1Department of Physiology, University of Oklahoma College of Medicine, 2Department of Geriatric Medicine, University of Oklahoma College of Medicine
Bisulfite amplicon sequencing (BSAS) is a method for quantifying cytosine methylation in targeted genomic regions of interest. This method uses bisulfite conversion paired with PCR amplification of target regions prior to next-generation sequencing to produce absolute quantitation of DNA methylation at a base-specific level.
Published February 24, 2015. Keywords: Molecular Biology, Epigenetics, DNA methylation, next-generation sequencing, bioinformatics, gene expression, cytosine, CpG, gene expression regulation
1Department of Medicine, Weill Cornell Medical College, 2Institute for Computational Biomedicine, Weill Cornell Medical College, 3Department of Physiology and Biophysics, Weill Cornell Medical College, 4Department of Pathology, University of Michigan
Enhanced Reduced Representation Bisulfite Sequencing is a method for the preparation of sequencing libraries for DNA methylation analysis based on restriction enzyme digestion combined with cytosine bisulfite conversion. This protocol requires 50 ng of starting material and yields base pair resolution data at GC-rich genomic regions.
Published February 24, 2015. Keywords: Genetics, Epigenetics, bisulfite sequencing, DNA methylation, genomic DNA, 5-methylcytosine, high-throughput
1Cytogenetics Department, Guy's & St Thomas' NHS Foundation Trust, 2Cytogenetics Department, Viapath Analytics
Array CGH for the detection of genomic copy number variants has replaced G-banded karyotype analysis. This paper describes the technology and its application in a diagnostic service laboratory.
Published February 21, 2015. Keywords: Molecular Biology, array CGH, aCGH, copy number variant, CNV, deletion, duplication, hybridization, fluorescent labeling.
1Genetics and Genomic Medicine, Institute of Child Health, University College London, 2Molecular Cell Science Research Centre, St. George's University of London
The zebrafish is a popular tool to model chronic kidney disease (CKD). However, their small size makes it impossible to evaluate renal function using traditional methods. We describe a fluorescent dye kidney clearance assay1 that allows quantitative analysis of zebrafish kidney function in CKD.
Published February 20, 2015. Keywords: Developmental Biology, Kidney function, Danio rerio, pronephric tubules, renal disease, kidney clearance assay, cilia, Nephronophthisis. Chronic Kidney Disease, Bardet Biedl Syndrome, pericardial injection.
1Department of Biology, Ball State University, 2Division of Nephrology, Cincinnati Children's Hospital
This article describes a yeast growth-based assay for the determination of genetic requirements for protein degradation. It also demonstrates a method for rapid extraction of yeast proteins, suitable for western blotting to biochemically confirm degradation requirements. These techniques can be adapted to monitor degradation of a variety of proteins.
Published February 16, 2015. Keywords: Molecular Biology, Ubiquitin-proteasome system, Saccharomyces cerevisiae, budding yeast, growth assay, protein extracts, western blotting, yeast genetics, mutants, endoplasmic reticulum-associated degradation, protein degradation
1W. Harry Feinstone Department of Molecular Microbiology and Immunology, Johns Hopkins University Bloomberg School of Public Health, 2School of Life Sciences, Chinese University of Hong Kong, 3Center for Cell Dynamics, Department of Biological Chemistry, Johns Hopkins University School of Medicine
The term anastasis refers to the phenomenon in which dying cells reverse a cell suicide process at a late stage, repair themselves, and ultimately survive. Here we demonstrate protocols for detecting and tracking cells that undergo anastasis.
Published February 16, 2015. Keywords: Cellular Biology, Anastasis, apoptosis, apoptotic bodies, caspase, cell death, cell shrinkage, cell suicide, cytochrome c, DNA damage, genetic alterations, mitochondrial outer membrane permeabilization (MOMP), programmed cell death, reversal of apoptosis
1Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center
Synthesis of custom plasmids is labor and time consuming. This protocol describes the use of Gibson assembly cloning to reduce the work and duration of custom DNA cloning procedure. The protocol described also produces reliable tagged protein constructs for mammalian expression at similar cost to the traditional cut-and-paste DNA cloning.
Published February 9, 2015. Keywords: Molecular Biology, human EF1α, promoter, SV40 promoter, CMV promoter, Gibson assembly, embryonic stem cells, protein expression, FLAG-tag, synthetic biology
1Clarendon Laboratory, Department of Physics, University of Oxford, 2Wellcome Trust Sanger Institute, Genome Center
Studies of biomolecules in vivo are crucial for understanding molecular function in a biological context. Here we describe a novel method allowing the internalization of fluorescent biomolecules, such as DNA or proteins, into living microorganisms. Analysis of in vivo data recorded by fluorescence microscopy is also presented and discussed.
Published February 8, 2015. Keywords: Microbiology, Electroporation, fluorescence, FRET, in vivo, single-molecule imaging, bacteria, Escherichia coli, yeast, internalization, labeled DNA, labeled proteins
1Department of Pharmacy and Biotechnology, University of Bologna, 2CSGI, Department of Chemistry, University of Firenze
Protein co-expression is a powerful alternative to the reconstitution in vitro of protein complexes, and is of help in performing biochemical and genetic tests in vivo. Here we report on the use of protein co-expression in Escherichia coli to obtain protein complexes, and to tune the mutation frequency of cells.
Published February 5, 2015. Keywords: Biochemistry, Escherichia coli, protein co-expression, compatible plasmids, complementation test, DNA polymerase III, mutator strains
1LOEWE Excellence Cluster for Integrative Fungal Research (IPF), Senckenberg Research Institute
A convenient and powerful method for studying autophagy in Penicillium chrysogenum by using starvation pads (mixtures of agarose and tap water in a microscope slide containing a central cavity) is presented here.
Published February 1, 2015. Keywords: Microbiology, starvation, degradation, autophagy, microscopy, microscope slide, cavity, fungi, Penicillium chrysogenum