| Amplification of minute amounts of oral bacterial DNA for real-time quantitative PCR analysis. | |
| | |
MedLine Citation:
|
PMID: 20881394 Owner: NLM Status: In-Process |
Abstract/OtherAbstract:
|
BACKGROUND: High-throughput technologies for typing caries or health-associated bacterial populations including PCR, DNA microarrays and next-generation sequencing techniques require significant amounts of bacterial DNA. In clinical settings, the amount of sampled DNA is often limited and amplification is therefore essential. Protocols should be able to reproducibly amplify sequences in order to maintain initial sequence ratios and should not bias the representation of particular DNA sequence types. METHODS: A linear amplification protocol using DNA polymerase I was modified to permit the amplification and subsequent analysis of small amounts of bacterial DNA. The protocol was tested on human oral bacterial biofilms from different sources, including carious dentine and plaque, and compared to amplification by degenerate PCR of 16S rDNA sequences. Real-time quantitative PCR of 24 bacterial species was used as a readout system to test amplified DNA against unamplified DNA. RESULTS: The amplification protocol reliably yielded 5-10 μg DNA from as little as 12.5 ng of template DNA. Correlation coefficients between real-time quantitative PCR results from amplified and unamplified DNA were between 0.78 and 0.98. CONCLUSION: The optimized protocol consistently produced amplification products from minute amounts of bacterial DNA from caries and plaque; the amplification products are suitable for downstream genetic analyses. |
| | |
Authors:
|
D Wolff; H J Staehle; B Wolff |
Related Documents
:
|
23288404 - Lqb-118, an orally active pterocarpanquinone, induces selective oxidative stress and ap... 8560754 - Applications of dna amplification techniques in veterinary diagnostics. 17317684 - Multiplex amplification of all coding sequences within 10 cancer genes by gene-collector. 1738604 - 7-deazapurine containing dna: efficiency of c7gdtp, c7adtp and c7idtp incorporation dur... 11369894 - Genes ia, ii, iii, iv and v of soybean chlorotic mottle virus are essential but the gen... 9722644 - Conserved dna sequences adjacent to chromosome fragmentation and telomere addition site... |
Publication Detail:
|
Type: Journal Article; Research Support, Non-U.S. Gov't Date: 2010-09-24 |
Journal Detail:
|
Title: Caries research Volume: 44 ISSN: 1421-976X ISO Abbreviation: Caries Res. Publication Date: 2010 |
Date Detail:
|
Created Date: 2010-11-16 Completed Date: - Revised Date: - |
Medline Journal Info:
|
Nlm Unique ID: 0103374 Medline TA: Caries Res Country: Switzerland |
Other Details:
|
Languages: eng Pagination: 498-504 Citation Subset: D; IM |
Copyright Information:
|
Copyright © 2010 S. Karger AG, Basel. |
Affiliation:
|
Department of Conservative Dentistry, School of Dental Medicine, Ruprecht Karls University, Heidelberg, Germany. Diana.Wolff@med.uni-heidelberg.de |
Export Citation:
|
APA/MLA Format Download EndNote Download BibTex |
| MeSH Terms | |
Descriptor/Qualifier:
|
|
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
Previous Document: Early improvement in peripheral vascular tone following smoking cessation using nicotine replacement...
Next Document: KIBRA genetic polymorphism influences episodic memory in Alzheimer's disease, but does not show asso...