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Retracted HIV Study Provides New Information about the Status of the in Vitro Inhibition of DNA Replication by Backbone Methylation  [PDF]
Henk M. Buck
Journal of Biophysical Chemistry (JBPC) , 2015, DOI: 10.4236/jbpc.2015.61003
Abstract: In this publication attention is given to a retracted article in Science at the end of 1990 concerning the HIV-1 inhibition by a modified backbone DNA as the phosphatemethylated DNA. A disproportion in the presented data resulted in a faulty generalization of the (bio)chemical characteristics of the phosphatemethylated DNA (18- and 20-nucleotides). In the confusion and the outside pressure a related study in Nucleic Acids Research on the in vitro dynamics of a regiospecific inhibition of DNA duplication with long (20- and 18-nucleotides) and short (8-nucleotides) phosphatemethylated DNA was completely ignored. A restoration will be given based on a comprehensive view demonstrating the unique molecular and conformational properties of phosphatemethylated DNA in their (bio)chemistry towards natural DNA and RNA (HIV-1 RNA loops).
Cleavage of Phosphorothioated DNA and Methylated DNA by the Type IV Restriction Endonuclease ScoMcrA  [PDF]
Guang Liu,Hong-Yu Ou,Tao Wang,Li Li,Huarong Tan,Xiufen Zhou,Kumar Rajakumar,Zixin Deng ,Xinyi He
PLOS Genetics , 2010, DOI: 10.1371/journal.pgen.1001253
Abstract: Many taxonomically diverse prokaryotes enzymatically modify their DNA by replacing a non-bridging oxygen with a sulfur atom at specific sequences. The biological implications of this DNA S-modification (phosphorothioation) were unknown. We observed that simultaneous expression of the dndA-E gene cluster from Streptomyces lividans 66, which is responsible for the DNA S-modification, and the putative Streptomyces coelicolor A(3)2 Type IV methyl-dependent restriction endonuclease ScoA3McrA (Sco4631) leads to cell death in the same host. A His-tagged derivative of ScoA3McrA cleaved S-modified DNA and also Dcm-methylated DNA in vitro near the respective modification sites. Double-strand cleavage occurred 16–28 nucleotides away from the phosphorothioate links. DNase I footprinting demonstrated binding of ScoA3McrA to the Dcm methylation site, but no clear binding could be detected at the S-modified site under cleavage conditions. This is the first report of in vitro endonuclease activity of a McrA homologue and also the first demonstration of an enzyme that specifically cleaves S-modified DNA.
Assessing the efficiency and significance of Methylated DNA Immunoprecipitation (MeDIP) assays in using in vitro methylated genomic DNA
Jinsong Jia, Aleksandra Pekowska, Sebastien Jaeger, Touati Benoukraf, Pierre Ferrier, Salvatore Spicuglia
BMC Research Notes , 2010, DOI: 10.1186/1756-0500-3-240
Abstract: We performed MeDIP assays using in vitro methylated DNA, with or without previous DNA amplification, and hybridization to a human promoter array. We observed that CpG content at gene promoters indeed correlates strongly with the MeDIP signal obtained using in vitro methylated DNA, even when lowering significantly the amount of starting material. In analyzing MeDIP products that were subjected to whole genome amplification (WGA), we also revealed a strong bias against CpG-rich promoters during this amplification procedure, which may potentially affect the significance of the resulting data.We illustrate the use of in vitro methylated DNA to assess the efficiency and accuracy of MeDIP procedures. We report that efficient and reproducible genome-wide data can be obtained via MeDIP experiments using relatively low amount of starting genomic DNA; and emphasize for the precaution that must be taken in data analysis when an additional DNA amplification step is required.DNA methylation at CpG dinucleotides is a major epigenetic modification with direct implications in many aspects of mammalian biology, including development and disease [1]. In normal tissues, most promoter-associated CpGs remain unmethylated, although DNA methylation does occur at promoters of a small set of genes where it generally leads to transcriptional silencing. On the other hand, cancer cells undergo dramatic changes in the level and distribution of DNA methylation [2]. Indeed, the DNA methylation-dependent silencing of many tumor suppressor genes is now recognized as a major mechanism of gene inactivation that complements genetic lesions. Recent technological advances have allowed the comprehensive analysis of DNA methylation profiles in normal and disease-associated cells [3-6]. In particular, the Methylated DNA ImmunoPrecipitation (MeDIP) assay appears to be an efficient, reproducible and cost-effective approach to characterize the methylome of large collections of DNA samples [7-10]. The overall
Synthesis of DNA/RNA and Their Analogs via Phosphoramidite and H-Phosphonate Chemistries  [PDF]
Subhadeep Roy,Marvin Caruthers
Molecules , 2013, DOI: 10.3390/molecules181114268
Abstract: The chemical synthesis of DNA and RNA is universally carried out using nucleoside phosphoramidites or H-phosphonates as synthons. This review focuses on the phosphorus chemistry behind these synthons and how it has been developed to generate procedures whereby yields per condensation approach 100% with very few side products. Additionally the synthesis and properties of certain DNA and RNA analogs that are modified at phosphorus will also be discussed. These analogs include boranephosphonates, metallophosphonates, and alkylboranephosphines.
Use of Specific Chemical Reagents for Detection of Modified Nucleotides in RNA  [PDF]
Isabelle Behm-Ansmant,Mark Helm,Yuri Motorin
Journal of Nucleic Acids , 2011, DOI: 10.4061/2011/408053
Abstract: Naturally occurring cellular RNAs contain an impressive number of chemically distinct modified residues which appear posttranscriptionally, as a result of specific action of the corresponding RNA modification enzymes. Over 100 different chemical modifications have been identified and characterized up to now. Identification of the chemical nature and exact position of these modifications is typically based on 2D-TLC analysis of nucleotide digests, on HPLC coupled with mass spectrometry, or on the use of primer extension by reverse transcriptase. However, many modified nucleotides are silent in reverse transcription, since the presence of additional chemical groups frequently does not change base-pairing properties. In this paper, we give a summary of various chemical approaches exploiting the specific reactivity of modified nucleotides in RNA for their detection. 1. Introduction Native cellular RNAs contain numerous modified residues resulting from specific action of various RNA modification enzymes. These RNA modifications are ubiquitous in nature, but the specific modification profile varies depending on the organism. Over 100 chemically distinct modified nucleotides have been identified so far mostly in tRNAs, rRNAs, snRNAs and some snoRNAs. From the chemical point of view, these modifications are highly diverse and almost any position of the nucleobases as well as the 2′-OH of the ribose has been found to be a target of modification enzymes (see Table 1 and below) [1–3]. Table 1: Known RNA modifications and their abbreviations and symbols (modified nucleosides mentioned in the text are indicated in bold). Identification of the chemical nature and localization of the modified nucleotides even in highly abundant RNAs represents a laborious and time-consuming task. Moreover, the analysis of low abundant cellular RNAs is extremely difficult due to limited access to highly purified RNA species required for most types of analysis, like HPLC or mass spectrometry [4–7]. One alternative to this consists in direct analysis of underrepresented RNA species in total cellular RNA by reverse transcription (RT) using specific DNA primers [8]. This generally allows the sequencing of a given RNA, but the information on its modified nucleotide content is still missing. The use of specific chemical reagents reviewed in this survey explores the particular reactivity of a given modified residue and may considerably help in the interpretation of an RT profile. Another area for the use of specific chemical reactions is RNA analysis and sequencing by various types of mass
A convenient method to generate methylated and un-methylated control DNA in methylation studies
Mehdi Manoochehri, Mojgan Bandehpour, Bahram Kazemi
Molecular Biology Research Communications , 2013,
Abstract: Methylated and un-methylated control DNA is an important part of DNA methylation studies. Although human and mouse DNA methylation control sets are commercially available, in case of methylation studies on other species such as animals, plants, and bacteria, control sets need to be prepared. In this paper a simple method of generating methylated and un-methylated control DNA is described. Whole genome amplification and enzymatic methylation were performed to generate un-methylated and methylated DNA. The generated DNA were confirmed using methylation sensitive/dependant enzymes, and methylation specific PCR. Control reaction assays confirmed the generated methylated and un-methylated DNA.
Research on Corporate DNA and Corporate RNA  [cached]
Xianbai Li
International Journal of Business and Management , 2009,
Abstract: Noel M. Tichy and other scholars in the commercial college of University of Michigan in American have proposed the whole concept of corporate DNA, regarding it is structured by enterprise decision-making configuration and social relationship configuration. Later, many scholars have explored deeply on the corporate DNA. This paper has presented the corporate RNA concept, regarding technology as the corporate RNA. The enterprise central dogma includes the corporate DNA, the corporate RNA and product ,which is similar to the biological central dogma. In the enterprise central dogma, the corporate DNA, corporate RNA (technology), product information transferring model is a whole circular process.
Different denaturation rates between methylated and non-methylated genomic DNA can result in allele-specific PCR amplification  [PDF]
David J. Bunyan, Hilary M. S. Bullman, Margaret Lever, Sasi D. Saminathan, Wee Teik Keng, Roziana Araffin, David O. Robinson
Open Journal of Genetics (OJGen) , 2011, DOI: 10.4236/ojgen.2011.12003
Abstract: We analysed a DNA sample from a father and child who were both heterozygous for a 7 base pair insertion in the MEST gene differentially-methylated promoter region, previously shown by PCR analysis of bisulphite-treated DNA to be on the methylated allele in the unaffected father and the unmethylated allele in the affected child. PCR from genomic DNA was then carried out using a commercial PCR kit with its recommended initial DNA denaturation step of 2 minutes. Subsequent sequence analysis showed that only the non-methylated allele had been amplified, the father appearing to be homozygous normal and the child appearing to have a homozygous 7 b.p. insertion. The PCR protocol was then modified in order to use a longer DNA denaturation stage prior to the addition of the polymerase enzyme. Upon doing so, both the methylated and non-methylated alleles were then identifiable by sequencing with the mutation appearing in its expected heterozygous form. These results highlight the fact that the methylation status of DNA can affect the denaturation rate prior to PCR and result in allele drop-out, showing that the standard protocols of commercial kits should be used with caution when working with methylated regions of DNA.
Methylated DNA and microRNA in Body Fluids as Biomarkers for Cancer Detection  [PDF]
Yanning Ma,Xian Wang,Hongchuan Jin
International Journal of Molecular Sciences , 2013, DOI: 10.3390/ijms140510307
Abstract: Epigenetic alterations including DNA methylation and microRNAs (miRNAs) play important roles in the initiation and progression of human cancers. As the extensively studied epigenetic changes in tumors, DNA methylation and miRNAs are the most potential epigenetic biomarkers for cancer diagnosis. After the identification of circulating cell-free nuclear acids, increasing evidence demonstrated great potential of cell-free epigenetic biomarkers in the blood or other body fluids for cancer detection.
RNA-DNA杂交反应分析
陈建华,林元藻
生物化学与生物物理进展 , 1984,
Abstract: 转录水平的调节是真核生物基因调控的主要方式,它对研究杂交反应有重要意义。RNA-DNA杂交反应对于决定基因组中DNA序列和转录产物RNA的关系是很重要的手段。杂交体的形成有二种方式,即DNA驱动的反应和RNA驱动的反应,从中可以得到相互补充的信息。一、DNA驱动反应动力学在DNA驱动反应中,DNA是过剩的,而RNA是少量的,同时存在下列二种反应: DNA+DNA→(k) DNA:DNA RNA+DNA→(k) RNA:DNA 其中k是双分手反应常数,其大小由溶液
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