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Identification of signature and primers specific to genus Pseudomonas using mismatched patterns of 16S rDNA sequences.

Purohit HJ, Raje DV, Kapley A - BMC Bioinformatics (2003)

Bottom Line: The signature region was used to design PCR primers, which yielded a PCR product of 150 bp whose specificity was validated through a sample experiment.The developed approach was successfully applied to genus Pseudomonas.It could be tried in other bacterial genera to obtain respective signature patterns and thereby PCR primers, for their rapid tracking in the environmental samples.

View Article: PubMed Central - HTML - PubMed

Affiliation: Environmental Modeling and Genomics Division, National Environmental Engineering Research Institute, Nehru Marg, Nagpur - 440020 (MS) India. hemantdrd@hotmail.com

ABSTRACT

Background: Pseudomonas, a soil bacterium, has been observed as a dominant genus that survives in different habitats with wide hostile conditions. We had a basic assumption that the species level variation in 16S rDNA sequences of a bacterial genus is mainly due to substitutions rather than insertion or deletion of bases. Keeping this in view, the aim was to identify a region of 16S rDNA sequence and within that focus on substitution prone stretches indicating species level variation and to derive patterns from these stretches that are specific to the genus.

Results: Repeating elements that are highly conserved across different species of Pseudomonas were considered as guiding markers to locate a region within the 16S gene. Four repeating patterns showing more than 80% consistency across fifty different species of Pseudomonas were identified. The sub-sequences between the repeating patterns yielded a continuous region of 495 bases. The sub-sequences after alignment and using Shanon's entropy measure yielded a consensus pattern. A stretch of 24 base positions in this region, showing maximum variations across the sampled sequences was focused for possible genus specific patterns. Nine patterns in this stretch showed nearly 70% specificity to the target genus. These patterns were further used to obtain a signature that is highly specific to Pseudomonas. The signature region was used to design PCR primers, which yielded a PCR product of 150 bp whose specificity was validated through a sample experiment.

Conclusions: The developed approach was successfully applied to genus Pseudomonas. It could be tried in other bacterial genera to obtain respective signature patterns and thereby PCR primers, for their rapid tracking in the environmental samples.

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Related in: MedlinePlus

The consensus pattern obtained for the 495 bp region. Each dash indicates that the IC value at that position is less than 1.9, while the bases indicate absolute conservation at the position.
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Figure 4: The consensus pattern obtained for the 495 bp region. Each dash indicates that the IC value at that position is less than 1.9, while the bases indicate absolute conservation at the position.

Mentions: Thus the conservation analysis for sub-sequences enclosed between the four selected consistent repeats yielded a consensus pattern of conserved and variable positions for the 495 bp region as shown in Figure 4. The region might be having part of the ancestral information as well as some information specific to genus Pseudomonas. The 5' and the 3'-ends of this consensus pattern have elongated variable stretches. The alignment of sub-sequences flanked by the repeating pattern CAGCAG enclosing the stretch of maximum variation, as shown in Figure 3B, was focused for identifying target specific patterns and thereby the primers.


Identification of signature and primers specific to genus Pseudomonas using mismatched patterns of 16S rDNA sequences.

Purohit HJ, Raje DV, Kapley A - BMC Bioinformatics (2003)

The consensus pattern obtained for the 495 bp region. Each dash indicates that the IC value at that position is less than 1.9, while the bases indicate absolute conservation at the position.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC161812&req=5

Figure 4: The consensus pattern obtained for the 495 bp region. Each dash indicates that the IC value at that position is less than 1.9, while the bases indicate absolute conservation at the position.
Mentions: Thus the conservation analysis for sub-sequences enclosed between the four selected consistent repeats yielded a consensus pattern of conserved and variable positions for the 495 bp region as shown in Figure 4. The region might be having part of the ancestral information as well as some information specific to genus Pseudomonas. The 5' and the 3'-ends of this consensus pattern have elongated variable stretches. The alignment of sub-sequences flanked by the repeating pattern CAGCAG enclosing the stretch of maximum variation, as shown in Figure 3B, was focused for identifying target specific patterns and thereby the primers.

Bottom Line: The signature region was used to design PCR primers, which yielded a PCR product of 150 bp whose specificity was validated through a sample experiment.The developed approach was successfully applied to genus Pseudomonas.It could be tried in other bacterial genera to obtain respective signature patterns and thereby PCR primers, for their rapid tracking in the environmental samples.

View Article: PubMed Central - HTML - PubMed

Affiliation: Environmental Modeling and Genomics Division, National Environmental Engineering Research Institute, Nehru Marg, Nagpur - 440020 (MS) India. hemantdrd@hotmail.com

ABSTRACT

Background: Pseudomonas, a soil bacterium, has been observed as a dominant genus that survives in different habitats with wide hostile conditions. We had a basic assumption that the species level variation in 16S rDNA sequences of a bacterial genus is mainly due to substitutions rather than insertion or deletion of bases. Keeping this in view, the aim was to identify a region of 16S rDNA sequence and within that focus on substitution prone stretches indicating species level variation and to derive patterns from these stretches that are specific to the genus.

Results: Repeating elements that are highly conserved across different species of Pseudomonas were considered as guiding markers to locate a region within the 16S gene. Four repeating patterns showing more than 80% consistency across fifty different species of Pseudomonas were identified. The sub-sequences between the repeating patterns yielded a continuous region of 495 bases. The sub-sequences after alignment and using Shanon's entropy measure yielded a consensus pattern. A stretch of 24 base positions in this region, showing maximum variations across the sampled sequences was focused for possible genus specific patterns. Nine patterns in this stretch showed nearly 70% specificity to the target genus. These patterns were further used to obtain a signature that is highly specific to Pseudomonas. The signature region was used to design PCR primers, which yielded a PCR product of 150 bp whose specificity was validated through a sample experiment.

Conclusions: The developed approach was successfully applied to genus Pseudomonas. It could be tried in other bacterial genera to obtain respective signature patterns and thereby PCR primers, for their rapid tracking in the environmental samples.

Show MeSH
Related in: MedlinePlus