Dataset Information

Amplification of GC-rich Putative Mouse PeP Promoter using Betaine and DMSO in Ammonium Sulfate Polymerase Chain Reaction Buffer.

ABSTRACT: BACKGROUND:Recently, we have shown that peroxisomal protein expression was induced upon retinoic acid treatment in mouse embryonic stem cells during the process of neurogenesis. Thus, characterization of the respective promoter could elucidate the molecular aspects of transcriptional regulation of this gene. METHODS:Using the conventional software programs for promoter prediction, a putative promoter region was identified approximately 561 bp upstream of the peroxisomal protein coding sequence. In order to clone this region with a GC-content of 71.01%, a cocktail of ammonium sulfate buffer supplied with two additive components, betaine and dimethyl sulfoxide, and a high concentration of MgCl(2) was used. RESULTS:The modulated polymerase chain reaction composition significantly improved the amplification of GC-rich DNA target sequences. Improved amplification of this region was due to reduction in the formation of secondary structures by the GC-rich region. CONCLUSION:Therefore, this polymerase chain reaction composition could be generally used to facilitate the amplification of other GC-rich DNA sequences as verified by amplification of different GC rich regions.

SUBMITTER: Seifi T

PROVIDER: S-EPMC3558226 | biostudies-literature | 2012 Oct

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Amplification of GC-rich Putative Mouse PeP Promoter using Betaine and DMSO in Ammonium Sulfate Polymerase Chain Reaction Buffer.

Seifi Tahere T Ghaedi Kamran K Salamian Ahmad A Tanhaei Sommayeh S Safari Forouzan F Hojati Zohreh Z Tavassoli Manuchehr M Baharvand Hossein H Esfahani Mohammad-Hossein Nasr MH

Avicenna journal of medical biotechnology 20121001 4

<h4>Background</h4>Recently, we have shown that peroxisomal protein expression was induced upon retinoic acid treatment in mouse embryonic stem cells during the process of neurogenesis. Thus, characterization of the respective promoter could elucidate the molecular aspects of transcriptional regulation of this gene.<h4>Methods</h4>Using the conventional software programs for promoter prediction, a putative promoter region was identified approximately 561 bp upstream of the peroxisomal protein co ...[more]

PMID: 23408119

Similar Datasets

Project description:Polymerase chain reaction (PCR) has extensive bioanalytical applications in molecular diagnostics and genomic research studies for rapid detection and precise genomic amplification. Routine integrations for analytical workflow indicate certain limitations, including low specificity, efficiency, and sensitivity in conventional PCR, particularly towards amplifying high guanine-cytosine (GC) content. Further, there are many ways to enhance the reaction, for example, using different PCR strategies such as hot-start/touchdown PCR or adding some special modifications or additives such as organic solvents or compatible solutes, which can improve PCR yield. Due to the widespread use of bismuth-based materials in biomedicine, which have not yet been used for PCR optimization, this attracts our attention. In this study, two bismuth-based materials that are inexpensive and readily available were used to optimize GC-rich PCR. The results demonstrated that ammonium bismuth citrate and bismuth subcarbonate effectively enhanced PCR amplification of the GNAS1 promoter region (∼84% GC) and APOE (75.5% GC) gene of Homo sapiens mediated by Ex Taq DNA polymerase within the appropriate concentration range. Combining DMSO and glycerol additives was critical in obtaining the target amplicons. Thus, the solvents mixed with 3% DMSO and 5% glycerol were used in bismuth-based materials. That allowed for better dispersion of bismuth subcarbonate. As for the enhanced mechanisms, the surface interaction of PCR components, including Taq polymerase, primer, and products with bismuth-based materials, was maybe the main reason. The addition of materials can reduce the melting temperature (Tm), adsorb polymerase and modulate the amount of active polymerase in PCR, facilize the dissociation of DNA products, and enhance the specificity and efficiency of PCR. This work provided a class of candidate enhancers for PCR, deepened our understanding of the enhancement mechanisms of PCR, and also explored a new application field for bismuth-based materials.

Dataset Information

Amplification of GC-rich Putative Mouse PeP Promoter using Betaine and DMSO in Ammonium Sulfate Polymerase Chain Reaction Buffer.

Publications

Amplification of GC-rich Putative Mouse PeP Promoter using Betaine and DMSO in Ammonium Sulfate Polymerase Chain Reaction Buffer.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets