Curr Microbiol (2014) 68:486–494 DOI 10.1007/s00284-013-0488-1
A Simple One-Step PCR Walking Method and Its Application of Bacterial rRNA for Sequencing Identification Hongfa Zhang • Chunping You • Jing Ren Dan Xu • Mei Han • Wenyan Liao
Received: 4 January 2013 / Accepted: 28 September 2013 / Published online: 21 November 2013 Ó Springer Science+Business Media New York 2013
Abstract There are many PCR walking methods applied currently, and they all have examples of successful application in organisms which are more complex than bacteria. However, to a certain extent, it will be more convenient for researchers if the complicated operation and poor specificity for bacteria can be improved. Here, we introduced an improved one-step PCR walking method of bacteria. Using a specific primer of the known sequence together with a universal semirandom primer, the unknown sequence adjacent to a known sequence can be obtained easily by just one ordinary round PCR. The products can be gel purified and directly sequenced. Specific primers were designed according to the gene sequence of bacterial rRNA, and the variable and adjacent gene sequences were obtained by this method. The sequence analysis of the product showed that it can improve the resolution of bacterial identification to the species level.
Introduction It is an important task in microbial genome-related research to obtain unknown sequence adjacent to a known sequence using gene walking . There are many PCR-based gene
H. Zhang (&) C. You J. Ren M. Han W. Liao State Key Laboratory of Dairy Biotechnology, Technology Center of Bright Dairy & Food Co., Ltd, Shanghai 200436, China e-mail: [email protected] D. Xu Pass College of Chongqing Technology and Business University, Chongqing 400060, China
walking methods. Although there are examples of successful application of these methods, most of them are complicated in operation. For example, self-formed adaptor PCR is efficient to get unknown sequence in complex genome, but the cycling conditions are complicated, and the annealing temperatures of several cycles are ranged from 35 to 45 °C ; sequential hybrid primer PCR has two rounds of PCR, and the annealing temperature of one of the cycles is 35 °C ; two-step PCR has 61 cycles, and the annealing temperature for one of the cycles is 40 °C ; easy gene walking has to use three rounds of PCR to get the target sequence, although the annealing temperature is higher . In short, to some extent, almost all the gene walking methods currently used have inconveniences of complicated operation and poor specificity for bacteria which genome is relatively small. Because the lower annealing temperature likely to increasing nonspecific binding of the primer with the template, and increasing the nonspecific products of amplification. Therefore, it is urgent that we need to explore a simple, efficient, and universal gene walking method for bacterial identification. Sequences of rRNA genes have been well-applied in microbial identification . However, since their sequences are relatively conserved [5, 14], it is almost impossible to discriminate between highly related species . Also, as rRNA genes in bacteria often occur in multiple copies , PCR products of rRNA genes are often mixed, and hence they have to be cloned and sequenced. In this study, a one-step PCR walking method was established, and primarily used to amplify variable regions of multiple copies gene which are adjacent to conserved regions of rRNA in bacteria. After being gel purified, they were directly sequenced and analyzed for bacteria identification.
H. Zhang et al.: A Simple One-Step PCR Walking Method
Table 1 Primers Primer name
Primer sequence(50 –30 )
Materials and Methods Primers Primers used in this study are shown in Table 1. The primers of rRNA were designed for members of bacteria, but not for those of archaea. EU27F and 1492R were designed according to references of [1, 7], 357R was designed according to references of [13, 14], 474F and 474R were designed according to reference of . The semirandom primer (AP) consists of three parts (50 –30 ): (i) DQW primer, (ii) 6 bases of degenerate sequence, and (iii) 6 bases of a specific sequence. DQW was the sequence of the nonbacterial strains. The specificity of the primers was checked using BLAST in NCBI. One-Step PCR Walking The principle of one-step PCR walking method is shown in Fig. 1 [10, 11]. Firstly, specific primers (SP) only amplify the single chain of the target gene sequence at the higher annealing temperature. When single chain accumulates to a certain number, they combine with the AP, and are amplified with SP and AP. The nonspecific pairing of AP and SP is reduced at higher annealing temperature, and that reduce nonspecific amplifications. SP or AP which containing the sequence of DQW can
form panhandle structure to inhibit the nonspecific amplification. Genomic DNA of single colonies of bacterial strains was isolated from bacteria using TaKaRa minibest bacterial genomic DNA extraction kit ver. 2. 0 (Takara Biotechnology (Dalian) Co., Ltd). Approximately, 50 ng of template DNA was used for PCR amplification with SP (0.5 lmol/L) and AP (0.5 lmol/L) in a 50 lL reaction volume, 5u Ex-Taq (Takara Biotechnology (Dalian) Co., Ltd). The cycling program was as follows: 94 °C for 5 min; 35 cycles at 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min; and extended reaction at 72 °C for 5 min. The clear bands of the remaining PCR products were gel purified and sequenced (Sagon, Shanghai, China) directly by using the primer of either SP or DQW. The results were analyzed by BLAST in NCBI.
Results Selection of the Primers of rRNA for Bacterial Identification Genomic DNA of 23 strains bacteria of 18 species was used as templates for PCR walking to get sequences about rRNA. 357R primer was used to get upstream sequence of 16S rRNA, 474R primer was used to get intergenic sequence of 16S-23S rRNA, and 474F primer was used to get sequence of 23S rRNA. The results (Fig. 2; Table 2) prove that one-step PCR walking method can be used for different primers and bacteria. The sequences obtained from primer 357R of the 23 bacteria show that only one species of bacteria was the highest homologous, and can be used for bacterial identification. Identification of Different Bacteria The primer 357R was used to obtain upstream sequence of 16S rRNA. Using the primers of EU27F and 1492R, to obtain the sequence of 16S rRNA. The results of
Fig. 1 Basic principle of the AP gene-walking PCR. The known sequence stretch is shown as solid line, the unknown sequence stretch is shown as dotted line, SP is shown as black arrow, and AP is shown as black and white arrow. After 35 cycles, the target sequences were the products of the double primers of SP and AP; the nontarget sequences were the products of a single prime of SP or AP
H. Zhang et al.: A Simple One-Step PCR Walking Method
Fig. 2 Walking PCRs of different bacterial rRNA. Lane M: DL2, 000 DNA Marker; Lanes 1–3:357R, 474F, 474R. The bands shown by arrow were purified and sequenced
BLAST are shown in Table 3. For the BLAST analysis of the sequence upstream 16S rRNA, the bacterium with the highest score is corresponding to the bacterium of detection, and is significantly different from the other bacteria with the second highest score. All 41 selected strains of 31 species can be identified at the species level.
Discussion Compared with the ordinary walking PCR methods, the one-step PCR walking in this study has the advantages of convenient operation. Furthermore, the higher annealing temperature in the entire PCR procedure ensures the specificity and accuracy of the amplification. This is due to the fact that the AP primer consists of three parts (50 –30 ): 1.
The DQW primer, which was used as sequencing primer, can increase the pairing length of the APs to amplify the target sequence in the first round; DQW also can help forming panhandle structure
between primers of AP to inhibit the nonspecific amplification. 2. Six bases of degenerate sequence add more complementary sites to increasing annealing temperature. 3. Six bases of a specific sequence have high G/C % which can increase annealing temperature. As sequences of rRNA genes are obtained the universality of bacterial sequencing primers, and the variability of amplified sequences is achieved. It can also solve the problem that the mixed PCR products of multiple gene copies cannot be directly sequenced. The resolution of bacterial identification by using the primer 357R was increased to the species level. In practical applications, we also found that some bacteria sequences do not contain the corresponding upstream sequences of the 16S rRNA genes deposited in public databases. This leads to difficulties in identifying highly related sequences by BLAST analysis as often several sequences show the highest BLAST scores. With the speed at which genomes of bacterial strains are sequences it will one day be unnecessary to do PCR at all as relationships will be determined by IT programs.
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494 Acknowledgments This work was supported by the Major Project of Chinese National Programs for Fundamental Research and Development (973 Program) (grant no. 2012CB723706).
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