Primer design for a prokaryotic differential display RT-PCR

Fislage, Rainer; Berceanu, M.; Humboldt, Y.; Wendt, Maria; Oberender, H

doi:10.1093/nar/25.9.1830

Cited by 64 publications

(50 citation statements)

References 22 publications

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“…The sequence of MPRP2 was selected based upon analysis of the complete 6n9 kbp pPS41 sequence (Powers et al, 2000) and partial sequences of four additional marine plasmids : p09022, p0329, Salt marsh diazotroph plasmid diversity p23023 and p0908 (P. Sobecky & J. Eisen, unpublished). The plasmid sequences were analysed using OligoCalc (Fislage et al, 1997) to calculate the most frequently occurring oligomers of a given length (6-mer and 8-mer).…”

Section: Rapd Primersmentioning

confidence: 99%

Differentiation of plasmids in marine diazotroph assemblages determined by randomly amplified polymorphic DNA analysis

et al. 2002

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Nitrogen fixation by diazotrophic bacteria is a significant source of new nitrogen in salt marsh ecosystems. Recent studies have characterized the physiological and phylogenetic diversity of oxygen-utilizing diazotrophs isolated from the rhizoplanes of spatially separated intertidal macrophyte habitats. However, there is a paucity of information regarding the traits encoded by and the diversity of plasmids occurring in this key ecological functional group. Five-hundred and twenty-one isolates cultivated from the rhizoplanes of Juncus roemarianus, Spartina patens and different growth forms (short-form and tall-form) of Spartina alterniflora were screened for the presence of plasmids. One-hundred and thirty-four diazotrophs carrying plasmids that ranged in size from 2 to S 100 kbp were identified. The majority of the marine bacteria contained one plasmid. Diazotrophs from the shortform S. alterniflora rhizoplane contained significantly fewer plasmids relative to isolates from tall-form S. alterniflora, J. roemarianus and S. patens. Although some plasmids exhibited homology to a nifH gene probe, the majority of the plasmids were classified as cryptic. Two oligonucleotide primers were developed to facilitate genotypic typing of the endogenously isolated marine plasmids by the randomly amplified polymorphic DNA (RAPD)-PCR technique. These primers proved to be more effective than 21 commercially available primers tested to generate RAPD-PCR patterns. Analysis of the RAPD-PCR patterns indicated as many as 71 different plasmid genotypes occurring in diazotroph bacterial assemblages within and between the four different salt marsh grass rhizoplane habitats investigated in this study.

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Section: Rapd Primersmentioning

confidence: 99%

Differentiation of plasmids in marine diazotroph assemblages determined by randomly amplified polymorphic DNA analysis

et al. 2002

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“…These methods rely on arbitrary primers for cDNA synthesis and\or subsequent amplification, and are therefore subject to the poorer reproducibility conferred by low-stringency PCR conditions. Suitable primers have to be designed from a statistical evaluation of coding genomic sequences (Fislage et al, 1997) and many random primers are required to cover the whole bacterial genome.…”

Section: Introductionmentioning

confidence: 99%

Restriction fragment differential display of pediocin-resistant Listeria monocytogenes 412 mutants shows consistent overexpression of a putative β-glucoside-specific PTS system The EMBL accession numbers for the sequences reported in this paper are AJ251202, AJ251203 and AJ251204.

Gravesen¹,

Warthoe²,

Knøchel³

et al. 2000

Microbiology

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Pediocin PA-1, which is a bacteriocin produced by lactic acid bacteria, has potential as a biopreservative of food. However, such use may lead to the development of resistance in the target organism. Gene expression in two independent pediocin-resistant mutants of Listeria monocytogenes 412 was compared to the original isolate by restriction fragment differential display PCR (RFDD-PCR). This method amplifies cDNA restriction fragments under stringent PCR conditions, enabled by the use of specific primers complementary to ligated adaptor sequences. RFDD-PCR was very well suited for analysis of listerial gene expression, giving reproducible PCR product profiles. Three gene fragments having increased expression in both resistant mutants were identified. All three had homology to components of β-glucoside-specific phosphoenolpyruvate-dependent phosphotransferase systems (PTS), one fragment having homology to enzyme II permeases, and the two others to phospho-β-glucosidases. Overexpression of the putative PTS system was consistently observed in 10 additional pediocin-resistant mutants, isolated at different pH, salt content and temperature. The results suggest that RFDD-PCR is a strong approach for the analysis of prokaryotic gene expression and that the putative β-glucoside-specific PTS system is involved in mediating pediocin resistance.

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“…The application of techniques such as differential display and subtractive hybridization, commonly used for the analysis of eukaryotic gene expression, has been limited in prokaryotes, owing to the generally accepted view that 3h-polyadenylylation of mRNA is a eukaryotic feature. Differential display in prokaryotes therefore requires the use of non-specific, random arbitrary primers (RAP) for cDNA synthesis, often involving extensive testing and optimization of primers (Kwaik & Pederson, 1996 ;Fislage et al, 1997 ;Rivera-Marrero et al, 1998). In addition, reamplification by PCR and subcloning of the isolated cDNA fragments is often difficult and results in failure.…”

Section: Discussionmentioning

confidence: 99%

Polyadenylylation in mycobacteria: evidence for oligo(dT)-primed cDNA synthesis

2000

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The ability of mRNA to direct synthesis of cDNA in the presence of oligo(dT) was analysed using a novel application of fluorescein-11-dUTP incorporation into cDNA by reverse transcriptase. Evidence is provided for the first time that a majority of the mycobacterial mRNA pool is polyadenylylated. mRNA transcripts of hsp65 were also amplified with specific primers from the oligo(dT)-primed cDNA preparation in Mycobacterium bovis BCG, M. smegmatis and M. vaccae. Furthermore, PCR amplication of cDNAs for genes entD, entC and trpE2 from M. bovis BCG yielded the expected products when reverse transcription was primed with oligo(dT), suggesting that polyadenylylation is a general phenomenon in mycobacteria.

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Primer design for a prokaryotic differential display RT-PCR

Cited by 64 publications

References 22 publications

Differentiation of plasmids in marine diazotroph assemblages determined by randomly amplified polymorphic DNA analysis

Differentiation of plasmids in marine diazotroph assemblages determined by randomly amplified polymorphic DNA analysis

Restriction fragment differential display of pediocin-resistant Listeria monocytogenes 412 mutants shows consistent overexpression of a putative β-glucoside-specific PTS system The EMBL accession numbers for the sequences reported in this paper are AJ251202, AJ251203 and AJ251204.

Polyadenylylation in mycobacteria: evidence for oligo(dT)-primed cDNA synthesis

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