PCR amplification of highly GC-rich DNA template after denaturation by NaOH

Agarwal, Rajeev; Perl, András

doi:10.1093/nar/21.22.5283

Cited by 30 publications

(19 citation statements)

References 6 publications

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“…These included: boiling the template before ampli®cation (1±10 min) [19]; denaturing with 0.4 M NaOH and 0.4 mM EDTA for 10 min at room temperature followed by DNA puri®cation with a commercial DNA clean-up kit (Promega) [20]; adding For enumeration of total bacteria present in plaque samples, the PCR reaction master mix was added separately to 1 ìl of each boiled plaque sample and to 1 ìl of 10-fold serial dilutions of 10 6 E. coli cells (strain INFáF') in separate tubes. The basic PCR conditions were modi®ed as follows; 3 mM MgCl 2 and 22 repeated cycles of 958C (2 min), 508C (2.5 min) and 728C (2 min).…”

Section: Enumeration Of Total Bacteria In Samplesmentioning

confidence: 99%

Enumeration of Porphyromonas gingivalis, Prevotella intermedia and Actinobacillus actinomycetemcomitans in subgingival plaque samples by a quantitative-competitive PCR method

Doungudomdacha

Rawlinson

Douglas³

2000

Journal of Medical Microbiology

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Porphyromonas gingivalis, Prevotella intermedia and Actinobacillus actinomycetemcomitans are believed to play an important role in adult periodontitis, but the signi®cance of their relative numbers and progress of the disease is still unclear. Traditional quantitative methods are generally time-consuming and inaccurate. The aim of this study was to develop a sensitive, quantitative PCR technique that would be useful for enumerating P. gingivalis, Pr. intermedia and A. actinomycetemcomitans in subgingival plaque samples from subjects with adult periodontitis. Primers to the following genes were employed: the ®mbrial gene ( ®mA) of P. gingivalis, the 16S rRNA gene of Pr. intermedia and the leukotoxin-A (lktA) gene of A. actinomycetemcomitans. Competitive templates were constructed either by sequence deletion between primer binding sites or by annealing of the primer binding sites to an appropriate DNA core so as to yield products of a different size from that obtained with the target template. Coampli®cation of target and competitive templates yielded products of expected size and non-speci®c recognition by the primers was not found. The sensitivity of the designed primers was 100 cells of P. gingivalis, 100 cells of Pr. intermedia and 10 cells of A. actinomycetemcomitans. The three species were found in subgingival plaque samples collected from both healthy and diseased sites by the quantitative-competitive (QC)-PCR method and the technique was more sensitive than cultural methods. For determining the proportions of each of the three periodontopathogens, the total number of bacteria in the samples was enumerated by quantitative-PCR with 16S rRNA universal primers (27f and 342r). The ®ndings indicate that QC-PCR is a useful method for enumerating bacteria in clinical oral specimens and the technique could play a role in the investigation of disease progression.

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Section: Enumeration Of Total Bacteria In Samplesmentioning

confidence: 99%

Enumeration of Porphyromonas gingivalis, Prevotella intermedia and Actinobacillus actinomycetemcomitans in subgingival plaque samples by a quantitative-competitive PCR method

Doungudomdacha

Rawlinson

Douglas³

2000

Journal of Medical Microbiology

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“…The S. cinnamonensis DNA was prepared by digesting genomic DNA (10 g) with EcoRI, BamHI, PstI, BglII, and SmaI, precipitating with EtOH, and redissolving in TE (100 l). DNA (1 g) was then denatured in water (32 l) by addition of 4 M NaOH, 4 mM EDTA (8 l) for 10 min (22). After addition of sodium acetate (3 M, 7 l, pH 4.8) and water (4 l), the DNA was precipitated with EtOH and redissolved in water (100 l).…”

Section: Pcr Amplification Of An Icma Gene Fragmentmentioning

confidence: 99%

Cloning, Sequencing, Expression, and Insertional Inactivation of the Gene for the Large Subunit of the Coenzyme B12-dependent Isobutyryl-CoA Mutase from Streptomyces cinnamonensis

Zerbe-Burkhardt¹,

Ratnatilleke²,

Philippon³

et al. 1998

Journal of Biological Chemistry

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Purification of the coenzyme B12-dependent isobutyryl-CoA mutase (ICM) from Streptomyces cinnamonensis gave a protein of approximately 65 kDa by SDS-polyacrylamide gel electrophoresis, whose gene icmA was cloned using sequences derived from tryptic peptide fragments. The gene encodes a protein of 566 residues (62, 487 Da), with 43-44% sequence identity to the large subunit of methylmalonyl-CoA mutase (MCM) from S. cinnamonensis and Propionibacterium shermanii. Targeted disruption of the icmA gene yielded an S. cinnamonensis mutant devoid of ICM activity. The IcmA protein is approximately 160 residues shorter than the large subunit of the bacterial MCMs, corresponding to a loss of the entire C-terminal coenzyme B12 binding domain. The sequence of the (beta/alpha)8-barrel comprising residues A1-A400 in P. shermanii MCM is highly conserved in IcmA. The protein was produced in Streptomyces lividans and Escherichia coli with an N-terminal His6 tag (His6-IcmA), but after purification His6-IcmA showed no ICM activity. In the presence of coenzyme B12, protein from S. lividans and S. cinnamonensis of approximately 17 kDa by SDS-polyacrylamide gel electrophoresis could be selectively eluted with His6-IcmA from a Ni2+ affinity column. After purification, this small subunit showed no ICM activity but gave active enzyme when recombined with coenzyme B12 and IcmA or His6-IcmA.

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“…As seen in Table 1, two other conditions known to denature ds-DNA (9), namely the temperature of 95-C (28,29) and the alkaline pH of 12.5 (30), were also able to completely solubilize aggregated DNA. These observations confirm a predominant role played by hydrogen bonding in the moisture-induced aggregation.…”

Section: Solubilization Of Dna Aggregatesmentioning

confidence: 98%

Moisture-Induced Aggregation of Lyophilized DNA and its Prevention

Sharma

Klibanov

2006

Pharm Res

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The moisture-induced aggregation of lyophilized DNA is caused mainly by non-covalent cross-links between disordered, single-stranded regions of DNA. At high RH levels, renaturation and aggregation of DNA compete with each other. The aggregation is minimized at low RH levels, at optimal solution pH and salt concentration prior to lyophilization, and by co-lyophilizing with excipients capable of forming multiple hydrogen bonds, e.g., dextran and sucrose.

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PCR amplification of highly GC-rich DNA template after denaturation by NaOH

Cited by 30 publications

References 6 publications

Enumeration of Porphyromonas gingivalis, Prevotella intermedia and Actinobacillus actinomycetemcomitans in subgingival plaque samples by a quantitative-competitive PCR method

Enumeration of Porphyromonas gingivalis, Prevotella intermedia and Actinobacillus actinomycetemcomitans in subgingival plaque samples by a quantitative-competitive PCR method

Cloning, Sequencing, Expression, and Insertional Inactivation of the Gene for the Large Subunit of the Coenzyme B12-dependent Isobutyryl-CoA Mutase from Streptomyces cinnamonensis

Moisture-Induced Aggregation of Lyophilized DNA and its Prevention

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