Large segments of DNA can vary in copy number between individuals. Such copy number variations (CNVs) contribute greatly to genetic diversity and are also thought to be associated with susceptibility or resistance to some diseases, including cancer. Simple Copy Number Determination with Reference Query Pyrosequencing (RQPS), featured in the September issue of Cold Spring Harbor Protocols, provides an assay for determining the copy number of any allele in the genome. The method, from Raphael Kopan and colleagues at Washington University, takes advantage of the fact that pyrosequencing can accurately measure the ratio of DNA fragments in a mixture that differ by a single nucleotide. A reference allele with a known copy number and a query allele with an unknown copy number are engineered with single nucleotide variations, and the ratio seen between these probes and genomic DNA reflects the copy number. RQPS can be used to measure copy number of any transgene, differentiate homozygotes from heterozygotes, detect the CNV of endogenous genes, and screen embryonic stem cells targeted with bacterial artificial chromosome (BAC) vectors. RQPS is rapid, inexpensive, sensitive, and adaptable to high-throughput approaches. As one of our featured articles, the protocol is freely available to subscribers and non-subscribers alike.

Improvements in automation and acquisition time have made the microscope a viable platform for performing hundreds of concurrent parallel experiments. Using these sorts of tools, it is now possible to run high-throughput screens for protein function and interaction in living cells, examining dynamic cellular processes to distinguish between primary and secondary phenotypes, and to study the phenotype kinetics. In the August issue of Cold Spring Harbor Protocols, Jan Ellenberg and colleagues from the EMBL present High-Throughput Microscopy Using Live Mammalian Cells, an overview of how to screen live cells using imaging technologies. The article examines each aspect of the general screening process and considers specific examples in the processing of time-lapse experiments. The techniques discussed are based on the use of cultured mammalian cells, but the concepts are easily transferred to cultured cells from other species like Drosophila and small organisms such as C. elegans.

Zinc finger nucleases (ZFNs) are artificial restriction enzymes made by fusing an engineered zinc finger DNA-binding domain to the DNA cleavage domain of a restriction enzyme. ZFNs can be used to generate targeted genomic deletions of large segments of DNA in a wide variety of cell types and organisms. In the August issue of Cold Spring Harbor Protocols, Jin-Soo Kim and colleagues present Analysis of Targeted Chromosomal Deletions Induced by Zinc Finger Nucleases, a detailed protocol for the detection and analysis of large genomic deletions in cultured cells introduced by the expression of ZFNs. The method described allows researchers to detect and estimate the frequency of ZFN-induced genomic deletions by simple PCR-based methods. As one of our featured articles, the protocol is freely available to subscribers and non-subscribers alike.

The zebrafish (Danio rerio) has rapidly become a favored model organism for studying developmental biology. One of the most commonly used methods for genetic manipulation in the zebrafish is the delivery of plasmids or oligonucleotides to cells within the living embryo via electroporation. When cells are exposed to brief electrical fields, transient membrane destabilization occurs and nucleic acids can cross the plasma membrane. When the electrical field is removed, the membrane seals and the nucleic acids are trapped inside the cell. In vivo electroporation has proven particularly effective for delivering fluorescent protein expression vectors for imaging and loss-of-function reagents such as morpholinos or RNA interference (RNAi) constructs for the knockdown of gene function. In the July issue of Cold Spring Harbor Protocols, Jack Horne and colleagues present Targeting the Zebrafish Optic Tectum Using In Vivo Electroporation, a modification of the technique that can be used to specifically target the developing optic tectum, the midbrain’s visual processing center. Instructions are given for the construction of electroporation electrodes, preparation and injection of DNA, and electroporation of the DNA into the embryonic brain.

Using a promoter that can drive expression at an appropriate level is crucial in designing constructs for gene expression. Promoters can be tested via transient or stable transfection. But transfection efficiency in such assays can be low, so promoters are commonly fused to heterologous reporter genes that encode enzymes that can be quantified using highly sensitive assays. The reporter protein’s activity or fluorescence within a transfected cell population is approximately proportional to the steady-state mRNA level. The May issue of Cold Spring Harbor Protocols includes updated versions of three commonly used assays for promoter strength.

The Luciferase Assay uses a gene from the firefly Photinus pyralis. This gene encodes a 61-kDa enzyme that oxidizes D-luciferin in the presence of ATP, oxygen, and Mg++, yielding a fluorescent product that can be quantified by measuring the released light with a luminometer. The luciferase assay is extremely rapid, simple, relatively inexpensive, sensitive, and possesses a broad linear range.

The Chloramphenicol Acetyltransferase Assay utilizes an Escherichia coli chloramphenicol acetyltransferase (CAT) reporter gene. CAT catalyzes the acetylation of [14C]chloramphenicol which is monitored by autoradiography following thin-layer chromatography (TLC). The percent conversion of [14C]chloramphenicol to acetyl-[14C]chloramphenicol can be measured by PhosphorImager analysis of the TLC plate, counting in a scintillation counter, or by densitometry analysis of an autoradiograph.

The Beta-Galactosidase Assay uses the E. coli lacZ gene which encodes a beta-galactosidase. Beta-gal activity is measured through a simple and inexpensive colorimetric assay. Cells are lysed and extracts are mixed with O-nitrophenyl-beta-D-galactopyranoside (ONPG), which results in a yellow product. The optical densities of the samples are then determined spectrophotometrically.

While 454-based pyrosequencing has led to great advances, an intrinsic artifact of the process leads to artificial over-representation of more than 10% of the original DNA sequencing templates. This is particularly problematic in metagenomic studies, where the abundance of any sequence in a dataset is often used for comparative community analysis. It’s important to remove these artificial replicates before analysis. This phenomenon can skew data interpretation when making comparisons between datasets. As metagenome datasets become more plentiful, the ability to apply more robust statistical tests becomes increasingly important, and the validity of the input datasets becomes more crucial. Tools such as MG-RAST (covered in the January issue of Cold Spring Harbor Protocols in Using the Metagenomics RAST Server (MG-RAST) for Analyzing Shotgun Metagenomes) have the capability to remove exact duplicates, but this captures only a subset of the artificial replicates. In the April issue of Cold Spring Harbor Protocols, Tracy Teal and Thomas Schmidt from Michigan State University present an instruction set for Identifying and Removing Artificial Replicates from 454 Pyrosequencing Data. Their 454 Replicate Filter is a web-based tool that incorporates the algorithm cd-hit. This protocol provides details on how to use the replicate filter and obtain a file of unique sequences for use in metagenomic or transcriptomic analyses. This allows users to obtain a more accurate quantitative representation of the sequence diversity in a dataset.

The incorporation of thymidine analogues, such as 5-bromo-2′-deoxyuridine (BrdU), into newly synthesized DNA is a powerful tool for analysis of DNA replication, repair and other aspects of DNA metabolism. In Genome-Wide Analysis of DNA Synthesis by BrdU Immunoprecipitation on Tiling Microarrays (BrdU-IP-chip) in Saccharomyces cerevisiae, Oscar Aparicio and colleagues from the University of Southern California couple BrdU immunoprecipitation with DNA microarrays to enable genome-wide identification of BrdU-labeled chromosomal DNA. BrdU-IP-chip has many potential applications and has already been used to identify replication origins, make quantitative comparisons of origin firing between strains, and examine replication fork progression. As one of February’s featured articles in Cold Spring Harbor Protocols, the protocol is freely available to subscribers and non-subscribers alike.

Metagenomics, the study of DNA isolated from naturally occurring populations and samples, is rapidly growing. Improvements to cloning and sequencing techniques are allowing researchers to study organism in environmental samples, and new knowledge of species interactions and community dynamics is emerging. The identification of microorganisms in these samples is of vital importance to their interpretation. In the January issue of Cold Spring Harbor Protocols, Annelie Wendeberg of the Helmholtz Centre for Environmental Research presents a protocol for Fluorescence In Situ Hybridization for the Identification of Environmental Microbes. The methods described allow the phylogenetic identification of microorganisms in environmental samples (e.g., water and sediments) by means of fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes followed by signal amplification with catalyzed reporter deposition (CARD). The protocol is one of January’s featured articles, and like all featured articles in Cold Spring Harbor Protocols, it is freely accessible to subscribers and non-subscribers alike.

With the recent progress in understanding epigenetic mechanisms, methods for profiling patterns of DNA modification have become important tools for analysis of gene regulation. DNA methylation, in which cytosine is modified to form 5-methylcytosine, is a well-characterized epigenetic modification essential for normal development in plants and mammals. In the December issue of Cold Spring Harbor Protocols, Jon Reinders presents Amplification of Bisulfite-Converted DNA for Genome-Wide DNA Methylation Profiling. This method utilizes the treatment of DNA with sodium bisulfite, which converts unmethylated cytosine to uracil (5-methylcytosine is not converted). This is followed with PCR amplification, where the uracil amplifies as thymine, creating a C-to-T transition. The genome can then be analyzed for these transitions using an array-based platform. Reinders protocol mitigates the major issues with bisulfite conversion (DNA fragmentation and poor reproducibility) and reduces bias during the amplification step. While the protocol is optimized for use in Arabidopsis, it can potentially be adapted for use in other organisms.

We’re getting toward the end of the second volume of our Emerging Model Organisms series in Cold Spring Harbor Protocols, and November’s issue brings us a look at the Hawaiian Bobtail Squid and the genus Dioscorea, or True Yams.

Euprymna scolopes, the Hawaiian Bobtail Squid (our cover model this month, see below) is a cephalopod that’s well-suited for study in the laboratory. E. scolopes is primarily studied in three contexts:
1) as a model for cephalopod development–the embryos and protective chorions are clear, making it amenable for the observations and manipulations common in other studied model systems
2) as a model of animal-bacteria symbioses with the luminous marine bacterium Vibrio fischeri
3) as a system for studying the interaction of tissues with light, as the squid features a specialized light organ.

Heinz Gert de Couet and colleagues supply an overview of the Hawaiian Bobtail Squid as a model system, along with protocols for Preparation of Genomic DNA, Confocal Immunocytochemistry, Whole-Mount In Situ Hybridization (parts 1 and 2), and Culture and Observation.

Dioscorea is a large genus of plants that are monocots but that look like dicots, and are closely related to the phylogenetically derived group containing the grasses. It’s interesting evolutionarily because of the position it occupies, as a link between the eudicots and grasses–groups that contain all the model flowering plant species. The true yam is also important as a food crop. R. Geeta and colleagues provide an overview of the genus, and protocols for husbandry, culturing tissues, management of plantlets, controlled crosses, and DNA extraction.

CSH Protocols November Cover

CSH Protocols November Cover

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