KR20110066018A - Analysing methods of bacteria community using 16s rdna-dgge and real-time pcr - Google Patents

Abstract

PURPOSE: A method for analyzing bacteria community using 16S rDNA-DGGE and real-time PCR is provided to minimize analysis error. CONSTITUTION: A method for analyzing bacteria community using 16S rDNA-DGGE and real-time PCR comprises: a step of performing PCR amplification using 0.25-0.375 mM of dNTP concentration; or a step of performing real-time PCR using bacteria DNA as a template and primers; a step of performing DGGE of PCR products to measure PCR band intensity; and a step of calculating ratio of bacteria species based on the number and intensity of the bands.

Description

Analytical methods of bacteria community using 16S rDNA-DGGE and real-time PCR} using 16S rDNA-DVE and PCR-amplification PCR

The present invention relates to a method for analyzing bacterial populations using 16S rDNA-DGGE and real-time PCR amplification, and more specifically, in bacterial colonization analysis using PCR amplification and modified gradient gel electrophoresis (DGGE) of bacteria. In the PCR amplification, the concentration of dNTP is 0.25 to 0.375 mM, or in the PCR amplification, the number of PCR repetitions is the inflection point of the PCR amplification using real-time PCR, or the acrylic of the DGGE gel in the DGGE. And a concentration of amide / bis of at least one of 12% to 14% of the conditions.

The structure of a bacterial community is expressed by the number of species and the proportion of each species within that community. An assay such as 16S rDNA DGGE is frequently used to identify these two factors. However, it is reported that many factors can distort these two factors. The ratio of PCR products to 1: 1 convergence (Suzuki and Giovannoni, 1996), the binding energy difference of primers during PCR (Polz and Cavanaugh, 1998), or sequence mismatch (Hongoh et al., 2003) Bias amplification of specific sequences, micro-changes of similar sequences during PCR and cloning (Speksnijder et al., 2001), shifting of different sequences to the same position on the DGGE gel (Vallaeys et al., 1997), further generation of bands due to heteroduplex molecules on DGGE gels (Ferris and Ward, 1997), and the occurrence of two DGGE bands in one sequence (Zhu et al., 1997). Reported. In order to accurately evaluate the effects of these various factors on 16S rDNA DGGE analysis, 16S rDNA PCR should be performed on a sample containing a mixture of 16S rDNA of various bacteria, and the results analyzed. Very few In a previous study, we showed that when 16S rDNA of representative soil bacteria were mixed with the same copy number and 16S rDNA DGGE was performed, a specific 16S rDNA did not appear on the DGGE gel due to sequence mismatch between the primer and 16S rDNA. This problem could be solved by designing the primers to have one base sequence mismatch with each base sequence (Ahn et al., 2006).

In the present invention, by identifying the causes of various errors of the existing DGGE method, it is possible to reduce the DNA band smear appearing in the DGGE gel and develop a new DGGE technique that can detect and analyze the bacterial community qualitatively and quantitatively and accurately. It was.

Therefore, the main object of the present invention is 16S rDNA- which can detect the bacterial population qualitatively and quantitatively through optimization of PCR primer, optimization of dNTP concentration, optimization of acrylamide / bis concentration and optimization of real-time PCR repeats. It is to provide a bacterial community analysis method using DGGE and real-time PCR amplification.

According to an aspect of the present invention, the present invention provides a bacterial community characterized by applying one or more of the following conditions in bacterial community analysis using PCR amplification and denaturation gradient gel electrophoresis (DGGE) of bacteria 16S rDNA. Provide an analysis method:

a) in the PCR amplification, the dNTP concentration is 0.25 to 0.375 mM, or

b) In the PCR amplification, the PCR repetition number is used as the inflection point of the PCR amplification using Rreal-time PCR, or

c) In the DGGE, the concentration of acrylamide / bis in the DGGE gel is 12% to 14%.

The principle of the 'Denaturing Gradient Gel Electrophoresis' assay used in the present invention is that the double helix structure and denaturation structure of the nucleic acid according to the concentration gradient of the denaturant present in the gel during electrophoresis, that is, the base sequence By using the difference in the Tm (melting temperature) value of the movement speed of the nucleic acid is used. Therefore, DNA having a low Tm value is detected at the top of the gel having a low denaturant, and DNA having a high Tm value is detected at the bottom of the gel having a high denaturant. In this case, since DNAs having different nucleotide sequences form bands at different positions, the number of bands increases as the n-value of the nucleotide sequence increases, and the brightness of the band appears as the number of identical nucleotide sequences increases. Therefore, there is an advantage that the numerical and quantitative changes to the overall DNA can be observed on one gel.

The 16S rDNA DGGE analysis process, which has been used in the conventional bacterial community analysis, may cause various errors due to various factors, thereby distorting the analysis result of the bacterial community structure. The present invention has been developed a method for minimizing the error of the analysis result by studying the method of finding the cause of the error in the 16S rDNA DGGE analysis method for conventional bacteria analysis and solving it.

In the present invention, the condition a) may minimize errors in the bacterial population analysis result by optimizing the concentration of dNTP in PCR amplification of 16S rDNA. In Experiment 2 of the present invention, PCR amplification was performed separately at a dNTP concentration of 0.2 to 0.4 mM, and compared with the resulting band brightness, it was confirmed that the increase in the dNTP concentration in the above range can reduce the DNA polymerase error. In the present invention, 0.25 to 0.375 mM of the dNTP concentration range was determined to be the preferred dNTP concentration, because when the increase in the dNTP concentration range of 0.375 mM or more showed a severe inhibition on the PCR reaction. Most preferably 0.325 mM (see FIG. 3).

In the present invention, the condition b) is to use the inflection point of the PCR amplification as a method for determining the number of PCR repeats in PCR amplification. In Experiment 4 of the present invention, PCR amplification was performed to the inflection point at an optimal dNTP concentration of 0.325 mM. When the relative brightness of the four bands was 0.95 to 1.05, the most uniform band brightness was shown, and this condition was selected as the optimal condition (see FIG. 6).

In the present invention, the c) condition can minimize the error of the bacterial population analysis results by optimizing the concentration of the diacrylamide / bis of the DGGE gel. In Experiment 3 of the present invention, it was confirmed that by increasing the concentration of acrylamide / bis of the DGGE gel to reduce the length of the DNA in which partial melting occurs to improve the separation between bands (see Figure 4).

In the bacterial colony analysis method of the present invention, i) preparing a primer capable of specifically PCR amplifying 16S rDNA of bacteria; ii) using DNA of bacteria as a template and performing real-time PCR using the primers; iii) electrophoresis of the PCR product in DGGE to measure the intensity of the bands; And iv) characterized in that the bacterial population analysis method comprising the step of calculating the number of species and the number of bacteria occupy in the community based on the number and intensity of the bands.

In the bacterial colony analysis method of the present invention, two PCR amplifications are performed using two pairs of primers complementary to target region sequences of different 16S rDNA of bacteria in the PCR amplification, and then amplification products are mixed in the same volume. It is desirable to make a DGGE sample. In Experimental Result 5 of the present invention, in the optimization condition of the present invention, when one PCR amplification was performed using one primer, the target site sequences of different 16S rDNAs of bacteria could not be effectively amplified. Therefore, by performing two PCR amplifications using two pairs of primers complementary to the target region sequence of 16S rDNA in one sample, the target region sequence could be effectively amplified.

In the bacterial colony analysis method of the present invention, the bacteria are OP11, Verrucomicrobia, Bacteroidetes, Gammaproteobacteria, Actinobacteria and Genebank accession no. This study is aimed at bacterial community of various environmental samples including representative soil bacteria such as bacteria having AY930457 16S rDNA. As described above, 16S rDNA-DGGE and real-time PCR can be used to analyze various bacterial populations included in environmental samples. In particular, in the embodiment of the present invention, 16S rDNA-DGGE analysis method using the representative soil bacteria. We developed an optimization method and minimized the error of the analysis result.

In the present invention, primers capable of specifically PCR amplifying the 16S rDNA of the bacteria are characterized in that the F352AT primer of SEQ ID NO: 3 and F352TA primer of SEQ ID NO: 4. The F352AT and F352TA primers of the present invention can amplify a 353-514 region based on the E. coli 16S rDNA nucleotide sequence, and use the 515r primer of SEQ ID NO: 5 as a reverse primer. The primer is a case in which the bases are A and T (AT type) or T and A (TA type) in E. coli 16S rDNA sequences 340 and 349. As shown in Table 3, most soil bacteria are 90 Since it accounts for more than%, it can be used as a primer specific for 16S rDNA of soil bacteria. In addition, the 16S rRNA gene is a conserved region of the phylogenetic sequence, and the bacterial community analysis method of the present invention targeting the gene can perform a cluster analysis on a large number of microorganisms.

In the present invention, when the real-time PCR amplification of 16S rDNA, the bacterium was most accurately used when the F352AT primer of SEQ ID NO: 3 and the F352TA primer of SEQ ID NO: 4 were used as primers capable of PCR amplifying 16S rDNA of bacteria. Clusters could be detected (see Table 3 and FIG. 8).

In the experimental result 5 of the present invention, in order to amplify both the TA type and AT type 16S rDNA of the bacteria with the same efficiency, each of the two PCR amplification using the primers, respectively, and then mixed the amplification products in the same volume DGGE Samples were made. It was demonstrated that the method using these two primers and the PCR reaction can well reflect the microbial community structure of the sample (see FIG. 8). FIG. 8 shows the relative brightness of six bands representing 16S rDNA of the soil bacteria in DGGE gels. The relative brightness of each band is generally shown in the order of concentration between 16S rDNA in the sample, indicating that the method proposed in the present invention can well reflect the original microbial community structure of the sample.

Hereinafter, the present invention will be described in more detail with reference to Examples. These embodiments are only for illustrating the present invention, and thus the scope of the present invention is not construed as being limited by these embodiments.

Example 1 DNA Quantitation

Relative amounts of DNA were estimated from the band brightness seen in agarose or acrylamide / bisbis stained with ethidium bromide (EtBr). The band brightness was measured using Bionumerics softaware version 4.00 (Applied Maths). In some cases, the band brightness is expressed as the brightness relative to the average band brightness. When relative brightness was in the range of 0.9 to 1.1, each band was considered to have the same amount of DNA. This is based on the results obtained when the same sample was electrophoresed on agarose gel.

Example 2. 16S rDNA sequence

16S rDNA nucleotide sequence used in the present invention is shown in Table 1 below. 16S rDNA used for PCR was prepared by the following method. DNA obtained from bacteria or environmental samples was PCR amplified using primers 27mf (5'-AGAGTTTGATCMTGGCTCAG-3 ': SEQ ID NO: 1) and 1492r (5'-GGYTACCTTGTTACGACTT-3': SEQ ID NO: 2), followed by extraction kit (QIAquick gel). Purification using an extraction kit (QIAGEN). The purified DNA was cloned using a vector (pGEM-T Easy vector, Promega) and plasmid DNA was extracted using an Accuprep plasmid mini extraction kit (Bioner). The extracted plasmid DNA was linearized with restriction enzymes. S1, S2, S3, S4, S5 were treated with ScaI (Takara) and S6 was treated with SphI (Takara). The linearly converted DNA was again purified using the QIAquick gel extraction kit (QIAGEN) and each DNA was adjusted to the same amount using the method presented above. The concentration of each DNA was 38.5 ± 3.2 μg / ml as a result of spectroscopy comparing the DNA concentration with the standard solution.

TABLE 1 16S rDNA sequence

Example 3. PCR Primers

The primer used in the present invention amplifies the 353-514 region based on the nucleotide sequence of E. coli 16S rDNA. The base sequence in the forward primer and the corresponding 16S rDNA is shown in Table 2 below. 515r (5'-ACCGCGGCTGCTGGCAC-3 ': nucleotide 5) (Crump et al., 2004) was used as a reverse primer.

TABLE 2 Forward Primer and Corresponding 16S rDNA Sequence

The 16S rDNAs used in the experiments are classified into E. coli 16S rDNA SEQ ID NOs: 340 and 349, where the base sequences are A and T (AT type) and T and A (TA type). S1 and S2 are AT types, and S3 to S6 are TA types.

Example 4. PCR Conditions

Unless otherwise specified, PCR was performed under the following conditions. The volume of the total PCR mixture was 50 μl, including 1 × PCR buffer (Takara) containing 1.5 mM MgCl ₂ , 1 μl of 16S rDNA sample, 0.5 μM forward and reverse primer, 0.2 mM each of four dNTPs (Takara) , 2.5 U Taq DNA polymerase (Takara) was included. The PCR program was first repeated 5 minutes at 94 ° C., 30 seconds at 94 ° C., 30 seconds at 51 ° C., 30 seconds at 72 ° C., and repeated 30 times. The reason why it was kept at 72 ° C. for 30 minutes in the last step was to prevent the formation of double bands in DGGE analysis afterwards (Janse et al., 2004).

In real-time PCR, 1 mg / ml BSA (bovine serum albumin, Q-BIO Gene) and 0.5 × SYBR Green I (Cambrex) were added in addition to the PCR mixture, and were performed using a Roche LightCycler 480 instrument (Roche). .

The Phusion ^TM high-fidelity PCR kit (Finnzymes) was used to investigate the effect of using a polymerase with proofreading on DGGE analysis, and the error rate of the polymerase measured by the manufacturer was 4.4 × 10- ^{. 7} was. The PCR reaction was carried out with a total volume of 20 μl with the PCR mixed solution composition as above except for polymerase, and the PCR program followed the method suggested by the manufacturer.

Example 5 DGGE Analysis

DGGE analysis was performed using the DCode mutation detection system (Bio-Rad). Acrylamide / bis concentration, denaturant concentration and electrophoresis time of the DGGE gel are shown in FIG. 4. As a result, each DGGE band strength is shown in FIG. 5. 100% denaturant corresponds to 7 M urea and 40% (v / v) formamide. The gel was cured for about 2 hours and electrophoresed in 0.5 × TAE buffer at 60V, 60 ° C. After electrophoresis, the gel was stained with ethidium bromide (0.75 μg / ml) and observed using UV (302 nm).

Experimental Results 1.Reduction of Band Brightness Difference on DGGE Gel

When several 16S rDNA sequences obtained in Examples 1 and 2 were mixed in the same amount, PCR amplification was performed, and the PCR product was subjected to DGGE analysis, and the brightness of the bands appearing in the lower part of the DGGE gel (high denaturant region). Was lower than the brightness of the band appearing at the top of the DGGE gel (low denaturant area). This will make it difficult to analyze quantitative bacterial populations in the analysis of the actual sample, so studies were conducted to improve it.

First, to determine whether this difference in band brightness is due to non-uniform PCR amplification between 16S rDNA sequences, the six 16S rDNA sequences shown in Table 1 can be identified by F352TA-515r (SEQ ID NOs: 3 and 5) or F352AT-515r (SEQ ID NO: 6). PCR amplification using Nos. 4 and 5) was performed separately, and then adjusted so that the relative band brightness was 0.9-1.1 on the agarose gel. As a result of mixing the six samples in the same amount and electrophoresing the DGGE gel, it was confirmed that the band brightness was weaker than the upper part of the gel in general (FIG. 1). This indicates that the difference in band brightness is not due to heterogeneous PCR amplification between 16S rDNA sequences.

Next, the inventors assumed that the difference between the DNA polymerase error occurring in the PCR process and the melting profile of the PCR product caused the difference in the band brightness. During PCR amplification, Taq DNA polymerase can produce PCR products with erroneous sequences, and these PCR products are PCR products (PCR products with exactly the same sequence as the template) with the correct sequence on the DGGE gel. It has been found in previous studies that it can be isolated from (Keohavong and Thilly, 1989). PCR products with DNA polymerase errors will be separated on the DGGE with PCR products with the correct sequence and dispersed on the DGGE gel, which will weaken the band brightness of the correct PCR product. The bands at the bottom of the DGGE gel then assume more DNA polymerase errors during PCR than the bands at the top, thus making the bottom band brighter. In general, PCR products appearing at the bottom of the DGGE gel have a higher GC (guanine + cytosine) content than PCR products appearing at the top (see Table 1 and FIG. 1). This is because the higher the GC content, the higher denaturant concentration is required to melt the DNA. It is known that DNAs with high GC content have more secondary structures, which tend to increase DNA polymerase errors during PCR amplification (Loewen and Switala, 1995). In order to reduce the secondary structure of the 16S rDNA sequence during PCR amplification, betaine or DMSO (Shen and Hohn) are known to decrease the annealing temperature (55-65 ° C) and reduce the DNA secondary structure. , 1992; Henke et al., 1997; Rees et al., 1993), but it was not effective in reducing the difference in band brightness.

As these two methods do not succeed, the inventors have found that PCR polymerase errors between different 16S rDNAs are similar, but due to differences in DNA melting profiles, PCR products and errors with correct sequences can be identified. It is assumed that the degree of separation of the PCR products is different. Melting profiles of the PCR products used in FIG. 1 are shown in FIG. 2. Except for the melting domain (melting domain) generated by the GC clamp, all PCR products are composed of a higher melting domain (R1) and a lower melting domain (R2). The rate of DNA migration on the DGGE gel is reduced by DNA melting and the location of the melting on the DGGE gel is determined by the sequence of the melting region. DNA melting occurs first in the low melting region. In previous studies, it was found that only when the temperature difference between DNA melting regions is below 1 ° C., sequence changes located in high melting regions can be detected by DGGE analysis (Wu et al., 1998). This is because the melting occurs not only in the low melting region but also in the high melting region only when the temperature difference between the two regions is small, and thus the base sequence of this region affects the position where the melting occurs on the DGGE. If the temperature difference between the two melting regions is large, when melting occurs in the low melting region, the DNA no longer moves and the melting of the high melting region does not occur. As a result, the change in the nucleotide sequence of the high melting region does not affect the speed of DNA movement and thus the change in the nucleotide sequence of this region will not be detected.

DNA polymerase errors that occur during PCR are expected to occur randomly over the entire region of the PCR product. Then, PCR products with a smaller temperature difference between the two melting regions will increase the rate at which the erroneous PCR products are separated from the PCR products with the correct sequences, which will lower the brightness of the PCR products with the correct sequences. You can expect it. This assumption is supported by the close correlation between the relative band brightness of the six PCR products and the temperature difference between the melting regions (FIG. 1B). There was a significant correlation between the linear regression models (P = 0.00461).

In order to reduce the polymerase error generated during the amplification process, the analysis was performed under the same experimental conditions using PhusionTM DNA polymerase with proofreading function, but the difference in band brightness was not reduced.

Experimental Results 2. Effect of dNTP Concentration on DGGE Analysis

Since DNA polymerase with correction did not reduce the difference in band brightness, other factors that could reduce DNA polymerase error were examined. Eckert and Kunkel found that polymerase errors were reduced when MgCl ₂ and dNTP were present in the same molar concentration in PCR reactions (Eckert and Kunkel, 1990). Under typical PCR conditions, concentrations of 0.2 to 0.25 mM (total 0.8-1 mM dNTP) per dNTP in 1.5 mM MgCl ₂ are used. High concentrations of dNTP inhibit the PCR reaction by decreasing the effective Mg ²⁺ concentration. Known (Sambrook and Russell, 2001).

To investigate the effect of dNTP concentration on the difference in band brightness, S3 and S6 were PCR amplified separately at various dNTP concentrations. Two PCR products obtained at specific dNTP concentrations were adjusted to have a relative band brightness of 0.9-1.1 and then mixed in equal amounts. Figure 3 shows the change in the ratio of the band brightness of S6 and S3 with the change of dNTP concentration. As the concentration of each dNTP increased from 0.2 to 0.4 mM, the ratio of the band brightness of S6 and S3 became closer to 1 but did not exceed 0.7. This indicates that increasing dNTP concentration reduced DNA polymerase error. Higher concentrations of dNTP could not be used because increasing the concentration of each dNTP above 0.375 mM showed severe inhibition in the PCR reaction.

Experimental results 3. Effect of acrylamide / bis concentration on DGGE analysis

If the hypothesis that the difference in band brightness between PCR products comes from the difference in the melting profile is correct, then the melting of the high brightness area (R1) does not occur in all PCR products and only the low melting area (R2) causes melting to occur. You can reduce the difference. It is thought that the cause of the decrease in the DNA transfer rate in the DGGE analysis is due to the increase in the volume of the DNA due to partial melting of the DNA, thereby increasing the contact with the acrylamide / bis-gel. It was then expected that by increasing the concentration of acrylamide / bis in the DGGE gel, it was possible to reduce the length of the DNA where partial melting occurred.

In order to confirm this hypothesis, PCR amplification of S1, S2, S3, S4, S5, and S6, respectively, and the relative band brightness was adjusted to 0.9 to 1.1. These samples were mixed at a volume ratio of 1: 1: 1: 1: 1: 1, 32: 16: 8: 4: 2: 1, and 1: 2: 4: 8: 16: 32, followed by 8% Electrophoresis was performed until each band was sufficiently separated in, 10%, 12%, 14% acrylamide / bis gel (FIG. 4). Electrophoresis of 1: 1: 1: 1: 1: 1: 1 samples on 8-12% acrylamide / bisgel showed that the band brightness of S5 and S6 was lower than half of the other band brightnesses. However, when electrophoresis on 14% acrylamide / bis gel, the relative band brightness of S2 ~ S5 was between 0.8 ~ 1.0 (FIG. 5A). For the sample of 32: 16: 8: 4: 2: 1, the band brightnesses were generally in their original order (FIG. 5B). In addition, in the case of 1: 2: 4: 8: 16: 32, the band brightness of S5 in the 8-12% acrylamide / bis-gel decreases compared to the original ratio, so that the concentration of the band brightness of S4 having a concentration of 1/2 of S5 is reduced. Although small or equal, the original concentration order was restored in 14% acrylamide / bis gel (FIG. 5C).

In summary, the relatively low band brightness of S5 and S6 could only be recovered in 14% acrylamide / bisgel. In addition, increasing concentrations of acrylamide / bis have been shown to improve interband separation (FIG. 4).

Experimental Results 4. Effect of dNTP Concentration and PCR Repeat Count on DGGE Analysis Using Six 16S rDNA Mixtures as Samples

When a 1: 1: 1: 1 mixture of S3, S4, S5, and S6 was used as the sample and 14% acrylamide / bis was used as the DGGE gel, the effect of dNTP concentration on the DGGE analysis was evaluated. At this time, the effect of PCR repeat count on DGGE analysis was also evaluated using real-time PCR system. That is, the PCR reaction was terminated at the number of repetitions corresponding to the inflection point of the PCR amplification or at the number of repetitions where the amount of PCR product no longer increased. Figure 6 shows the PCR amplification curve at 0.3 ~ 0.375 mM each dNTP concentration. The amount of PCR product slightly decreased when each dNTP concentration was above 0.35 mM. PCR was performed again under the same conditions to obtain a PCR product terminated in 21 or 35 replicates.

Figure 7 shows the relative band brightness of S3, S4, S5, S6 when the DGGE analysis of the PCR product obtained in the above experiment. As dNTP concentration increased, the relative band brightness of S5 and S6 increased, and the increase of PCR repeats decreased the relative brightness of S4. When PCR amplification was performed to an inflection point at a dNTP concentration of 0.325 mM, the relative brightness of the four bands was 0.95 to 1.05, showing the most uniform band brightness. Therefore, this condition was selected as the optimum condition.

Experimental Results 5. Two PCR Amplifications per Sample to Amplify AT and TA Sequences with the Same Efficiency

In the previous study, the inventors showed that in order to amplify AT and TA 16S rDNA sequences with the same efficiency, primer F352T (Table 1) having one type sequence mismatch with each type sequence should be used (Ahn et al. al., 2006). However, primer F352T did not amplify the two types of sequences with the same efficiency under the optimized conditions in the present invention. Therefore, one sample was subjected to PCR using primer F352AT (SEQ ID NO: 3) and the other using primer F352TA (SEQ ID NO: 4), and a method of mixing two PCR products in the same volume was selected. When two primers were used simultaneously in one reaction, this method could not be used because two DGGE bands were generated from one type of 16S rDNA. In order to examine whether the method using the two PCR reactions can reflect the microbial community structure of the sample, a sample in which ATS and TA type 16S rDNA sequences were mixed at various ratios was used as a model sample. S1, S2, S3, S4, S5 S6 1: 1: 1: 1: 1: 1: 1 (M1), 32: 16: 8: 4: 2: 1 (M2), and 1: 2: 4: 8: Mixed at 16:32 (M3), each sample was subjected to PCR amplification using F352AT or F352TA as a forward primer. Each dNTP concentration was fixed at 0.325 mM and PCR amplification was terminated at the inflection point. The earliest repeats of the inflection points obtained from the two PCR amplifications were chosen as the final repeats. For example, when PCR was performed using M2 as a sample, the inflection point was 24 repetitions in PCR amplification using F352AT, and the inflection point in PCR amplification using F352TA was 26 repetitions. In this case two PCR amplifications were performed again up to 24 replicates. After PCR amplification, both PCR products were mixed to the same volume and concentrated about 9-fold using standard ethanol precipitation (Sambrook and Russell, 2001).

8 shows the relative brightness of six bands in the DGGE gel. The relative brightness of each band is generally shown in the order of concentration between 16S rDNA in the sample, indicating that the method proposed in the present invention can well reflect the original microbial community structure of the sample.

conclusion

From previous studies (Ahn et al., 2006) and the results of the present invention, the errors occurring in the 16S rDNA PCR DGGE assays were determined by sequence mismatch between primer-16S rDNA, DNA polymerase error, differences in melting profile of PCR products, It can be seen that due to excessive PCR repeats. These errors could be reduced by using primers, dNTP concentrations, acrylamide / bis concentrations, and PCR repeats (inflection points) different from the existing experimental conditions.

If a sample obtained in a real environment is used, the PCR reaction for determining the inflection point is considered to be sufficient only by using the forward primer F352TA. The reason is that the 16S rDNA sequences included in all the soil clone libraries examined so far contain overwhelmingly many TA type sequences. Therefore, the number of repetitions to the inflection point will be faster PCR reaction using F352TA than the number of repetitions using F352AT. Table 3 below shows the distribution of TA type and AT type sequences in several soil clone libraries. The nucleotide sequence of TA type and AT type occupies 90.6 ~ 98.9% of each clone library, indicating that most nucleotide sequences belong to this type. The nucleotide sequence of the AT type occupies only 0.0 to 13.0% in each clone library, but this type of nucleotide sequence has some important qualities such as Chloroflexi (38.0%), Planctomycetes (60.9%), Verrucomicrobia (88.7%), and OP11 (70.1%). A significant proportion of the soil phylum was found in the Probe match program of the Ribosoaml Database Project (Cole et al., 2005).

TABLE 3 Distribution of TA Type and AT Type 16S rDNA in Soil Clone Library

16S rDNA DGGE analysis method of the microbial community structure derived from the present invention proceeds to the following process.

(1) real-time PCR amplification

① Inflection point determination PCR

50 μl total

PCR buffer 1 × with 1.5 mM MgCl ₂

1 μl sample

F352TA 0.5 μM

515r 0.5 μM

0.325 mM each dNTP (1.3 mM total)

Bovine serum albumin 1 mg / ml

SYBR Green I 0.5 ×

Taq DNA polymerase 2.5 U

Real-time PCR system

step 1.94 ℃ 5 minutes

step 2. 94 ℃ 30sec

step 3. 51 ℃ 30 seconds

step 4.72 ℃ 30 seconds

repeat step 5.step 2 ~ step 4, 35

Inflection point determination.

② F352TA PCR

50 μl total

PCR buffer 1 × with 1.5 mM MgCl ₂

1 μl sample

F352TA 0.5 μM

515r 0.5 μM

0.325 mM each dNTP (1.3 mM total)

Bovine serum albumin 1 mg / ml

SYBR Green I 0.5 ×

Taq DNA polymerase 2.5 U

Real-time PCR system

step 1.94 ℃ 5 minutes

step 2. 94 ℃ 30sec

step 3. 51 ℃ 30 seconds

step 4.72 ℃ 30 seconds

step 5. Step 2 ~ step 4, repeat until the inflection point number determined above

step 6. 72 ℃ 30min

③ F352AT PCR

50 μl total

PCR buffer 1 × with 1.5 mM MgCl ₂

1 μl sample

F352AT 0.5 μM

515r 0.5 μM

0.325 mM each dNTP (1.3 mM total)

Bovine serum albumin 1 mg / ml

SYBR Green I 0.5 ×

Taq DNA polymerase 2.5 U

Real-time PCR system

step 1.94 ℃ 5 minutes

step 2. 94 ℃ 30sec

step 3. 51 ℃ 30 seconds

step 4.72 ℃ 30 seconds

step 5. Step 2 ~ step 4, repeat until the inflection point number determined above

step 6. 72 ℃ 30min

④ PCR product mixing and concentration

Take 45 μL of each PCR product from ② and ③, mix, and concentrate to 12 μL using standard ethanol precipitation (Sambrook and Russell, 2001) or a commercial DNA enrichment kit.

(2) DGGE analysis

14% acrylamide / bis (37.5: 1)

43-63% denaturant

Harden gel for about 2 hours.

Electrophoresis for 28 h at 60 V, 60 ° C. in 0.5 × TAE buffer.

As described above, according to the present invention, an error occurring in 16S rDNA-DGGE and PCR analysis may be caused by sequence mismatch between primer-16S rDNA, DNA polymerase error, difference in melting profile of PCR product, and excessive PCR repeat count. It can be seen that. These errors could be reduced by using primers, dNTP concentrations, acrylamide / bis concentrations, and PCR repeats different from the existing experimental conditions. Therefore, the present invention will enable a more accurate bacterial community analysis by providing a new DGGE technique that can accurately analyze the bacterial community through the combination of 16S rDNA-DGGE and real-time PCR.

[references]

Ahn, JH, Kim, MC, Shin, HC, Choi, MK, Yoon, SS, Kim, T., Song, HG, Lee, GH, Ka, JO, 2006. Improvement of PCR amplification bias for community structure analysis of soil bacteria by denaturing gradient gel electrophoresis. J. Microbiol. Biotechnol. 16, 1561-1569.

Chan, OC, Yang, XD, Fu, Y., Feng, ZL, Sha, LQ, Casper, P., Zou, XM, 2006. 16S rRNA gene analyses of bacterial community structures in the soils of evergreen broad-leaved forests in south-west China. FEMS Microbiol. Ecol. 58, 247-259.

Cole, JR, Chai, B., Farris, RJ, Wang, Q., Kulam, SA, McGarrell, DM, Garrity, GM, Tiedje, JM, 2005.The ribosomal database project (RDP-II): Sequences and tools for high-throughput rRNA analysis. Nucleic Acids Res. 33, D294-D296.

Crump, B.C., Hopkinson, C.S., Sogin, M.L., Hobbie, J.E., 2004. Microbial biogeography along an estuarine salinity gradient: Combined influences of bacterial growth and residence time. Appl. Environ. Microbiol. 70, 1494-1505.

Eckert, K.A., Kunkel, T.A., 1990. High fidelity DNA-synthesis by the Thermus aquaticus DNA-polymerase. Nucleic Acids Res. 18, 3739-3744.

Ferris, M.J., Ward, D.M., 1997. Seasonal distributions of dominant 16S rRNA-defined populations in a hot spring microbial mat examined by denaturing gradient gel electrophoresis. Appl. Environ. Microbiol. 63, 1375-1381.

Hartmann, M., Widmer, F., 2006. Community structure analyzes are more sensitive to differences in soil bacterial communities than anonymous diversity indices. Appl. Environ. Microbiol. 72, 7804-7812.

Henke, W., Herdel, K., Jung, K., Schnorr, D., Loening, S.A., 1997. Betaine improves the PCR amplification of GC-rich DNA sequences. Nucleic Acids Res. 25, 3957-3958.

Hongoh, Y., Yuzawa, H., Ohkuma, M., Kudo, T., 2003. Evaluation of primers and PCR conditions for the analysis of 16S rRNA genes from a natural environment. FEMS Microbiol. Lett. 221, 299-304.

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

Keohavong, P., Thilly, W. G., 1989. Fidelity of DNA-polymerases in DNA amplification. Proc. Natl. Acad. Sci. U.S.A. 86, 9253-9257.

Kim, MS, Ahn, JH, Jung, MK, Yu, JH, Joo, D., Kim, MC, Shin, HC, Kim, T., Ryu, TH, Kweon, SJ, Kim, DH, Ka, JO, 2005. Molecular and cultivation-based characterization of bacterial community structure in rice field soil. J. Microbiol. Biotechnol. 15, 1087-1093.

Lerman, L.S., Silverstein, K., 1987. Computational simulation of DNA melting and its application to denaturing gradient gel-electrophoresis. Methods Enzymol. 155, 482-501.

Lesaulnier, C., Papamichail, D., McCorkle, S., Ollivier, B., Skiena, S., Taghavi, S., Zak, D., van der Lelie, D., 2008.Elevated atmospheric CO2 affects soil microbial diversity associated with trembling aspen. Environ. Microbiol. 10, 926-941.

Loewen, P.C., Switala, J., 1995.Template secondary structure can increase the error frequency of the DNA-polymerase from Thermus aquaticus. Gene 164, 59-63.

Niederberger, T.D., McDonald, I.R., Hacker, A.L., Soo, R.M., Barrett, J.E., Wall, D.H., Cary, S.C., 2008.Microbial community composition in soils of Northern Victoria Land, Antarctica. Environ. Microbiol. 10, 1713-1724.

Polz, M. F., Cavanaugh, C. M., 1998. Bias in template-to-product ratios in multitemplate PCR. Appl. Environ. Microbiol. 64, 3724-3730.

Rees, W. A., Yager, T. D., Korte, J., Vonhippel, P. H., 1993. Betaine can eliminate the base pair composition dependence of DNA melting. Biochemistry 32, 137-144.

Sambrook, J., Russell, D. W., 2001. Molecular cloning: a laboratory manual, 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

Shen, W.H., Hohn, B., 1992. DMSO improves PCR amplification of DNA with complex secondary structure. Trends Genet. 8, 227-227.

Speksnijder, A., Kowalchuk, G.A., De Jong, S., Kline, E., Stephen, J.R., Laanbroek, H.J., 2001.Microvariation artifacts introduced by PCR and cloning of closely related 16S rRNA gene sequences. Appl. Environ. Microbiol. 67, 469-472.

Suzuki, M.T., Giovannoni, S.J., 1996.Bias caused by template annealing in the amplification of mixtures of 16S rRNA genes by PCR. Appl. Environ. Microbiol. 62, 625-630.

Vallaeys, T., Topp, E., Muyzer, G., Macheret, V., Laguerre, G., Rigaud, A., Soulas, G., 1997. Evaluation of denaturing gradient gel electrophoresis in the detection of 16S rDNA sequence variation in rhizobia and methanotrophs. FEMS Microbiol. Ecol. 24, 279-285.

Wu, Y., Hayes, V.M., Osinga, J., Mulder, I.M., Looman, M.W.G., Buys, C., Hofstra, R.M.W., 1998. Improvement of fragment and primer selection for mutation detection by denaturing gradient gel electrophoresis. Nucleic Acids Res. 26, 5432-5440.

Zhu, D., Zhou, J. S., Keohavong, P., 1997. Taq-amplified fragments appear as doublets in denaturing gradient gels. Anal. Biochem. 244, 404-406.

1 is a result of DEEG analysis of the six PCR products of S1 ~ S6. A: DGGE assay of 6 PCR products. Six PCR products were diluted appropriately to have the same DNA concentration and mixed in equal amounts, followed by electrophoresis for 18 hours in 8% acrylamide / bis (37.5: 1), 45-65% denaturant. B: Temperature difference between the relative brightness of the six bands shown in A and the melting area. This temperature difference was calculated from FIG. 2.

2 is a melting profile of six PCR products calculated using the MELT94 program (Lerman and Silverstein, 1987).

3 shows the change of the band brightness ratio of S6 and S3 according to the change of dNTP concentration. DGGE analysis was performed for 18 hours in 8% acrylamide / bis (37.5: 1), 45-65% denaturant.

4 shows the results of DGGE analysis at 8-14% acrylamide / bis concentration. Samples of each lane were prepared by mixing six PCR products, and the six PCR products were PCR amplified for each of S1, S2, S3, S4, S5, and S6 (using each dNTP concentration of 0.375 mM), and the relative band brightness was 0.9 ~. 1.1, and by volume ratio 1: 1: 1: 1: 1: 1 (lane 1), 32: 16: 8: 4: 2: 1 (lane 2), 1: 2: 4: 8: 16: 32 It mixed so that it might become (lane 3). ^a concentration of acrylamis / bis (37.5: 1). ^b Maximum denaturant concentration. ^c Minimum denaturant concentration. ^d Electrophoresis time.

5a to 5c show the relative band brightness calculated from FIG. 4, respectively. The dashed line represents the original ratio of each mixture. Each mixture has a ratio of 1: 1: 1: 1: 1: 1 (A), 32: 16: 8: 4: 2: 1 (B), 1: 2: 4: 8: 16: 32 (C) Mixed.

Figure 6 shows the PCR amplification curves at each dNTP concentration of 0.3 ~ 0.375 mM. PCR was performed using a 1: 1: 1: 1 mixture of S1, S2, S3, and S4 as a sample and F352TA-515r as a primer. The dotted line represents the inflection point (21 repetitions) of each amplification curve.

7A and 7B show two repeats at a concentration of 0.3, 0.325, 0.35, 0.375 mM dNTP using a 1: 1: 1: 1 mixture of S1, S2, S3, and S4 as samples and F352TA-515r as a primer. Relative brightness of each band obtained by PCR with Repetition-A, Repetition-B) and DGGE analysis is shown. DGGE analysis was performed for 28 hours in 14% acrylamide / bis (37.5: 1), 43-63% denaturant.

8 shows S1, S2, S3, S4, S5, S6 1: 1: 1: 1: 1: 1 (M1), 32: 16: 8: 4: 2: 1 (M2) 1: 2: 4: 2A PCR is performed on a sample mixed with 8:16:32 (M3), followed by DGGE analysis (A). DGGE analysis was performed for 28 hours in 14% acrylamide / bis (37.5: 1), 43-63% denaturant. B represents the relative band brightness of six bands.

<110> SNU R & DB FOUNDATION <120> Analysing methods of bacteria community using 16S rDNA-DGGE and          real-time PCR <160> 5 <170> KopatentIn 1.71 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for genomic DNA <400> 1 agagtttgat cmtggctcag 20 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for genomic DNA <400> 2 ggytaccttg ttacgactt 19 <210> 3 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> Forward primer of AT type for PCR <400> 3 acacctacgg gtggc 15 <210> 4 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> Forward primer of TA type for PCR <400> 4 actcctacgg gaggc 15 <210> 5 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for PCR <400> 5 accgcggctg ctggcac 17  

Claims (5)

In the bacterial colonization method using PCR amplification and denaturation gradient gel electrophoresis (DGGE) of 16S rDNA of bacteria, the bacterial colony analysis method characterized by applying one or more of the following conditions: a) in the PCR amplification, the dNTP concentration is 0.25 to 0.375 mM, or b) In the PCR amplification, the PCR repetition number is used as the inflection point of the PCR amplification using real-time PCR, or c) The DGGE, wherein the concentration of acrylamide / bis in the DGGE gel is from 12% to 14%. The method of claim 1, further comprising: i) preparing a primer capable of specifically PCR amplifying 16S rDNA of bacteria; ii) using DNA of bacteria as a template and performing real-time PCR using the primers; iii) electrophoresis of the PCR product in DGGE to measure the intensity of the bands; And iv) calculating a species number of bacteria and a ratio of various kinds in the population based on the number and intensity of the bands. The method of claim 1, wherein in the PCR amplification, at least two PCR amplifications are performed by using two or more primers complementary to target region sequences of different 16S rDNAs of bacteria, and then, amplification products are mixed to make samples. Bacterial community analysis method characterized in that. The method of claim 1, wherein the bacteria are OP11, Verrucomicrobia, Bacteroidetes, Gammaproteobacteria, Actinobacteria and Genebank accession no. A method for analyzing bacterial communities of various environmental samples including representative soil bacteria such as bacteria having AY930457 16S rDNA. The method of claim 1, wherein the primers capable of specifically PCR amplifying the 16S rDNA of the bacteria are bacterial population analysis method characterized in that the F352AT primer of SEQ ID NO: 3 and F352TA primer of SEQ ID NO: 4.

Images