News Flow - Edition 1 - 2011 - The Benelux
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In this edition of BIOKÉ NEWS FLOW
Welcome to our first edition of the BIOKÉ NEWS FLOW newsletter! The BIOKÉ NEWS FLOW is a quarterly publication dedicated to providing you with the most recent application- and product updates, interview and our current special offers. In this edition, we have highlighted three hot products: SimpleChIP Enzymatic Chromatin IP kit, Fluidigm Access Array™ System and NextGENe. Additionally, we focus on the new NEBNext RNA sample prep reagents from New England Biolabs. Techniques such as next-generation sequencing, microarrays and library construction all require efficient, uniform and unbiased processing of nuclear material for accurate analysis. As a result, the demand for high quality sample preparation reagents has grown, as well as the need for novel reagents, novel formats of existing reagents and more stringent quality controls.
For this edition of the BIOKÉ NEWS FLOW, we had an interview with Prof. dr. Johan den Dunnen - head of a research group at the Center for Human & Clinical Genetics (LUMC) – about the BioMark nanolitre-PCR-machine from Fluidigm. Furthermore, find out more about our latest special offers!
Highlighted productsClick on one of the following titles to view the article:
MACHEREY-NAGEL: Nucleospin FFPE DNA
4titude: Framestar® Break-a-way plates
PCR Reagents from New England Biolabs
MACHEREY-NAGEL: Nucleospin FFPE DNA
Improved DNA quality from formalin-fixed, paraffin-embedded samples made easy
The NucleoSpin® FFPE DNA kit provides a convenient, reliable, and fast method to isolate DNA from formalin-fixed, paraffin-embedded (FFPE) tissue specimen. The procedure omits the use of flammable and malodorous xylene or d-limonene commonly used for deparaffinization. Further, the procedure omits the difficult removal of organic solvent from often barely visible tissue pellets. NucleoSpin® FFPE DNA employs the odorless Paraffin Dissolver (patent pending) and allows efficient lysis in a convenient two-phase system.#9002) and one based on protein G magnetic beads (#9003). Both can be used with any ChIP-validated antibody. These are the same products we use in house to validate the ChIP protocol and will save you time for your optimization.
Fluidigm Access Array™ System
Next generation Sequencing Target Enrichment
Sample Barcoding and Library preparation for multiplex NGS
The Access Array™ System
The Access Array system comprises a standalone thermal cycler and two IFC-controllers; one Pre-PCR loader for loading samples and primers and one Post-PCR loader for harvesting PCR products.
The Access Array IFC automatically assembles 2,304 unique PCR reactions, each reaction is a combination of one of the 48 samples and one of the 48 primer inlets. The Post PCR product of 48 reactions per sample is harvested out of the Access Array IFC for downstream sequencing.
Example of Amplicon tagging for 454 sequencing library preparation. A+B; Amplicon tagging (Tag1/2) introduces sequencer-specific adaptors (454A/B) and a sample-specific barcodes (BC). C; The end product can directly be used for 454 emulsion PCR.
Next generation Sequencing Target Enrichment
Figure 1: NextGENe viewer/browser screenshot of ChIP sequencing in Drosophila. (1) Coverage sequence reads (grey), annotated gene locations (blue arrows), codons (yellow) and mRNA (green). Target enrichment compared to genomic background is indicated with the brown lines. (2) Reference and consensus sequences. (3) Zoom view of sequences at position of the blue cross in window 1. Novel identified SNPs are highlighted in light blue. (4) Information of the mutations are presented in the mutation report.
Next generation RNA sequencing
The emergence of next-generation sequencing technologies has not only facilitated genome sequencing but also allowed development of new approaches like RNA sequencing. The RNA-Seq technology allows researchers to have a more comprehensive understanding of the transcriptome as RNA-Seq accesses both single-base and gene expression levels at the genome scale.
From the beginning New England Biolabs (NEB) has been involved in this rapidly developing technology. Recently, NEB has expanded its line of reagents for sample preparation upstream of next generation sequencing to include NEBNext® Small RNA Sample Prep Set 1 for sequencing on the Illumina® platforms, NEBNext® mRNA sample Prep Reagent Set 2 for sequencing on the Roche 454™ platforms, and NEBNext Small RNA Sample Prep Set 3 for sequencing on LIFE's SOLiD™ platforms. These new products offer a new and unique protocol, which results in higher yields and lower adaptor-dimer formation, as compared to other protocols. With oligos, enzymes and buffers included, the sets provide substantial cost savings, and the master mix format streamlines the workflow by reducing the number of vials and pipetting steps required.Advantages
Available as reagent sets, master mixes or modules, for the three leading next generation sequencing platforms, the full range of NEBNext reagents offer convenience and customization of workflow. Additionally, NEB offers NEBNext dsDNA Fragmentase™, an enzyme-based solution for fragmentation of DNA.
Nanoscale equipment brings about big changesinterview with Prof. dr. Johan den Dunnen
BIOKÉ uses the BioMark nanolitre-PCR-machine from Fluidigm in collaboration with the Leiden Genome Technology Center (LGTC) at the Leiden University Medical Center (LUMC). The machine performs thousands of PCR’s at the same time, with only a tiny amount of sample. It has the potential to radically affect the field of medical genetics.
Prof. dr. Johan den Dunnen, heads a research group at the Center for Human & Clinical Genetics (LUMC) working on muscle diseases. He acquired the new BioMark nanolitre-PCR-machine that is going to be used through the LGTC by researchers in and outside the LUMC. Den Dunnen: "It's up to me to make sure that LGTC has cutting edge lab equipment and this equipment is accessible to the scientists. When we buy a new system, we train LGTC employees in its operation and familiarize ourselves with its possibilities and limitations. Once established we educate the scientific community at the LUMC about the new system and how to use it."
The BioMark is a nanolitre-PCR-machine. A special controller primes and loads a cartridge that contains two times 48 wells. Fluidigm's Integrated Fluidic Circuit (IFC) Controller divides the sample over 48 wells, loads the other 48 wells with primers and, mixes these in a small matrix. The BioMark then processes 2,304 PCR's (48 times 48) simultaneously on that cartridge.
Den Dunnen: "Suppose that array analysis indicated that a set of genes is correlated with high cholesterol and you want to confirm this in a large group of samples. With arrays and traditional PCR, you can analyze and confirm only a limited number of samples and/or genes due to time and budget constraints. Using the Fluidigm technology, cost and effort for such studies, can be reduced significantly."
With the BioMark, researchers can perform real-time PCR, digital PCR and SNP typing. Den Dunnen: "One collaborating group just published on the use of the BioMark in a study of soil pollution (de Boer et al., 2011 BMC Mol Biol 12:11). Using high-throughput quantitative PCR it could be shown that changes in gene expression in a small soil living arthropod (Folsomia candida) can be used to detect and partially characterise pollution. Digital PCR is an attractive method to exactly count the number of molecules in your sample, something which is difficult and laborious using other technologies. High-throughput SNP typing is required to confirm correlations found in genome wide association studies (GWAS) using SNP-arrays between specific sequence variants and certain conditions/phenotypes. The fact that the BioMark requires just a few microlitres of sample and SNP-assay makes the PCR less expensive."
When Den Dunnen first heard of the BioMark, he knew it would be a valuable addition to his laboratory. Den Dunnen: "As far as I know, it’s the only machine of its kind. However, Fluidigm is a company located in the United States. On-site technical support would thus be difficult and costly if it had to be performed by Fluidigm itself. Luckily, we found BIOKÉ prepared to help us acquiring the machine and to provide us with support. Until now that hasn’t been necessary, the BioMark is a robust machine. But it’s good to know that with BIOKÉ we have a competent and reliable partner close at hand."