[29] Identification of archaea in objects of art by denaturing gradient gel electrophoresis analysis and shotgun cloning

Piñar, Güadalupe; Gurtner, Claudia; Lubitz, Werner; Rölleke, Sabine

doi:10.1016/s0076-6879(01)36601-6

Cited by 19 publications

(12 citation statements)

References 33 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…PCR screening was used to confirm the presence of inserted DNA on white colony (Pinar et al 2001;Suharsono et al 2009). PCR colony has been proven to be the easiest and most convenient way to amplify target DNA sequence in several yeast cells without any DNA extraction and purification prior to PCR (Mirhendi et al 2007).…”

Section: Pcr Colonymentioning

confidence: 99%

“…Sequencing and sequence data processing DNA sequencing is the determination and order of the nucleotide in a DNA strand. Sequencing is done to predict the function of sequence and manipulate it molecularly or to perform phylogenetic analysis and identification (Pinar et al 2001). In this research, DNA sequencing was conducted after the plasmid was successfully isolated.…”

Section: Plasmid Isolation and Pcr Plasmidmentioning

confidence: 99%

See 1 more Smart Citation

Development of protocols for genomic library construction of Agarwood (Aquilaria malaccensis)

Siregar

Suharsono

2017

Biodiversitas

View full text Add to dashboard Cite

Abstract. Siregar UJ, Maulana MI, Suharsono UW. 2017. Development of protocols for genomic library construction of Agarwood (Aquilaria malaccensis). Biodiversitas 18: 1150Biodiversitas 18: -1158. Agarwood is one of non timber forest products (NTFP) which has high economic value. Diminishing agarwood trees in natural forest due to intensive harvesting has shifted the production paradigm to forest plantation. In order to support agarwood tree improvement, investigation on the agarwood tree genomes and genes involved in agarwood production is highly needed through genomic library construction. This research aimed at establishing cloning protocols for genomic library construction of agarwood tree (Aquilaria malaccensis) until sequencing process. Genomic DNA was isolated using DNeasy Plant Mini Kit from Qiagen, then was digested with EcoR1, and was ligated to pGem®-T Easy vector system before it was finally transformed and was cloned to E.coli strain DH5α. Confirmation of DNA inserts was done using PCR colony with universal primer of SP6 and T7, plasmid isolation and also PCR plasmid and plasmid DNA digestion used Quick Plasmid Miniprep Kit from Thermofisher. Cloned A. malaccensis DNA fragments were sequenced and BLAST at NCBI site. The cloning successfully obtained five white colonies which contain inserted A. malaccensis DNA fragments with the size of 136-225 bp. PCR colony, plasmid isolation, PCR plasmid, and plasmid DNA digestion had confirmed the existence of inserted A. malaccensis DNA genome inside the bacteria cells. Sequencing and BLAST showed that DNA inserts from colony number 6, 8, and 9 were not similar with A. malccensis sequence that was recorded in NCBI, indicating that the genomic region in this library construction is different from the genomic DNA sequences of A. malaccensis in NCBI.

show abstract

Section: Pcr Colonymentioning

confidence: 99%

Section: Plasmid Isolation and Pcr Plasmidmentioning

confidence: 99%

Development of protocols for genomic library construction of Agarwood (Aquilaria malaccensis)

Siregar

Suharsono

2017

Biodiversitas

View full text Add to dashboard Cite

show abstract

“…Molecular techniques take advantage of the specificity provided by nucleic acid sequences for the identification of microorganisms and their independence of culturing microorganisms. Different, mainly PCR-based, genotyping techniques have been developed and adapted for the fingerprinting of microbial communities on biodeteriorated cultural heritage (Piñar et al, 2001a; Schabereiter-Gurtner et al, 2001; González, 2003; González and Saiz-Jimenez, 2004, 2005; Michaelsen et al, 2006). …”

Section: Introductionmentioning

confidence: 99%

Quantification of fungal abundance on cultural heritage using real time PCR targeting the Î²-actin gene

et al. 2014

View full text Add to dashboard Cite

The traditional methodology used for the identification of microbes colonizing our cultural heritage was the application of cultivation methods and/or microscopy. This approach has many advantages, as living microorganisms may be obtained for physiological investigations. In addition, these techniques allow the quantitative and qualitative assessment of the investigated environment. Quantitative analyses are done by plate count and the determination of abundance by the colony forming unit (CFU). Nevertheless, these techniques have many drawbacks that lead to an underestimation of the cell numbers and do not provide a comprehensive overview of the composition of the inhabiting microbiota. In the last decades, several molecular techniques have been developed enabling many advantages over the cultivation approach. Mainly PCR-based, fingerprinting techniques allow a qualitative detection and identification of the microbiota. In this study, we developed a real time PCR method as a simple, rapid and reliable tool to detect and quantify fungal abundance using the β-actin gene, which is known to appear as a single-copy gene in fungi. To this end, five different indoor thermal insulation materials applied for historical buildings that were previously tested for their bio-susceptibility against various fungi were subjected to qPCR analyses. The obtained results were compared with those obtained from a previous study investigating the bio-susceptibility of the insulation materials using classical cultivation experiments. Both results correlated well, revealing that Perlite plaster was the most suitable insulation material, showing the lowest fungal CFU and qPCR values. In contrast, insulations made of wood showed to be not recommendable from the microbiological point of view. In addition, the potential of qPCR was tested in other materials of cultural heritage, as old parchments, showing to be a suitable method for measuring fungal abundance in these delicate materials.

show abstract

“…16S ribosomal DNA analysis showed that the bacteria are most closely related to Halobacillus litoralis. DNA-DNA reassociation experiments identified the isolates as a population of hitherto unknown Halobacillus species.Recently, a number of investigations based on molecular techniques (9,22,24,25) as well as on cultivation techniques (10,16,17,27) have demonstrated that objects of art and building materials may be habitats for extremely salt tolerant and moderately halophilic bacteria. In fact, this group of microorganisms has been detected by molecular means on the mural paintings from the 14th century located in the Catherine chapel of the castle of Herberstein (Styria, Austria) (24, 25), the subject of this study.…”

mentioning

confidence: 99%

“…Amplification of 16S ribosomal DNA (rDNA) using specific archaeal primers (22) did not give any positive results, indicating that no Archaea were present in the enrichment cultures. However, 16S rDNA amplification using specific eubacterial primers (29) gave positive results.…”

mentioning

confidence: 99%

Detection of Indigenous Halobacillus Populations in Damaged Ancient Wall Paintings and Building Materials: Molecular Monitoring and Cultivation

Piñar

Ramos

Rölleke

et al. 2001

Appl Environ Microbiol

Self Cite

View full text Add to dashboard Cite

Several moderately halophilic gram-positive, spore-forming bacteria have been isolated by conventional enrichment cultures from damaged medieval wall paintings and building materials. Enrichment and isolation were monitored by denaturing gradient gel electrophoresis and fluorescent in situ hybridization. 16S ribosomal DNA analysis showed that the bacteria are most closely related to Halobacillus litoralis. DNA-DNA reassociation experiments identified the isolates as a population of hitherto unknown Halobacillus species.Recently, a number of investigations based on molecular techniques (9,22,24,25) as well as on cultivation techniques (10,16,17,27) have demonstrated that objects of art and building materials may be habitats for extremely salt tolerant and moderately halophilic bacteria. In fact, this group of microorganisms has been detected by molecular means on the mural paintings from the 14th century located in the Catherine chapel of the castle of Herberstein (Styria, Austria) (24, 25), the subject of this study. The aim of this work was the selective isolation of halophilic bacteria from wall paintings and building materials.Two samples were collected from different areas delimited in squares of around 20 cm 2 from the wall paintings of the chapel of the castle. Sample H1 was taken below the chancel's east wall window, where a brownish biofilm was observed; sample H6 was taken from the chancel's north wall from a zone of the painting with an intense rosy discoloration. An additional sample (m7) was taken on the outside of the chapel from a lime wall also showing an intense rosy discoloration. All samples were collected with a sterile scalpel by scraping off surface material and plaster to a depth of 1 to 3 mm. Small amounts of samples were used immediately for cultivation.Enrichment cultures were set up in 300-ml Erlenmeyer flasks containing 30 ml of M2 medium (20% [wt/vol] NaCl) (34) and maintenance medium (MM) medium (10% [wt/vol] NaCl) (31). To avoid fungal growth, media were supplemented with 50 g of cycloheximide (Sigma) ml Ϫ1 . All enrichments were incubated aerobically at room temperature (22 Ϯ 3°C) and at 37 o C in a water bath using magnetic stirring. Aliquots of the same enrichment cultures were used for denaturing gradient gel electrophoresis (DGGE) analysis, whole-cell hybridization, and isolation of bacteria by plating as described below.Genomic DNA from enrichment cultures was extracted according to the protocol described by Ausubel et al.(1). Amplification of 16S ribosomal DNA (rDNA) using specific archaeal primers (22) did not give any positive results, indicating that no Archaea were present in the enrichment cultures. However, 16S rDNA amplification using specific eubacterial primers (29) gave positive results. For DGGE analysis, 200-bp fragments of the 16S rDNA were amplified using the forward primer 341f GC (19) and the reverse primer 518r (19). PCR and DGGE were performed as described by SchabereiterGurtner et al. (29) and Muyzer et al. (19), respectively. DGGE profiles revealed only on...

show abstract

[29] Identification of archaea in objects of art by denaturing gradient gel electrophoresis analysis and shotgun cloning

Cited by 19 publications

References 33 publications

Development of protocols for genomic library construction of Agarwood (Aquilaria malaccensis)

Development of protocols for genomic library construction of Agarwood (Aquilaria malaccensis)

Quantification of fungal abundance on cultural heritage using real time PCR targeting the Î²-actin gene

Detection of Indigenous Halobacillus Populations in Damaged Ancient Wall Paintings and Building Materials: Molecular Monitoring and Cultivation

Contact Info

Product

Resources

About