Advantages of a New
            <i>Taq</i>
            DNA Polymerase in Multiplex PCR and Time-Release PCR

Kebelmann-Betzing, Christian; Seeger, K.; Dragon, Serge; G, Schmitt; Möricke, Anja; Schild, Thomas A.; Henze, Günter; Beyermann, Birgit

doi:10.2144/98241pf01

Cited by 67 publications

(36 citation statements)

References 16 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This may be due to the production of more specific products by AmpliTaq Gold DNA polymerase than by the conventional Taq DNA polymerase. DNA polymerase enzymes, like AmpliTaq Gold or Hotstart, that need to be activated at elevated temperature are known to enhance the specificity of a PCR assay (27). Reviewing the literature on the use of AmpliTaq Gold DNA polymerase in C. pneumoniae PCR assays, we found that in the majority of the studies, the type of DNA polymerase enzyme used was not identified.…”

Section: Discussionmentioning

confidence: 99%

Is the Perceived Association between Chlamydia pneumoniae and Vascular Diseases Biased by Methodology?

Maraha

Berg²,

Kerver³

et al. 2004

J Clin Microbiol

View full text Add to dashboard Cite

Inter-and intralaboratory inconsistencies in detection rates of Chlamydia pneumoniae in vascular specimens have been demonstrated. In this study, 66 vascular tissue specimens from 66 patients with vascular disease were tested by three PCR assays: a 16S PCR-based reverse line blot (RLB) assay, a single-step PCR, and a nested PCR. Also, we explored the impacts of different DNA polymerase enzymes on the results based on gel electrophoresis and hybridization. The PCR results by gel electrophoresis in the single-step PCR depended on which DNA polymerase was used. All samples were negative with AmpliTaq Gold DNA polymerase, and 54.5% (36 of 66) were positive with the conventional Taq DNA polymerase. All samples were negative after hybridization with a C. pneumoniae-specific probe. In the nested PCR, all specimens were negative by gel electrophoresis and after hybridization. The RLB assay failed to detect C. pneumoniae in any specimen; however, 20 specimens were Chlamydia sp. positive. The sequence analysis of six of these samples demonstrated Chlamydialike organisms. RLB detected Chlamydia sp. DNA in water and in the elution buffer after passage of the Qiagen columns (11 of 40). This study identified factors that may influence the detection of C. pneumoniae DNA in vascular tissues and consequently bias the perception of a link between C. pneumoniae and vascular diseases. The following are strongly recommended: to use DNA polymerases that have to be activated, to decontaminate with dUTP-uracil-DNA glycosylase, to hybridize with specific probes, to include sufficient controls, and to use molecular grade water.

show abstract

Section: Discussionmentioning

confidence: 99%

Is the Perceived Association between Chlamydia pneumoniae and Vascular Diseases Biased by Methodology?

Maraha

Berg²,

Kerver³

et al. 2004

J Clin Microbiol

View full text Add to dashboard Cite

show abstract

“…Cycling was performed with a time-release protocol which activates the inactive polymerase AmpliTaq Gold only partially before cycling. Full activation is then achieved during the first cycles (Kebelmann-Betzing et al, 1998). In first-round PCR, the reaction mixtures were kept at 95 mC for 3 min and then cycled 55 times with 20 s denaturation at 95 mC, 30 s annealing at 46 mC and 30 s strand extension at 72 mC, followed by a final extension step at 72 mC for 10 min.…”

Section: Methodsmentioning

confidence: 99%

Detection of two novel porcine herpesviruses with high similarity to gammaherpesviruses.

Ehlers

Ulrich

Goltz

1999

Journal of General Virology

105

102

View full text Add to dashboard Cite

Evidence for the existence of porcine gammaherpesviruses was obtained by PCR and sequence analysis. Initially, samples of peripheral blood mononuclear cells (PBMC), spleens, lungs, kidneys and livers of pigs from Germany and Spain were tested with a PCR assay which targets conserved regions of the herpesvirus DNA polymerase gene with degenerate and deoxyinosine-substituted primers. Amplicons of identical sequence were obtained from one spleen and two PBMC samples. This sequence showed a high percentage of identity with the DNA polymerase genes of herpesviruses of the oncogenic subfamily Gammaherpesvirinae. Alignment of amino acid sequences showed the highest identity values with bovine gammaherpesviruses, namely alcelaphine herpesvirus type 1 (68 %), ovine herpesvirus type 2 (68 %) and bovine lymphotropic herpesvirus (67 %). Comparison with pseudorabies virus and porcine cytomegalovirus, which are the only porcine herpesvirus species presently known, showed values of only 41 %. PCR analysis of PBMC (n l 39) and spleen (n l 19) samples from German pigs, using primers specific for the novel sequence, revealed a prevalence of 87 and 95 %, respectively. In this analysis, three out of eight spleen samples from Spanish pigs were also positive. Subsequent sequencing of the amplicons revealed the presence of two closely related gammaherpesvirus sequences, differing from each other by 8 % at the amino acid level. The putative novel porcine herpesviruses, from which these sequences originated, were tentatively designated porcine lymphotropic herpesvirus type 1 and type 2 (PLHV-1 and PLHV-2). When using pig organs for xenotransplantation, the presence of these viruses has to be considered.

show abstract

“…DNAs obtained from mouth rinses was diluted to near-single molecule levels and amplified using time-release PCR (Kebelmann-Betzing et al, 1998;Slebos et al, 2004b). The resulting SP-PCR reflects the amplification of small numbers of molecules that can be separated by automated capillary gel electrophoresis as reported by others (Coolbaugh-Murphy et al, 2005).…”

Section: Microsatellite Marker Analysesmentioning

confidence: 96%

Microsatellite mutations in buccal cells are associated with aging and head and neck carcinoma

Slebos

Vadivelu

et al. 2008

Br J Cancer

View full text Add to dashboard Cite

Carcinogen exposure from tobacco smoking is the major cause of upper aerodigestive tract cancer, yet heavy smokers only have about a 10% life-time risk of developing one of these cancers. Current technologies allow only limited prediction of cancer risk and there are no approved screening methods applicable to the general population. We developed a method to assess somatic mutational load using small-pool PCR (SP-PCR) and analysed mutations in DNA isolated from cells obtained by mouth rinse. Mutation levels in the hypermutable tetranucleotide marker D7S1482 were analysed in specimens from 25 head and neck squamous carcinoma (HNSCC) cases and 31 controls and tested for associations with age, smoking history and cancer status. We found a significant association between mutation frequency and age (P ¼ 0.021, Generalized Linear Model (GLM), N ¼ 56), but no influence of smoking history. Cases had higher mutation frequencies than controls when corrected for the effects of age, a difference that was statistically significant in the subgroup of 10 HNSCC patients who were treated with surgery only (P ¼ 0.017, GLM, N ¼ 41). We also present evidence that cancer status is linked to levels of nonunique, and presumably clonally derived, mutations in D7S1482. Insertion mutations were observed in 833 (79%) of 1058 alleles, of which 457 (43%) could be explained by insertion of a single repeat unit; deletion mutations were found in 225 (21%) of tested alleles.In conclusion, we demonstrate that the sensitive detection of single molecule mutations in clinical specimens is feasible by SP-PCR. Our study confirms an earlier report that microsatellite mutations increase with age and is the first to provide evidence that these mutations may be associated with cancer status in individual subjects.

show abstract

Advantages of a New Taq DNA Polymerase in Multiplex PCR and Time-Release PCR

Cited by 67 publications

References 16 publications

Is the Perceived Association between Chlamydia pneumoniae and Vascular Diseases Biased by Methodology?

Is the Perceived Association between Chlamydia pneumoniae and Vascular Diseases Biased by Methodology?

Detection of two novel porcine herpesviruses with high similarity to gammaherpesviruses.

Microsatellite mutations in buccal cells are associated with aging and head and neck carcinoma

Contact Info

Product

Resources

About