COMPARISON OF THREE PROBE LABELLING METHODS TO DETECT PCR AMPLIFICATION PRODUCTS SURESH D. PILLAI,'.' KAREN L. JOSEPHSON and IAN L. PEPPER Department of Soil and Water Science Universiry of Arizona Tucson, AZ 85721 Accepted for Publication September 3, 1993 ABSTRACT Polymerase chain reaction amplijications are Jnding increased applications in environmental microbiology. l h e development of sensitive and specijic methods to detect amplijiedproducts is necessary especially when these amplifications are conducted in the presence of the environmental matrix. Gene probes specijic to the nptl 1 locus were prepared by nick translation, 5' end labelling and by a PCR driven amplijication. lhese probes were tested against a 300 bp PCR amplified segment of the nptl 1 region of the transposable element Tn5. Ihe nick translated probe was the most sensitive, though not as specijic as the other two types of probes. Sensitivity and specijicity were found to be dependent on the hybridiza- tionformat (Southern blots versus dot blots), the number of amplijication cycles and on the purity of the target sequence. INTRODUCTION The Polymerase Chain Reaction amplification (Saiki et al. 1988) has found applications in molecular biology (Rich and Willis 1990), in clinical medicine, epidemiology (Yolken et al. 1991), and for pathogen detection in the environ- ment (Josephson et al. 1991; Mahbubani et al. 1991). For commercialization of this technique, the manual operations involved in the reaction setup and product detection need to be simplified and made user friend- ly. Currently, the amplification products are detected by a variety of methods 'Presently at the Texas A&M University Research Center, El Paso, TX 79927. Torresponding author: EnvironmentalSciences Program, Texas A&M University Research Center, 1380 A&M Circle, El Paso, TX 79927. Tel: (915) 859-9111 Fax: (915) 859-1078. Journal of Rapid Methods and Automation in Microbiology 2 (1994) 299-309. All Rights Resented. 299 0 Copyright 1994 by Food & Nutrition Press, Inc., Trumbull, Connecticut
Transcript
COMPARISON OF THREE PROBE LABELLING METHODS TO DETECT PCR AMPLIFICATION PRODUCTS
SURESH D. PILLAI,'.' KAREN L. JOSEPHSON and IAN L. PEPPER
Department of Soil and Water Science Universiry of Arizona
Tucson, AZ 85721
Accepted for Publication September 3, 1993
ABSTRACT
Polymerase chain reaction amplijications are Jnding increased applications in environmental microbiology. lhe development of sensitive and specijic methods to detect amplijied products is necessary especially when these amplifications are conducted in the presence of the environmental matrix. Gene probes specijic to the nptl 1 locus were prepared by nick translation, 5' end labelling and by a PCR driven amplijication. lhese probes were tested against a 300 bp PCR amplified segment of the nptl 1 region of the transposable element Tn5. Ihe nick translated probe was the most sensitive, though not as specijic as the other two types of probes. Sensitivity and specijicity were found to be dependent on the hybridiza- tion format (Southern blots versus dot blots), the number of amplijication cycles and on the purity of the target sequence.
INTRODUCTION
The Polymerase Chain Reaction amplification (Saiki et al. 1988) has found applications in molecular biology (Rich and Willis 1990), in clinical medicine, epidemiology (Yolken et al. 1991), and for pathogen detection in the environ- ment (Josephson et al. 1991; Mahbubani et al. 1991).
For commercialization of this technique, the manual operations involved in the reaction setup and product detection need to be simplified and made user friend- ly. Currently, the amplification products are detected by a variety of methods
'Presently at the Texas A&M University Research Center, El Paso, TX 79927. Torresponding author: Environmental Sciences Program, Texas A&M University Research Center, 1380 A&M Circle, El Paso, TX 79927. Tel: (915) 859-9111 Fax: (915) 859-1078.
Journal of Rapid Methods and Automation in Microbiology 2 (1994) 299-309. All Rights Resented. 299 0 Copyright 1994 by Food & Nutrition Press, Inc., Trumbull, Connecticut
300 S.D. PILLAI, K.L. JOSEPHSON and I.L. PEPPER
including electrophoresis, HPLC and a silicon based potentiometric sensor system (Katz et al. 1990; Olson ef al. 1991). Detection of PCR products employing gel electrophoresis though relatively inexpensive and rapid, has drawbacks such as, the detection being a function of the amount of the product analyzed, the gel com- position and the staining efficiency.
The use of genetic probes to detect amplification products may be unnecessary when purified nucleic acid preparations are used as amplification targets, e.g., in cloning procedures. However, improved detection methods may be necessary when only crude nucleic acid targets are available such as in the food processing industry, clinical laboratories, plant nurseries and waste water treatment plants (Pillai ct al. 1992; Tsai et al. 1993). In these situations, genetic probes are used to detect extremely low amounts of the amplified product. The low product yield in these situations are due to primarily, the matrix effects of the sample rather than the efficacy of the primer and, or the reaction conditions. Under these cir- cumstances, a variety of approaches including “double PCR” (Pillai ef al. 1991), “magnetic immuno-PCR” (Fluit et al. 1993) and high specific activity probes are often employed to improve the amplification efficiency.
The primary objective of this study was to compare probes labelled in three different ways (viz., 5 ’ end label, nick translation and PCR generation), in terms of their sensitivity and specificity to detect a 300 bp PCR product of the npf l l region of the transposon Tn5. The targets for amplification were bacterial cell lysates and purified genomic DNA preparations. The aim was to determine which of the labelling methods provided a specific and sensitive probe to detect PCR products that may not be in optimal amounts.
MATERIALS AND METHODS
Target Sequences for PCR Amplification Neomycin phosphotransferase type I1 (npf l l ) DNA sequences were obtained
from the bacterium Rhizobium leguminosarum biovar phaseoli UAZ 304: : Tn5 (Pillai and Pepper 1991). The negative control used in these studies was CE-3, the wild type strain without the transposon. Total genomic DNA extraction was performed and the DNA concentrations expressed on the basis of the amount of the 300 bp target region (Pillai et al. 1991).
The DNA samples for PCR amplification were also obtained from crude cell lysates by a method described earlier by Sandhu et al. (1989). In this method, intact bacterial cells were added to the PCR reaction mixture and lysed by heating to 98C for 20 min (without the addition of Taq polymerase), and then cooled to mom temperature for 5-10 min to allow primers to anneal to the target region.
COMPARISON OF THREE PROBES 30 1
Taq polymerase was later added, and the PCR reactions were performed using the GeneAmp reagent kit and the DNA Thermal cycler (Perkin-Elmer Cetus Corp, Norwalk, CT).
PCR Reactions
Primers specific for the nptll region of T‘ (5 ’ CAC TGA AGC GGG AAA GGA CT 3 ’ and 5 AGA TCC TCG CCG TCG GGC AT 3 ’) were designed from the structural region of the gene encoding the enzyme neomycin phosphotransferase and synthesized using a DNA synthesizer (Applied Biosystems Inc., Foster City, CA). The primers had earlier been shown to be specific for the 300 bp nptll sequence (Pillai et al. 1991). The PCR reaction conditions were optimized using 3.5 mM MgC12, and cycling conditions were as follows: 94C, 1.5 min; 6OC, 1 min; 72C, 1 min. PCR amplification was conducted for 25 cycles. In order to evaluate the effect of performing a “double PCR” (Pillai er al. 1991) on the detection sensitivity using gene probes, 10 pl of the amplified product from 25 cycles was used as template for a further 25 cycles using the same conditions but fresh reagents. The products from this “double PCR” protocol were also analyzed in a similar manner to that of the initial 25 cycles.
Ten microliters of the reaction products were analyzed using 1.6% agarose gel electrophoresis in TBE buffer (0.089 M Tris; 0.089 M Boric acid; 0.002 M EDTA; pH 8.0) at 4 V/cm for a maximum of 2 h. The gels were stained for 15 min with ethidium bromide (1 pg/pl), and visualized using a UV trans- illuminator and the amplification products photographed using a PolaroidTM camera and Type 55 film.
Southern blots of Gene Screen Plus nylon membranes (Du Pont Wilmington, DE) were prepared using standard methods (Ausubel et al. 1987). For dot blots, 10 p1 of the product was denatured in 100 pl of 0.25N NaOH for 10 min. The denatured samples were blotted onto pretreated (0.4M Tris. HCl pH 7.5,30 min) nylon membranes using a MinifoldTM microsample filtration manifold (Schleicher and Schuell, Keene, NH). The DNA samples were fixed onto the membranes by baking the membranes under vacuum at 80C.
Preparation of Probes
as follows: Three types of gene probes specific to the nptll region of Tn5 were prepared
5 End Labelled Probe. A 5 ’ end labelled probe specific to an internal region of the amplified product was prepared using the forward reaction of the T,
302 S.D. PILLAI, K.L. JOSEPHSON and I.L. PEPPER
polynucleotide kinase (Ausubel et al. 1987). This end labelled probe was prepared using 200 ng of a 20 base oligomer (GGC TGA TGC AAT GCG GC) in a reac- tion mix containing 16 U of T4 kinase (Promega, Madison, WI), 5 p1 of 1OX T4 kinase buffer, and y32P ATP (3000 Ci/mol) (Amersham, Arlington Heights, IL). The reaction was performed at 37C for 45 min. The probe was purified us- ing a Sephadex G-50 spun column, and had specific activities averaging 1 x 105 cpdp l .
PCR Primed Probe. The PCR primed probe was prepared using 20 pCi of a! 3*P dCTP (NEN, DuPont, Wilmington, DE) along with 10 pM dATP, dTTP and dGTP. Template (plasmid pRz-l02::lh5), nptll specific primers, nucleotides and the Taq polymerase were added in a final volume of 100 pl. The PCR cycl- ing conditions were as those described above. At the end of 25 cycles, unincor- porated nucleotides, primers, etc., were separated from the labelled probe using Sephadex G-50 spun columns. The probes had specific activities averaging 1 X 106 cpdpl .
Nick Translated Probe. One microgram of plasmid pRZ-l02::Tn5 was nick translated using 100 pCi of C Y ~ ~ ~ C T P (NEN, DuPont, Wilmington, DE) along with 200 pM of each of dATP, dTTP, dGTP and 1 p1 of DNA PolI in a nick translation buffer for 60 min at 15C. The probe was purified on the spun column and the specific activity checked in a liquid scintillation counter. The nick translated probes averaged 1 X lo6 cpm/pl.
Hybridization and Washing Conditions
All hybridizations were conducted at 50C using a rotary hybridization chamber (Robbins Scientific, Mountain View, CA). The hybridizations were all conducted for at least 12 h in the hybridization buffer, which contained 1 % SDS, 1M NaCl and 10% dextran sulfate. On completion of the hybridization, the filters were washed as follows: 2 washes for 5 min each using 2X SSC at room temperature and one wash at 50C for 30 min using 0.2X SSC and 1 % SDS. Under these con- ditions it was empirically determined (Yang and Wu 1979; Howley et al. 1979) that only hybrids that had less than 3 1 % mismatch were stable. Thus, only those hybrids having 60% or greater homology (between the probe and the target) were stable. The hybridization results were recorded using autoradiography for a max- imum of 48 h on Kodak XR-5 film. In order to maintain standardized probing conditions, the filters were reprobed using the different probes. The probes were stripped off the filters by boiling the filters in distilled water containing 0.01 % SDS for 5 min. The filters were exposed to Xray film for a minimum of 16 h to confirm the complete removal of the probes before they were reprobed.
COMPARISON OF THREE PROBES 303
RESULTS
Genomic DNA Figure 1 shows the detection of PCR amplified nprll sequence from purified
genomic DNA preparations, when analyzed using ethidium bromide staining, and its corresponding Southern blot using a PCR primed probe. Lanes C through G are products from 25 amplification cycles while lanes I through 0 are from 50 cycles (“double PCR”). Ethidium bromide staining detected 0.12 pg of target DNA irrespective of whether 25 cycles (lane G) or 50 cycles (lane M) were per- formed. A smear of nonspecific primer dimer bands resulted with 50 cycles. It is evident that high concentrations of target DNA (350 pg) (lanes C and I) can inhibit formation of a specific product.
When the Southern blot was probed using the PCR primed probe, the detec- tion sensitivity was 0.12 pg of target. There was no significant difference in sen- sitivity when compared to ethidium bromide staining. Signals from the “double PCR” were relatively stronger than those after 25 cycles of PCR. The signals
A B C D E F G t i ! J K L 1.1 f J ! ?
FIG. 1. DETECTION OF nprZZ FROM PURIFIED GENOMIC DNA AFTER PCR AMPLIFICATION AND HYBRIDIZATION USING A PCR PRIMED PROBE
Lanes A through G represent products from 25 amplification cycles and Lanes H through 0 represent “double PCR” (50 cycles) products. Lane A: 300 bp size marker; B: CE-3 (-ve control); C: 350 pg; D: 24 pg; E: 2.4 pg; G: 0.12 pg. Lane H: CE-3 (-ve control); I: 350 pg; J: 24 pg;
were also much stronger than that of the end labelled probe (Fig. 2). When probed using the end labelled probe, the sensitivity was only 0.24 pg after a “double PCR” run (Lane L). Figure 3 shows the detection of the same products when probed using the nick translated probe. When this probe was employed, the detec- tion sensitivity improved to 12 fg (Lane N) after 50 cycles, but there was no in- crease in its sensitivity compared to the other probes when the products of only 25 cycles were compared.
When the same PCR products were dot blotted and probed using a 5 ’ end labelled probe, the product was just discernable at 0.24 pg (Lane E) (Fig. 4), which was similar to the Southern blot result. It was also evident that there was no signifi- cant difference in the sensitivity of detection regardless of whether 25 or 50 cycles were performed. When the same amplification products were probed with a nick translated probe, the detection limit increased to 0.12 fg but nonspecific signals were evident on the negative control (CE-3) samples (data not included).
FIG. 2. DETECTION OF nptU FROM PURIFIED GENOMIC DNA AFTER PCR AMPLIFICATION AND HYBRIDIZATION USING A 5 ’ END LABELLED PROBE Lanes A through G repreamt products from 25 amplibtion cycles and Lanes H through 0 represent “double PCR” (50 cycles) products. Lane A: 300 bp size marker; B: CE-3 (-ve control); C: 350 pg; D: 24 pg; E: 2.4 pg; F: 0.24 pg; 0: 0.12 pg. Lane H: CE-3 (-ve control); I: 350 pg; J: 24 pg; K: 2.4 pg; L 0.24 pg; M: 0.12 pg; N: 12 fg; 0: 1.2 fg.
COMPARISON OF THREE PROBES 305
Crude Cell Lysates
Figure 5 shows the ethidium bromide stained amplification products, and its corresponding Southern blot when probed using a 5 ’ end labelled probe. After 25 cycles, lo4 CFU were detectable (Lane B) while after the double PCR (50 cycles), a distinct band was evident even from lo3 CFU (Lane J). Use of the end labelled probe did not improve the sensitivity of detection. Corresponding data showing gene probe analysis of these products on a Southern blot by a nick translated probe gave similar results (figure not included).
When the products were dot blot analyzed, there was a significant difference between the nick translated and the other two types of probes (Fig. 6). When the end labelled and PCR primed probes (Panel A and Panel B) were employed, lo2 CFU were detected after 50 cycles (M). The nick translated probe (Panel C) produced nonspecific hybridization signals in the negative control (N).
‘t h I ) t I (, i( I ! i M t i ,
FIG. 3. DETECTION OF npfZ1 FROM PURIFIED GENOMIC DNA AFTER PCR AMPLIFICATION AND HYBRIDIZATION USING A NICK TRANSLATED PROBE
Lanes A through G represent products from 25 amplification cycles and Lanes H through 0 represent “double PCR” (50 cycles) products. Lane A: 300 bp size marker; B: CE-3 (0-ve control); C: 350 pg; D: 24 pg; E: 2.4 pg; G: 0.12 pg. Lane H: CE-3 (-ve control); I: 350 pg; J: 24 pg;
FIG. 4. DOT BLOT ANALYSIS OF PCR AMPLIFIED nptll FROM PURIFIED GENOMIC DNA USING A 5 ’ END LABELLED PROBE
A: -ve control (no DNA template); B: 350 pg; C: 24 pg; D: 2.4 pg; E: 0.24 pg; F: 120 fg; G: 12 fg; H: 1.2 fg; 1: 0.12 fg; J: 0.012 fg; K: -ve control (CE-3).
A B C D E F G H I J K L M N
a B C D E F G H I . J K L M N
FIG. 5. DETECTION OF nprll FROM CRUDE CELL LYSATES AFTER PCR AMPLIFICATION AND HYBRIDIZATION USING A 5 ’ END LABELLED PROBE Lana A through G represent pmducts from 25 amplification cycles and Lanes H through N
represent “double PCR’ (50 cycles) products from the same starting template concentrations as in the 25 cycles. A: 105 CFU; B: l(r CFU; C: 18 CFU; D: 102 CFU;
E: 10’ CFU; F: 100 CFU; G: -ve control (CE-3).
DISCUSSION
This study has shown that gene probe analysis of FCR products is not necessarily always more sensitive than ethidium bromide staining (Fig. 1 and Fig. 2). It is evident that the nick translated probe generally improved the detection sensitiv- ity; however, this probe exhibited nonspecific signals when employed in a dot
COMPARISON OF THREE PROBES 301
A B C D E F G H I J
FIG. 6. DOT BLOT ANALYSIS OF PCR AMPLIFIED nptll FROM CRUDE CELL LYSATES
Panel A: 5’ end labelled probe, Panel B: PCR primed probe, Panel C: Nick translated probe.
blot format (Fig. 3 and Fig. 6). When comparing the detection of PCR products in a Southern blot format to that of a dot blot format, it was observed that the dot blot format produced more intense signals than the Southern blot. This relative decrease in signal intensity and sensitivity in Southern blots can be attributed to the transfer efficiency from gel to membrane. Also, the dot blots concentrate more of the possible targets than a Southern blot filter.
The appearance of nonspecific signals in the negative controls in the dot blots (Fig. 6) can be due to the probe molecules nonspecifically hybridizing to the samples due to the increased concentration of nucleic acids rather than, hybridiza- tion due to purely base pair homologies. In addition, the weakly positive signals could also be due to the probes hybridizing to the primer dimers. The differences in sensitivities obtained by using the different probes could also be related to the specific activities, with the nick translated and the PCR primed probe having greater label incorporation than the end labelled probe, which has a terminal label.
Thus, the use of a particular labelling method and the type of analysis to detect PCR products need to be carefully evaluated. In cases where the presence or absence of a specific product needs to be evaluated, Southern blots using high specific activity probes are advantageous, whereas, in cases where all possible products are to be detected, the use of dot blots and high specific activity probes may be appropriate.
308 S.D. PILLAI, K.L. JOSEPHSON and I.L. PEPPER
ACKNOWLEDGMENT
This work was supported in part by the Arizona Disease Control Commission Contract No. 821693.
RETEZENCES
AUSUBEL, F.M., BRENT, R., KINGDOM, R.E., MOORE, D.D., SMITH, J.A., SIDEMAN, S.G. and STRUHL, K . 1987. Current Protocols in Molecular Biology. John Wiley & Sons, New York.
FLUIT, A.C., WIDJOJOATMODJO, M. N., BOX, A.T.A., TORENSMA, R. and VERHOEF, J. 1993. Rapid detection of Salmonellae in poultry with the magnetic immuno-polymerase chain reaction assay. Appl. Environ. Microbiol.
HOWLEY, P.M., ISRAEL, M.A., LAW, M-F. andMARTIN, M.A. 1979. A rapid method for detecting and mapping homology between heterologous DNA. Evaluation of polyomavirus genome. J. Biochem. 254, 48764883.
JOSEPHSON, K.L., PILLAI, S.D., WAY, J., GERBA, C.P. and PEPPER, I.L. 1991. Fecal coliforms in soil detected by polymerase chain reaction and DNA- DNA hybridizations. Soil Sci. Soc. Am. 3. 55, 1326-1332.
KATZ, E.D., HAFF, L.A. and EKSTEEN, R. 1990. Rapid separation, Quan- titation and purification of products of polymerase chain reaction by liquid chromatography. J. Chrom. 512, 433-444.
MAHBUBANI, M.H., BET, A.K., PERLIN, M., SCHAEFER 111, F.W., JAKUBOWSKI, W. and ATLAS, R.M. 1991. Detection of Giardia cysts by using the polymerase chain reaction and distinguishing live from dead cysts. Appl. Environ. Microbiol. 57, 3456-3467.
titation of DNA hybridization in a silicon sensor based system: application to PCR. Mol. and Cell. Probes 5, 351-358.
PER, I.L. 1991. Rapid method for processing soil samples for polymerase chain reaction amplification of specific gene sequences. AppE. Environ. Microbiol. 57, 2283-2286.
PILLAI, S.D., JOSEPHSON, K.L. and PEPPER, I.L. 1993. Specific detection of rhizobia in root nodules and soil using the polymerase chain reaction. Soil Biol. Biochem. 24, 885-891.
PILLAI, S.D. and PEPPER, I.L. 1991. Transposon Tn5 as an identifiable marker in rhizobia: survival and genetic stability of Tn5 mutant bean rhizobia under temperature stressed conditions in desert soils. Microb. Ecol. 21, 21-33.
PILLAI, S.D., JOSEPHSON, K.L., BAILEY, R.L., GERBA, C.P. and PEP-
COMPARISON OF THREE PROBES 309
RICH, J.J. and WILLIS, D.K. 1990. A single oligonucleotide can be used to rapidly isolate DNA sequences flanking a transposon Tn5 insertion by the polymerase chain reaction. Nuc. Acid. Res. 18, 6673-6676.
S A W , R.K. et al. 1988. Primer directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239, 487494.
SANDHU, G.S., PRECUP, J.W. and K I N E , B.C. 1989. Rapid one step characterization of recombinant vectors by direct analysis of transformed Escherichia coli colonies. Biotechniques 7, 689-690.
TSAI, Y-L., PALMER, C.J. and SANGERMANO, L.R. 1993. Detection of Escherichiu coli in sewage and sludge by polymerase chain reaction. Appl. Environ. Microbiol. 59, 353-357.
YANG, R.C.A. and WU, R. 1979. Bk virus DNA sequence: extent of homology with simian virus 40 DNA. Proc. Nat. Acad. Sci. 76, 1179-1183.
YOLKEN, R.H., LI, S., PERMAN, J. and VISCIDI, R. 1991. Persistent diar- rhea and fecal shedding of retroviral nucleic acids in children infected with human immunodeficiency virus. J. Infect. Dis. 164, 61-66.
