6 PM primer, 2.5 U AmpliTaq polymerase, and lo6 cells. Thermal cycler conditions: 94°C 15 s denaturation, 45°C 15 s annealing, and 72°C 30 s extension, for 35 cycles. Primer 615. Lanes: 1, negative control; 2, 0.2 PM; 3, 0.4 PM; 4, 0.8 PM; 5, 1.6 PM; 6, marker; Primer 682 Lanes: 7, negative control; 8, 0.2 PM; 9, 0.4 PM; 10, 0.8 PM; 11, 1.6 PM.

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3.3. Annealing

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temperature

The annealing temperature has been shown to have a noticeable effect on the resulting fragment size and number of products [4,5]. The annealing temperature determines the stringency of the PCR reaction by determining the primer’s ability or inability to bind to the template. Changes of 1 or 2 degrees can cause shifts in the overall size of fragments or result in no amplification of the region of interest [4]. Ideally, increasing the temperature should result in less mismatch between the primer and template. Fig. 2 shows the effect of increasing the annealing temperature on AP-PCR of E. coli using primer 615. An increase in temperature resulted in the primer annealing to sites that generated smaller fragments. Similar results were obtained with primer 620 (data not shown). However, increased annealing temperature for other primers resulted in: a loss of amplification products at 50°C and 55°C (primer 623), amplification of larger fragments (682), or no change (primer 625) (data not shown). Overall, the 45°C temperature was chosen as the annealing temperature for future amplifications because all primers gave unique amplification products at this temperature.

optimal amount of primer, since it generated most complex fingerprints.

the

3.5. Template concentration The template to primer ratio is key to the success of the PCR reaction. No amplification will occur if template concentrations are excessively low or high. Thus, for a reproducible AP-PCR reaction, the template to primer ratio must remain constant [4]. An increasing number of cells, 104-lo9 CFU per reaction, was used to test template/primer interactions for the three isolates using all five primers. Fig. 4 shows the results of increasing the template concentration for B. subtifis. Depending on template con cen-

12345678

3.4. Primer concentration The primer may have inherent characteristics such as excessively high or low percent GC content, or bases at the three prime end of the oligonucleotide, that can influence the generation of fragments [5]. Fig. 3 (primer 615 and 682) shows that as primer concentration increased there was a shift from larger to smaller fragments in the banding pattern from E. co/i. This change in size range of amplification products occurred up to a concentration of 0.4 PM, after which there was no change. Primers 620 and 623 gave results similar to those of primers 615 and 682 (data not shown). Primer 625 showed no bands at the 0.2 PM concentration and generated the same unique banding pattern for 0.4 PM and higher concentrations (data not shown). The 0.4 PM concentration of primer was chosen as the

Fig. 4. Template concentration as increasing number of per reaction. AP-PCR: 10 mM Tris-HCI, 50 mM KCI, 0.1 mg/ml gelatin, 3.5 mM MgCI,, 200 FM dNTP, 0.4 pM primer, 2.5 U AmpliTaq polymerase, and 104-10’ cells. Thermal cycler conditions: 94°C 15 s denaturation, 45°C 15 s annealing, and 72°C 30 s extension, for 35 cycles. B. subtilis Lanes: 1, Negative control, 2, 109, 3, 108, 4, lo’, 5, 106, 6, loS, 7, 104.

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tration, the degree of amplification varied from slight (lane 7) or robust (lanes 3-5). Excessive template resulted in smearing (lane 2). Results were similar for the other four primers tested in the study (data not shown). Assuming a bacterial cell contains approximately 9 fg of DNA [8], 10’ cells ml-’ are equivalent to 0.9 ng DNA pl-I. Fluorometer readings indicated that 10’ CFU ml-’ of lysed cells were equivalent to 6.3 ng p1-’ of DNA approximately 7 times the amount of DNA expected.. There are three possible sources for the extr,a DNA in solution; (1) dead cells in the culture which would have been excluded from viable plate counts; (2) CFUs which resulted from a clump of cells, thus representing more than one cell in the media; and (3) replication of the DNA which precedes cell division in bacteria resulting in multiple genomes in a single cell [9]. 3.6. Individual isolates Finally, the banding patterns resulting from individual isolates with single primers are presented in Fig. 5. ,E. coli, B. subtilis, and P. aeruginosa were amplified using the optimized

E.

coli

B.

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PCR conditions and electrophoresed using the optimized agarose gel concentration. As Fig. 5 illustrates, individual isolates produced unique patterns depending on the primer used. Banding ranged from nonexistent, to simple (1 band) to very complex (up to 9 bands). Some bands were faint while others were very intense. One primer, 615, resulted in a unique pattern of 4 or more bands for each isolate. Primer 682 resulted in a complex pattern for E. coli and simple patterns for B. subtilis and P. aeruginosa. One primer, 625, produced a simple pattern of less than 4 bands for each isolate.

4. Discussion When each parameter of the PCR reaction has been optimized, a primer will reproducibly generate a unique fingerprint [4]. Here, optimization of AP-PCR resulted in reproducible banding patterns for three cultured isolates tested. Each parameter of the reaction was shown to influence the amplification fragments generated. Artifactual variation was eliminated through optimization such that consistent, unique fingerprints were

subtilis

P.

aeruginosa

Fig. 5. Comparison of unique banding patterns generated from five different primers using three individual isolates final optimized conditions. Lanes alternating negative control, 615, 620, 623, 625, 682. 123 bp ladder as shown.

amplified

with

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produced from a particular bacterial isolate. The final optimized PCR reaction conditions were 1 X buffer (3.5 mM MgCl,, 10 mM Tris-HCl, 50 mM KC1 and 0.1 mg ml -’ gelatin), 200 FM dNTP, 0.4 PM primer, 2.5 U AmpliTaq@ (Perkin-Elmer Cetus) polymerase enzyme, and 5 ~1 of template (at least lo6 heat lysed bacterial cells). The Perkin-Elmer Gene-AmpTM 9600 PCR System was used with the following cycling conditions: a 94°C 15 s denaturation step, a 45°C 15 s annealing step, and 72°C 30 s extension step for a total of 35 cycles. AP-PCR reactions were electrophoresed in 1.4% agarose gels at 2.7 V cm-‘. One difference between optimal conditions for normal PCR and AP-PCR that was highlighted by this study is the response to annealing temperature. In PCR that uses two defined primers, increased annealing temperatures normally result in more specific amplification and less nonspecific amplification. In contrast, for AP-PCR, banding patterns showed an increase in small fragments in response to increased temperature. This may be due to more accessible annealing sites that result from structural changes in genomit DNA at increased temperature [5]. The subsequent banding patterns that resulted from a higher annealing temperature were reproducible. This temperature is higher than the 36°C annealing temperature previously reported in the literature for AP-PCR [2] and should result in less primer-template mismatches. It is clear that the influence of annealing temperature on arbitrarily primed PCR is complex and should be evaluated empirically for any given primer [lo]. The shift from large amplification fragments to smaller fragments with increasing primer concentration suggests that the primer was able to insert more frequently into the template when more primer was available. For an amplification product to be generated using a single primer, the primer must insert along both complementary stands of the template. A primer that inserts at only one strand of the template will not have a defined boundary and will increase arithmetically in the PCR reaction rather than exponentially, so that no banding product results from that primer insert.

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Some bacterial isolates resulted in low intensity fingerprints, for example, E. coli (primer 623), B. subtilis (primers 620, 623, 625 and 682), and Z? aeruginosa (primer 620). Ethidium bromide staining is semi-quantitative resulting in increased intensity with increased product. These low intensity banding fragments may be due to inefficient amplification that result in less amplification product being formed [3,6]. There are several factors that may have caused this. One may be inherent template characteristics or complex template/primer interactions. CaetanoAnolles [5] postulated that the formation of hairpin loops by the complementary ends of newly formed products inhibit primer binding. Another possibility for weak amplification of B. subtilis, for which 4 of the primers gave a weak pattern may have been due to the fact that Bacillus cells are Gram positive and the boiling lysis preparation used was not effective at removing interfering cell debris. Boiling lysis followed by a rapid centrifugation has been found to give reliable and reproducible banding patterns for Bacillus cells [12]. However, this was not necessary for the Gram negative bacteria used in this study. Although arbitrary primers with higher GC contents have been shown to result in a greater number of amplification products [6] B. subtilis DNA has a lower percent GC content, 30%) than either E. coli which is 51% or P. aeruginosa with 68%. All of the primers used had GC contents 70% or greater which could have resulted in less base complementation for Bacillus. These data show that for identification of unknown isolates by AP-PCR, it may be necessary to use a set of individual arbitrary primers to ensure that a reasonable fingerprint is obtained from one of the primers. In addition, it is critical that the operating AP-PCR parameters are strictly defined and adhered to when comparing DNA from different sources. This is also true when attempts are made to duplicate results from another laboratory, and may be the reason for inconsistent banding associated with a specific primer and bacterial species. AP-PCR is a useful diagnostic tool provided care and consistency are used. It may be par-

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titularly useful for identification of pure culture isolates of unknown origin. As shown here, each genus of bacteria generated a unique banding pattern. In future studies, these patterns could be scanned and digitized with a computer to allow characterization of the fingerprint. This may allow creation of a database consisting of a library of fingerprinted organisms that could subsequently be used to screen and identify unknown bacterial isolates. Alternatively, high performance liquid chromatography or capillary gel electrophoresis could be used to quantitate the amount of each amplified DNA fragment. If fingerprint patterns are additive, AP-PCR may also be of value to researchers to distinguish mixed populations of bacteria, particularly in environmental samples.

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141 Penner, G.A., A. Bush, R. Wise, W. Kim, L. Domier, K, Kasha, A. Laroche, G. Stoles, S.J. Moinar, and G. Fedak (1993) Reproducibility of arbitrary amplified polymorphic DNA (RAPD) analysis among laboratories. PCR Methods Applic. 9, 341-345. [51 Caetano-Anolles, G., B.J. Bassam, and P.M. Gresshoff (1992) Primer-template interactions during DNA amplification fingerprinting with single arbitrary oligonucleotides. Molec. Gen. Genet. 235, 157-165. 161 Caetano-Anollis, G., B.J. Bassam, and P.M. Gresshoff (1991) DNA amplification fingerprinting using very short arbitrary oligonucleotide primers. Bio/Technology 9, 553-557. [71 Saiki, R.K (1989) The design and optimization of the PCR. p 7-16. In: PCR Technology Ed. H.A. Erlich. Stockton Press, New York. PI Ingraham, J.L., 0. Maaloe, and F.C. Neidhardt (1983) Growth of the bacterial cell, p. l-48. Sinauer Associates, Inc., Sunderland, MA. 191 Krawiec, S. and M. Riley (1990) Organization of the bacterial chromosome. Microbial. Rev. 54, 502-539. WI Rychlik, W., W.J. Spencer, and R.E. Rhoads (1990) Optimization of the annealing temperature for DNA amplification in vitro. Nucleic Acids Res. 18, 64096412. WI Josephson, K.L., S.D. Pillai, J. Way, C.P. Gerba, and I.L. Pepper (1991) Fecal coliforms in soil detected by polymerase chain reaction and DNA-DNA hybridizations. Soil Sci. Sot. Am. J. 55, 1326-1332. L. WI Brousseau, R., A. Saint-Onge, G. Prefontaine, Masson, and J. Cabana (1993) Arbitrary primer polymerase chain reaction, a powerful method to identify Bacillus thuringiensis serovars and strains. Applied Environ. Microbial. 59, 114-119.