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A posit1ve control for detecting heteroduplexes in DGGE for microbial community fingerprinting.

Abstract.--Denaturing gradient gel electrophoresis in combination with PCR has found a wide application for analysis of genetic variants in many disciplines of bioscience, especially evaluation of microbial diversity in environmental samples. However, amplification of an environmental sample containing multiple DNA species can lead to formation of heteroduplexes (HDs), a PCR artifact. Appearance of HDs on the denaturing gradient gel can lead to a skewed interpretation of results and inaccurate conclusions. The present study is designed to develop a simple methodology to verify presence or absence of heteroduplexes in PCR samples which in conjunction with 'reconditioned' PCR can be used as an effective control to indicate presence and positions of HDs during DGGE analysis. Identification of HDs can allow effective design of reconditioned PCR by varying template and primer amounts and PCR cycle number.

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Denaturing gradient gel electrophoresis (DGGE) is a powerful technique for separation of double-stranded DNA molecules of nearly identical size according to their nucleotide composition. This technique has a wide range of applications, from detection of point mutations to evaluation of microbial community diversity. DGGE exploits the phenomenon of discrete melting behavior of double stranded DNA. PCR-amplified fragments are separated during electrophoresis according to their melting behavior on a polyacrylamide gel with a linearly increasing denaturant gradient. The resulting DGGE band patterns reflect sequence diversity in a given sample. However, during the final PCR cycles when the concentration of primers is low, heteroduplexes (HDs) can form. HDs result in "phantom bands" on the denaturing gradient gel and often are erroneously interpreted as additional DNA species or sequence variants (Acinas et al. 2005). This can lead to overestimation of sequence population diversity. Cloning of PCR products from a mixed DNA template sample can exacerbate the problem. The non-directed mismatch repair system of E. coli generates a number of novel sequences from a single HD clone (Ruano 1992; Qiu et al. 2001). Other screening methodologies which utilize PCR products such as RFLP and Thermal Gradient Gel Electrophoresis (TGGE) are prone to the same problem (Osborn & Moore 2000).

In the past there were a number of reports of PCR artifacts pertaining to DGGE. With the use of constant denaturing capillary electrophoresis, formation of HDs was demonstrated and a simple "reconditioning PCR" suggested (Jensen 1993; Thompson & Marcelino 2002) to reduce the number of HDs produced. In another study to eliminate HDs, samples were treated by T7 endonuclease (Qiu et al. 2001). Surprisingly, a recent literature search revealed neither evidence of research being conducted according to suggested improvements nor a general awareness of the problem within the DGGE community. This is probably due to a fact that in spite of recommended methods, researchers lack control measures to judge the absence or presence of HDs. Therefore, this study attempts to identify the presence and positions of HDs on DGGE gels and suggest a simple methodology which can serve as a positive control to indicate the presence of HDs on DGGE gels.

MATERIAL AND METHODS

DNA extraction and amplification.--Three groups of samples were prepared from 119bp fragments of 18S rDNA from the protistian parasites Eimeria tenella (A), Eimeria maxima (B) and Eimeria acervulina (C). Group I contained seven mixes: A only, B only, C only, AB, AC, BC and ABC. Group II contained group I mixes which were subjected to heating to 92[degrees]C and then allowed to cool to room temperature. The heating treatment was done in order to mimic one denaturing step of the PCR cycle in absence of primers. Group III was prepared by PCR (50 [micro]l final volume) using group I mixes as templates (0.5 [micro]l) with Eimeria species-specific primers (forward primer containing GC-clamp 5'-GCCCGCCGCGCCCGCGCCCGTCCGCCGCCCCCGCCCGGATT AGATACAAAACCAACCC-3', and reverse primer 5'-GCTGATA GGTCAGAAACTTG-3', 0.8 ng/[micro]l final concentration for both primers). The amplification process was carried out using 25 [micro]1 of JumpStart[TM] REDTaq[R] ReadyMix[TM] PCR Reaction Mix (Sigma Chemical Co., St. Louis, MO) in a Mastercycler (Eppendorf, Scientific Inc., Westbury, NY) according to the following program: initial denaturation at 94[degrees]C for 3 min followed by 35 cycles of denaturation at 92[degrees]C for 45 s, annealing at 60[degrees]C for 35 s, and an extension at 72[degrees]C for 2 min. The final extension was performed at 72[degrees]C for 7 min. To demonstrate formation of HDs as a function of PCR cycles, a sample containing all three fragments (ABC), was amplified using the same set of primers and amplification conditions as described above but with a reduced number of cycles (20, 16, 15, 14, 13 and 12 cycles). All PCR products were purified with a Wizard PCR purification kit (Promega, Inc., Madison, WI) and run on a 1% agarose gel to check for quality of amplification.

DNA extraction and amplification from a field sample.--Fecal field samples from a poultry farm located in east Texas were collected and used for DNA extraction. The birds were infected with several species of Eimeria. The extractions were carried out using the QIAamp DNA Stool Mini Kit QIAGEN (QIAGEN, Valencia, CA). The extracted DNA (0.5ul) was used as a template for PCR reactions with the same set of primers and PCR conditions as described above. The product of the PCR reaction (0.5ul) was used as a template to carry out three cycles of reconditioned PCR. One field sample was used to carry out a series of PCR reactions with an increasing number of cycles from 11 to 23.

DGGE analysis.--Polyacrylamide gels (8%, 0.75mm thick) were prepared using a 35-45% gradient of urea-formamide mix (Myers & Maniatis 1987). Electrophoresis was conducted in 1 x TAE buffer at 60[degrees]C using a DGGE-2001 apparatus (CBS Scientific Co., Del Mar, CA). After electrophoresis, the gels were stained with SYBR Green I and subsequently photographed using a BioRad Imager System equipped with a Gel Doc XR camera and Quantity-One software (BioRad Inc., Hercules, CA).

RESULTS

Formation of HDs.--We demonstrated the formation of HDs using a simple system on denaturing gradient (DG) gel. The model system contained the PCR products from the single A, B, and C fragments and their mixture ABC which we loaded on the DG gel (Group I) to show that no heteroduplexes were present in the original single template PCR products or in their mixtures. The heat-treated samples (Group II) along with the PCR products (Group III) were also loaded on the gel (Figure 1). As revealed by DGGE analysis, the PCR products from mixtures BC, AC, and AB contained in addition to the expected two original bands, two HD bands located higher on the gel than their parental bands (Group III, Figure 1). In the same group, the PCR product of the ABC mixture template showed five bands in addition to the three parental bands (Six HD bands were expected, however due to co-migration only five bands were visible). The HD bands represent HDs derived from all possible matches of the parental single strands. By matching the band patterns from the dual mixture samples (Group III) the origin of all the HD bands can be traced back to their corresponding parental bands. Group II samples which included the heat treated mixtures (ABC, BC, AC, and AB) showed striking similarity to their mixed template PCR counterparts from Group III. Moreover, by comparing bands from Group I and Group II or III it is possible to clearly identify HDs by a simple elimination of common bands.

[FIGURE 1 OMITTED]

Elimination of HDs.--The presence of identical HDs in Group II and Group III as shown on Figure 1 leads to a conclusion that a single denaturation step in absence of primers is sufficient for detection of HDs. After the denaturation step during the last cycles of PCR when the primer:template ratio is low, single strands can rehybridize and form all possible combinations of hetero- and homoduplexes (Ruano 1992; Jensen 1993). To determine the first PCR cycle when HDs can be detected, PCR products of the ABC mixture obtained from six amplification reactions with decreasing number of cycles were analyzed by DGGE (Figure 2). Loading Set I contained equal volumes of PCR products from every reaction. Loading Set II contained adjusted volumes of the same set of PCR products as in Loading Set I. The volumes were adjusted to emphasize that the disappearance of the HD bands was due to reduction of PCR cycles and not due to a lower amount of PCR product. Therefore, volumes of samples with less PCR product were increased to confirm the absence of HDs. Detection of HDs at different PCR cycles is complicated by different parameters such as PCR efficiency and initial copy number of template DNA therefore the proposed control measure is required to judge the presence, absence and position of HDs.

[FIGURE 2 OMITTED]

Environmental sample.--Elimination of HDs by reduction of PCR cycles was tested using a field sample of unknown template composition. A series of PCR reactions was performed with reduced numbers of cycles from 22 to 11 (Figure 3). Sixteen cycles were required to reach the detection limit on agarose gel (Figure 3, A). The DGGE analysis of the corresponding reactions demonstrated the gradual appearance of HDs in the upper part of the gel (Figure 3, B). No HD bands were detected before the 18th cycle. Thus, a controlled reduction in the number of PCR cycles demonstrates formation of HDs as a function of PCR cycles. However, elimination of HDs by the PCR cycle reduction strategy is not practical for a large number of samples.

[FIGURE 3 OMITTED]

Elimination of HDs by PCR reconditioning carried out with six field samples (Figure 4) showed a marked decrease in number of bands in reconditioned samples. Most of the HDs as expected were found in the upper part of the gel, however several were found in the lower part. In addition, we noted several bands in the lower part of the gel which were present in the reconditioned samples and absent in the heat treated samples pointing out that any given DNA species can disappear by forming HDs.

[FIGURE 4 OMITTED]

DISCUSSION

The ability of denatured nucleic acid molecules to renature or hybridize forms the basis for numerous methods and applications including southern blot, northern blot and PCR. This same property, if not accounted for, can lead to the misinterpretation of results (Thompson & Marcelino 2002). In the present study using DGGE, clear evidence was obtained of quantitative and qualitative parameters that effect HD formation. These are not the artifactual bands which can form during the late PCR cycles in a single template containing samples due to secondary structure formations (Janse et al. 2004).

This study demonstrates by using a simple model system where mixtures of double stranded homologous molecules were subjected to heating and cooling, that any two homoduplexes can form heteroduplexes in a single step. For instance [A.sup.-][A.sup.+], where [A.sup.-] designates the antisense and [A.sup.+] the sense strand of DNA respectively, and [B.sup.-][B.sup.+] require a single step of denaturation-renaturation to produce two HDs [A.sup.-][B.sup.+] and [A.sup.+][B.sup.-] (Group II, Figure 1). Quantitatively, the number of possible hybrids will depend on the number of original homoduplex molecules present in the mixture. The maximum number of possible HDs can be calculated according to the formula: [N.sub.ht]=[N.sub.hm]([N.sub.hm]-1]) where [N.sub.ht] is the number of HDs and [N.sub.hm] is the number of homo-duplexes. Thus, two homoduplex species during rehybridization will generate two additional heteromolecules; three will produce six, four--twelve and so on. Qualitatively, the structure of these hybrids as compared to the structure of homoduplexes has shape-distorting mismatch(es); thus, they melt under milder denaturing conditions and have different rates of migration on the denaturing gradient gel. It is therefore safe to say that HD bands on the denaturing gradient gel will always occur above the parental homoduplex bands. However, in a mixed template sample it is possible to have some homoduplex bands with lower melting temperature than some of the HDs. So, bands appearing on the upper part of the gel cannot always be discarded as HD bands. Therefore, to identify HDs on the gel use of a positive control is required.

Stability of the new hybrid molecules depends on the GC content of parental sequences and on their similarity. HDs can exhibit three different patterns of migration (Figure 1): (1) Co-migration (HDs migrate as a single band), (2) Double-band migration (HDs migrate close to each other), (3) Separate migration (HDs migrate as two separate bands). Due to the unstable structure of HDs, some may denature without forming a distinct band or may disappear after some time during the electrophoresis and leave smears. However, the number and amount of HDs can be significant and, if co-migrating, they can provide a strong fluorescent signal on a gradient gel and lead to false conclusions regarding total number of sequences and DNA species diversity. Using the A, B and C fragments in all possible combinations as a template for four PCR reactions this study has demonstrated that HD formation occurs in absence of primers during a single denaturation step (Group II, Figure 1).

During the first cycle of PCR, when different but closely related templates with identical priming sites are present, double the number of original homoduplexes is generated. As PCR progresses, the number of primers decreases exponentially at the same rate as the new strands are synthesized. Therefore, each consecutive PCR cycle creates an equilibrium shift from primer-single strand complex formation towards the formation of homo-and heteroduplexes. At a low concentration of primers or in their absence, there is an equal probability of formation of homo or heteroduplexes upon renaturation. The amount of heteromolecules depends entirely on the amount of primers, which are better competitors for hybridization sites. The presence of a GC clamp on one or both primers is required for DGGE resolution and provides favorable conditions for HD formation. As demonstrated in Figure 1 (Group II), a single denaturation-renaturation step is sufficient to form HDs. Such a condition repeatedly occurs during late cycles of PCR and it results in an even redistribution of homo and HDs. Formation of HDs in multi-template PCR is inevitable and it contributes to the '[C.sub.o]t effect' allowing amplification of less dominant DNA species during late cycles of PCR (Mathieu-Daude et al. 1996).

Several methods have been proposed to reduce or eliminate HDs to obtain a representative picture of DNA species diversity by DGGE analysis. Some of the unstable HDs could be eliminated by increasing denaturant concentration in the gel although this may not always remove all the hybrids (Qiu et al. 2001). Elimination of HDs by the T7 endonuclease I (Lowell 2000) demands strict experimental conditions with respect to enzyme concentration and incubation time which differ from sample to sample and can not be predicted (Qiu et al. 2001). PCR reconditioning (Thompson & Marcelino 2002) can be helpful, but with the limitation that a ten fold increase in primer concentration will only provide enough primers for three additional PCR cycles and a hundred-fold increase would allow for only five additional PCR rounds before depletion of primers. In addition, PCR amplification depends on many factors such as primer efficiency, concentration of inhibitors and initial concentration of the template. Therefore, conditions for reconditioning PCR must be determined empirically. This study proposes a simple positive control which can be used to control for the presence of HDs in a PCR sample containing multiple templates. Heating an aliquot of the individual PCR sample to 96[degrees]C followed by an on-bench cooling will generate all possible HDs. By comparing the DGGE band profiles of the original PCR product and the heat treated sample, a researcher can reach a grounded conclusion about the presence, absence and location of HD bands on the gel (Figure 4). Hopefully this finding will help the DGGE community to generate reliable data and make interpretation of DGGE results easier.

ACKNOWLEDGMENTS

We would like to thank Dr. Christian Zwieb (University of Texas Health Science Center at Tyler, Texas) and anonymous reviewers for critical review and comments on an earlier draft of this manuscript. This work was supported by Mr. Stan Decker and Stephen F. Austin State University ORSP grant.

LITERATURE CITED

Acinas, S. G., R. Sarma-Rupavtarm, V. Klepac-Ceraj & M. F. Polz. 2005. PCR induced sequence artifacts and bias: insights from two 16S rRNA clone libraries constructed from the same sample. Appl. Environ. Microbiol., 71(12):8966-8969.

Janse I., J. Bok & G. Zwart. 2004. A simple remedy against artifactual double bands in denaturing gradient gel electrophoresis. J. Microbiol. Methods, 57(2):279-281.

Jensen, M. A. & N. Straus. 1993. Effect of PCR conditions on the formation of heteroduplex and single-stranded DNA products in the amplification of bacterial ribosomal DNA spacer regions. PCR Methods Appl., 3:186-194.

Lowell J. L. & D. A. Klein. 2000. Heteroduplex resolution using T7 endonuclease I in microbial community analyses. Biotechniques, 28:676-678

Mathieu-Daude F., J. Welsh, T. Vogt & M. McClelland. 1996. DNA rehybridization during PCR: the 'Cot effect' and its consequences. Nucleic Acids Res., 24(11): 2080 2086.

Myers, R. M., T. Maniatis & L. S. Lerman. 1987. Detection and localization of single base changes by denaturing gradient gel electrophoresis. Meth. Enzymol., 155:501-527.

Osborn, A. M., E. R. B. Moore & K. Timmis. 2000. An evaluation of terminal-restriction fragment length polymorphism (T-RFLP) analysis for the study of microbial community structure and dynamics. Environ. Microbiol., 2:39-50.

Qiu, X. Y., L. Y. Wu, H. S. Huang, P. E. McDonel, A. V. Palumbo, J. M. Tiedje & J. Z. Zhou. 2001. Evaluation of PCR-generated chimeras: Mutations, and heteroduplexes with 16S rRNA gene-based cloning. Appl. Environ. Microbiol., 67:880-887.

Ruano, G. & K. K. Kidd. 1992. Modeling of heteroduplex formation during PCR from mixtures of DNA templates. PCR Methods Appl., 2:112-116.

Thompson, J., L. Marcelino & M. F. Polz. 2002. Heteroduplexes in mixed-template amplifications: formation, consequence and elimination by 'reconditioned PCR'. Nucleic Acids Res., 30(9): 2083-2088.

AMVK at: avankley@sfasu.edu

Andrew Syvyk, Armen Nalian, Michael Hume * and Alexandra Martynova-VanKley

Department of Biotechnology. Stephen F. Austin State University Nacogdoches. Texas 75965 and * USDA, ARS, SPARC, Food and Feed Safety Research Unit College Station, Texas 77845
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Author:Syvyk, Andrew; Nalian, Armen; Hume, Michael; Martynova-VanKley, Alexandra
Publication:The Texas Journal of Science
Date:Feb 1, 2008
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