DNA Precipitation Research Articles

Degradable starch nanoparticle assisted ethanol precipitation of DNA

Precipitation of DNA from a large volume of aqueous solution is an important step in many molecular biology and analytical chemistry experiments. Currently, this is mainly achieved by ethanol precipitation, where a long-term incubation (usually overnight) at low temperature of −20 to −80°C with high salt concentration is required. This method also requires a large quantity of DNA to form a visible pellet and was tested mainly for double-stranded DNA. To improve DNA precipitation, co-precipitating polymers such as linear polyacrylamide has been used. In this work, we report that starch nanoparticles (SNPs) can achieve convenient DNA precipitation at room temperature with a low salt concentration and short incubation time. This method requires as low as 0.01–0.1% SNPs and can precipitate both single- and double-stranded DNA of various lengths. The effect of salt concentration, pH and the crosslinking density of SNPs has been systematically studied. Compared to other types of precipitating agents, SNPs are highly biocompatible and can be degraded by a common enzyme (amylase). This work suggests a novel application of a bio-based material that is prepared in mass production.

Histone H3 Lysine 14 (H3K14) Acetylation Facilitates DNA Repair in a Positioned Nucleosome by Stabilizing the Binding of the Chromatin Remodeler RSC (Remodels Structure of Chromatin)

Histone H3 acetylation is induced by UV damage in yeast and may play an important role in regulating the repair of UV photolesions in nucleosome-loaded genomic loci. However, it remains elusive how H3 acetylation facilitates repair. We generated a strongly positioned nucleosome containing homogeneously acetylated H3 at Lys-14 (H3K14ac) and investigated possible mechanisms by which H3K14 acetylation modulates repair. We show that H3K14ac does not alter nucleosome unfolding dynamics or enhance the repair of UV-induced cyclobutane pyrimidine dimers by UV photolyase. Importantly, however, nucleosomes with H3K14ac have a higher affinity for purified chromatin remodeling complex RSC (Remodels the Structure of Chromatin) and show greater cyclobutane pyrimidine dimer repair compared with unacetylated nucleosomes. Our study indicates that, by anchoring RSC, H3K14 acetylation plays an important role in the unfolding of strongly positioned nucleosomes during repair of UV damage.

First identification of Sarcocystis hominis in Iranian traditional hamburger.

Zoonotic concerns of cattle sarcocystosis are of importance, because humans are the final host for Sarcocystis hominis. Therefore the meat products containing beef may encompass sarcocysts which endanger food safety. In this study, we described the first report of molecular identification of S. hominis in Iranian traditional hamburgers using PCR-RFLP. Throughout a pilot research that was carried out to setup a molecular approach to identify the Sarcocystis spp., using PCR-RFLP, a sample of raw Iranian traditional hamburger was purchased from a street food seller located in Yazd, central Iran in May 2013. DNA extraction was done, by salting out method; briefly, the sample was lysed with NET buffer. The DNA purification and precipitation was then performed. Amplicon and digestion results were analyzed, using gel agarose electrophoresis. The results showed a PCR product with 926bp in length after amplification and 376 and 550bp in length after digestion. This product was identified as S. hominis. To the best of our knowledge, this is the first report of S. hominis infection in Iranian hamburger.

Extraction and analysis of plasmid DNA from Listeria monocytogenes.

A plasmid preparation is a method used to extract and purify plasmid DNA. Methods developed to purify plasmid DNA from bacteria generally involve harvesting and alkaline lysis of the bacteria and precipitation of chromosomal DNA and protein, followed by purification of the plasmid DNA. Here, we describe the mini-preparation of plasmid DNA by a rapid small-scale method, adapted for Listeria monocytogenes. The quality of plasmid DNA isolated using this method is sufficient for analytical purposes but may be upscaled for further downstream analysis. Electrophoretic separation of the resultant lysate allows conclusions to be made on the presence, number, copy number, and size of the plasmids in the analyzed bacterial strains.

Focus Issue: Calcium Signaling

It has been my pleasure to act as Guest Editor for this Focus Issue about calcium signaling. Ca2+ is the fifth most abundant element in the Earth’s crust, yet high concentrations are cytotoxic because of precipitation of phosphates, proteins, and DNA. Homeostatic mechanisms to maintain low

FSH modulates the expression of inhibin‐alpha and the secretion of inhibins via orphan nuclear receptor NUR77 in ovarian granulosa cells

It has been previously reported that follicle-stimulating hormone (FSH) regulates the expression of inhibin-alpha in human granulosa cells, but the precise molecular pathway remains unknown. In the present study, we investigated the role of the orphan nuclear receptor, NUR77, in both the transcriptional regulation of the inhibin α-subunit gene and the secretion of inhibins. Our results showed that in a human granulosa cell tumor-derived cell line (KGN) and in human granulosa-lutein cells (hGL), FSH induced the expression of NUR77 and inhibin-alpha, although inhibin-alpha expression did not increased following FSH treatment if NUR77 was knocked down. Furthermore, simply overexpressing or reducing NUR77 levels affected inhibin-alpha expression, while NUR77 overexpression improved the secretion of inhibin A and B from human granulosa cells. In addition, chromatin immunoprecipitation-PCR, avidin-biotin-conjugated DNA precipitation, and luciferase reporter assays confirmed that NUR77 directly regulated the transcription of the inhibin-alpha gene through the specific NGFI-B response element located within its promoter. In the ovarian granulosa cells of the Nur77 knockout mice, the mRNA levels of inhibin-alpha were decreased relative to wild-type mice. These data indicate a role of NUR77 in the regulation of inhibin-alpha in ovarian granulosa cells.

Abstract 4789: Human papillomavirus genotyping in urine samples of women with squamous intraepithelial lesions of cervix.

Abstract Human papillomavirus (HPV) is the most frequent microorganism sexually transmitted and is the causal agent of cervical carcinoma were it could be found in 99.7% of cases. The detection of HPV en women with or without lesions of cervix is generally performed in biopsies and cytobrush, which are invasive samples; therefore many patients do not attend to health care to get HPV test. The aim of this study was to develop a technique capable of detect and genotype HPV in urine samples of women with squamous intraepithelial lesions of cervix. For this study, 87 urine samples from patients with normal epithelia of cervix (25), low grade squamous intraepithelial lesion (LSIL) (24), high grade squamous intraepithelial lesion (HSIL) (38) were collected from women who attended to gynecological care in a public health center in Temuco, Chile. DNA extraction was performed with a manual method. For protein precipitation, Phenol-Chloroform and ammonium acetate were used. DNA precipitation was performed with absolute ethanol. HPV detection and genotyping was performed with the kit HPV Typing (patent pending) which is capable of detect 18 more frequent HPV types and beta globin gen as control of DNA integrity. 20% of urines from normal epithelia women were positive for HPV. Three genotypes were found in normal samples: HPV52, HPV42 and HPV53. LSIL there was a 92% of HPV positivity and the most frequent genotypes were HPV16 and HPV18. In HSIL, 95% of urine sample were positive for HPV. HPV16, HPV18 and HPV31 were the most frequent genotypes in HSIL. Genotypes found in urine samples were similar to world reports. Using the DNA extraction described before and HPV easy typing kit it is possible to detect and genotype HPV in urine samples. These results suggest that is possible a HPV primary screening test in non-invasive sample, complementing PAP smear in order to determinate which women have a higher risk of developing preneoplastic and neoplastics lesions. Grant Support: Proyect CORFO N° 09CN14-5960 (CEGIN). CI is recipient of grants from FONDECYT Postdoctoral Proyect N° 3130630. PB is recipient of grants from FONDECYT Postdoctoral Proyect N° 3120141. Citation Format: Tamara Viscarra, Alejandra Andana, Priscilla Brebi, Doris Menzel, Carmen G. Ili, Ramon Silva, Juan C. Roa, Raul Sanchez. Human papillomavirus genotyping in urine samples of women with squamous intraepithelial lesions of cervix. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4789. doi:10.1158/1538-7445.AM2013-4789

High-throughput chromatin immunoprecipitation for genome-wide mapping of in vivo protein-DNA interactions and epigenomic states

Dynamic protein binding to DNA elements regulates genome function and cell fate. Although methods for mapping in vivo protein-DNA interactions are becoming crucial for every aspect of genomic research, they are laborious and costly, thereby limiting progress. Here we present a protocol for mapping in vivo protein-DNA interactions using a high-throughput chromatin immunoprecipitation (HT-ChIP) approach. By using paramagnetic beads, we streamline the entire ChIP and indexed library construction process: sample transfer and loss is minimized and the need for manually labor-intensive procedures such as washes, gel extraction and DNA precipitation is eliminated. All of this allows for fully automated, cost effective and highly sensitive 96-well ChIP sequencing (ChIP-seq). Sample preparation takes 3 d from cultured cells to pooled libraries. Compared with previous methods, HT-ChIP is more suitable for large-scale in vivo studies, specifically those measuring the dynamics of a large number of different chromatin modifications/transcription factors or multiple perturbations.

Development of a Method To Measure DNA Methylation Levels by Using Methyl CpG-Binding Protein and Luciferase-Fused Zinc Finger Protein

DNA methylation, which is an important epigenetic event for transcriptional regulation, is regarded as a biomarker for cancer. A rapid and sensitive method for measuring DNA methylation levels in target genomic regions may enable early diagnosis of cancer. To detect DNA methylation levels conveniently, we developed a detection system for DNA methylation, designated as methylated DNA precipitation combined luciferase-fused zinc finger assay (MELZA), which uses methyl CpG-binding domain (MBD) and luciferase-fused zinc finger protein. This system comprises the following 3 steps: (1) MBD-based methylated DNA precipitation, (2) PCR amplification of the target genomic region, and (3) detection of the PCR product quantity by using luciferase-fused zinc finger protein. Using this system, we have accurately measured methylation levels of the androgen receptor gene promoter region in LNCaP, PC3, Du145, and whole blood cells. This system does not require bisulfite treatment, and all the steps can be automated. Therefore, it might be useful for measuring DNA methylation levels in clinical cancer diagnoses.

DNA extraction from skins of wild (Hydrochoerus hydrochaeris and Pecari tajacu) and domestic (Sus scrofa domestica) species using a novel protocol

Sometimes, commercial products obtained from wild animals are sold as if they were from domestic animals and vice versa. At this point of the productive chain, legal control of possible wildlife products is difficult. Common in the commerce of northern Argentina, skins of two wild species, the carpincho and the collared peccary, look very similar to each other and to those of the domestic pig; it is extremely difficult to differentiate them after they have been tanned. Because there was no an adequate methodology to discriminate between leather of these three species, we developed a new methodology of DNA extraction from skin and leather. This new method involves digesting a leather sample using proteinase K, followed by precipitation of proteins with 5 M NaCl, cleaning with absolute isopropanol and DNA precipitation with 70% ethanol. DNA is hydrated in Tris-EDTA buffer. This protocol provided good-quality DNA suitable for analysis with molecular markers. This new protocol has potential for use in identifying leather products of these species using molecular markers based on RAPDs.

Methods for extraction of microorganism DNA from glacier surface snow

In order to thoroughly investigate the diversity of glacier microorganisms, four DNA extraction methods with different lysis patterns were tested and two screened methods (the Bosshard-Bano method and the Zhou method) were optimized for the most effective form of the filter membrane (cut vs. uncut), the DNA extraction method, and the precipitation method. The two optimized methods were then compared with the commercial Mo-Bio DNA extraction kit, and the results showed that the kit was generally suitable for extraction of microorganism DNA from glacier surface snow. Procedurally, it was found that a modified Bosshard-Bano method (i.e., cutting the filter membrane into pieces, using a specific lysis pattern [lysozyme (5 mg/mL)-protease K (1 mg/mL)-CTAB (1%)-SDS (1%)], performing the extraction only once by chloroform-isoamyl alcohol (24:1), and conducting DNA precipitation by pure ethanol) was also an effective and less expensive method for extraction of microorganism DNA from glacier surface snow.

Metal to ligand charge transfer induced DNA photobinding in a Ru(ii)–Pt(ii) supramolecule using red light in the therapeutic window: a new mechanism for DNA modification

The Ru(II)-Pt(II) supramolecular complex, [(Ph(2)phen)(2)Ru(dpp)PtCl(2)](2+), displays a new mechanism for DNA modification: photobinding through a (3)MLCT excited state. Gel shift analysis, selective DNA precipitation, and DNA melting point experiments support efficient covalent DNA binding following visible light excitation.

Global Analysis of DNA Methylation by Methyl-Capture Sequencing Reveals Epigenetic Control of Cisplatin Resistance in Ovarian Cancer Cell

Cisplatin resistance is one of the major reasons leading to the high death rate of ovarian cancer. Methyl-Capture sequencing (MethylCap-seq), which combines precipitation of methylated DNA by recombinant methyl-CpG binding domain of MBD2 protein with NGS, global and unbiased analysis of global DNA methylation patterns. We applied MethylCap-seq to analyze genome-wide DNA methylation profile of cisplatin sensitive ovarian cancer cell line A2780 and its isogenic derivative resistant line A2780CP. We obtained 21,763,035 raw reads for the drug resistant cell line A2780CP and 18,821,061reads for the sensitive cell line A2780. We identified 1224 hyper-methylated and 1216 hypomethylated DMRs (differentially methylated region) in A2780CP compared to A2780. Our MethylCap-seq data on this ovarian cancer cisplatin resistant model provided a good resource for the research community. We also found that A2780CP, compared to A2780, has lower observed to expected methylated CpG ratios, suggesting a lower global CpG methylation in A2780CP cells. Methylation specific PCR and bisulfite sequencing confirmed hypermethylation of PTK6, PRKCE and BCL2L1 in A2780 compared with A2780CP. Furthermore, treatment with the demethylation reagent 5-aza-dC in A2780 cells demethylated the promoters and restored the expression of PTK6, PRKCE and BCL2L1.

Factors affecting chemical‐based purification of DNA from Saccharomyces cerevisiae

Extraction of high molecular weight chromosomal DNA from yeast cells is a procedure that is performed frequently for experiments involving polymerase chain reaction (PCR), Southern blotting and other DNA analysis techniques. We have investigated several parameters affecting DNA yield and quality, using a simple chemical-based purification procedure that was modelled on alkaline lysis methods developed for bacterial cells. The three major steps of the procedure, cell lysis, protein removal and DNA precipitation, were optimized by testing the impacts of several chemicals, including sodium dodecyl sulphate (SDS), sodium hydroxide, Tris buffer, sodium acetate and potassium acetate. Other parameters, such as the effect of elevated temperatures on cell lysis, were also investigated. A rapid, optimized protocol was derived for the purification of DNA from small cell cultures that can be readily digested with restriction enzymes and used as a template for PCR. Average yield was calculated to be approximately 1.7 µg DNA/10(8) cells, which is similar to the theoretical maximum amount obtainable from haploid yeast cells.

Epigenetic Regulation of the Latency-Associated Region of Marek's Disease Virus in Tumor-Derived T-Cell Lines and Primary Lymphoma

Meq is the major Marek's disease virus (MDV)-encoded oncoprotein and is essential for T-cell lymphomagenesis. Meq and several noncoding RNAs, including three microRNA (MiR) clusters, are expressed from the repeats of the MDV genome during latent infection of T cells. To investigate the state of the chromatin in this and flanking regions, we carried out chromatin immunoprecipitation (ChIP) analysis of covalent histone modifications and associated bound proteins. T-cell lines and a lymphoma were compared. The chromatin around the promoters for Meq and the noncoding RNAs in both cell lines and the lymphoma were associated with H3K9 acetylation and H3K4 trimethylation, which are marks of transcriptionally active chromatin. These correlated with bound Meq-c-Jun heterodimers. The only binding site for Meq homodimers is located at the lytic origin of replication (OriLyt), next to the lytic gene pp38. This region lacked active marks and was associated with repressive histone modifications (H3K27 and H3K9 trimethylation). DNA CpG methylation was investigated using methylated DNA precipitation (MeDP). In cell lines, DNA methylation was abundant across the repeats but noticeably reduced or absent around the active promoters. In primary tumors, CpG methylation occurred less than 2 months after infection, focused within the ICP4 gene. These data suggest that nonrandom de novo DNA methylation occurs early in lymphomagenesis. In addition, the histone data indicate a role for Meq in the epigenetic regulation of the MDV genome repeats in transformed T cells and suggest that the OriLyt region and the Meq/MiR region might be separated by chromatin boundary elements, and preliminary data on CTCF binding are consistent with this.

Plasmid DNA precipitation in the presence of cationic surfactant/salt: effect of the surface charge on precipitation

Plasmid DNA precipitation in the presence of cationic surfactant/salt: effect of the surface charge on precipitation

Identification of a Replication-independent Replacement Histone H3 in the Basidiomycete Ustilago maydis

Ustilago maydis is a haploid basidiomycete with single genes for two distinct histone H3 variants. The solitary U1 gene codes for H3.1, predicted to be a replication-independent replacement histone. The U2 gene is paired with histone H4 and produces a putative replication-coupled H3.2 variant. These predictions were evaluated experimentally. U2 was confirmed to be highly expressed in the S phase and had reduced expression in hydroxyurea, and H3.2 protein was not incorporated into transcribed chromatin of stationary phase cells. Constitutive expression of U1 during growth produced ~25% of H3 as H3.1 protein, more highly acetylated than H3.2. The level of H3.1 increased when cell proliferation slowed, a hallmark of replacement histones. Half of new H3.1 incorporated into highly acetylated chromatin was lost with a half-life of 2.5 h, the fastest rate of replacement H3 turnover reported to date. This response reflects the characteristic incorporation of replacement H3 into transcribed chromatin, subject to continued nucleosome displacement and a loss of H3 as in animals and plants. Although the two H3 variants are functionally distinct, neither appears to be essential for vegetative growth. KO gene disruption transformants of the U1 and U2 loci produced viable cell lines. The structural and functional similarities of the Ustilago replication-coupled and replication-independent H3 variants with those in animals, in plants, and in ciliates are remarkable because these distinct histone H3 pairs of variants arose independently in each of these clades and in basidiomycetes.

Controlling nuclear JAKs and STATs for specific gene activation by IFNγ

We previously showed that gamma interferon (IFNγ) and its receptor subunit, IFNGR1, interacted with the promoter region of IFNγ-activated genes along with transcription factor STAT1α. Recent studies have suggested that activated Janus kinases pJAK2 and pJAK1 also played a role in gene activation by phosphorylation of histone H3 on tyrosine 41. This study addresses the question of the role of activated JAKs in specific gene activation by IFNγ. We carried out chromatin immunoprecipitation (ChIP) followed by PCR in IFNγ treated WISH cells and showed association of pJAK1, pJAK2, IFNGR1, and STAT1 on the same DNA sequence of the IRF-1 gene promoter. The β-actin gene, which is not activated by IFNγ, did not show this association. The movement of activated JAK to the nucleus and the IRF-1 promoter was confirmed by the combination of nuclear fractionation, confocal microscopy and DNA precipitation analysis using the biotinylated GAS promoter. Activated JAKs in the nucleus was associated with phosphorylated tyrosine 41 on histone H3 in the region of the GAS promoter. Unphosphorylated JAK2 was found to be constitutively present in the nucleus and was capable of undergoing activation in IFNγ treated cells, most likely via nuclear IFNGR1. Association of pJAK2 and IFNGR1 with histone H3 in IFNγ treated cells was demonstrated by histone H3 immunoprecipitation. Unphosphorylated STAT1 protein was associated with histone H3 of untreated cells. IFNγ treatment resulted in its disassociation and then re-association as pSTAT1. The results suggest a novel role for activated JAKs in epigenetic events for specific gene activation.

Extraction of genomic DNA from yeasts for PCR-based applications

We have developed a quick and low-cost genomic DNA extraction protocol from yeast cells for PCR-based applications. This method does not require any enzymes, hazardous chemicals, or extreme temperatures, and is especially powerful for simultaneous analysis of a large number of samples. DNA can be efficiently extracted from different yeast species (Kluyveromyces lactis, Hansenula polymorpha, Schizosaccharomyces pombe, Candida albicans, Pichia pastoris, and Saccharomyces cerevisiae). The protocol involves lysis of yeast colonies or cells from liquid culture in a lithium acetate (LiOAc)-SDS solution and subsequent precipitation of DNA with ethanol. Approximately 100 nanograms of total genomic DNA can be extracted from 1 × 10(7) cells. DNA extracted by this method is suitable for a variety of PCR-based applications (including colony PCR, real-time qPCR, and DNA sequencing) for amplification of DNA fragments of ≤ 3500 bp.

MicroRNA-155 is involved in the remodelling of human-trophoblast-derived HTR-8/SVneo cells induced by lipopolysaccharides

A low dose injection of lipopolysaccharides (LPS) may induce pre-eclampsia-like symptoms in rats, and microRNA-155 (miR-155) is elevated in the placentas of patients with pre-eclampsia. Our goal was to investigate the association of miR-155 with pre-eclampsia and the pathways involved using human-trophoblast-derived cell line (HTR-8/SVneo) stimulated with LPS. We measured miR-155 in HTR-8/SVneo cells treated with LPS (25-800 ng/ml) using real-time PCR. Western blotting was used to study transcription factor activated protein 1 (AP-1) (JunB and FosB subunits) and nuclear factor (NF)-κB p65 in the HTR-8/SVneo cells and placentas from patients with pre-eclampsia. DNA precipitation assays and luciferase reporter analysis were used to evaluate the regulation of miR-155 by AP-1 and NF-κB. Cell migration was determined by scratch assay. Syncytialization of HTR-8/SVneo cells was analysed following transfection with miR-155. miR-155 was increased together with AP-1 and NF-κB in HTR-8/SVneo cells incubated with low dose of LPS (≤100 ng/ml; P < 0.05 versus baseline). Both JunB/FosB and p65 were increased in placenta from women with severe pre-eclampsia versus a normal pregnancy, with elevated expression of miR-155 (P < 0.05). For specific DNA-binding sites upstream of BIC/miR-155 gene promoter, the AP-1 site was more important than the NF-κB site for increasing miR-155 in HTR-8/SVneo cells. The cells with enforced expression of miR-155 showed a reduced ability to migrate (P < 0.05) and an increased number of syncytiotrophoblast-like multinuclear cells (P < 0.05). LPS may induce remodelling of the human-trophoblast-derived HTR-8/SVneo cells by increasing miR-155, acting in part through the AP-1 and NF-κB pathways.