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!

Happy reading,
The BIOKÉ team

Highlighted products

Click 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.


Figure 1: ChIP workflow
The SimpleChIP Enzymatic Chromatin IP Kits of Cell Signaling Technology are available in 2 different formats: one based on protein G agarose beads (#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.

ChIPseq
ChIP combined with next generation sequencing (ChIPseq) allows to study DNA-protein interactions in a genomewide fashion. The DNA of interest is processed with the sample preparation sets (NEBNext) from New England Biolabs, or the Access Array System from Fluidigm before the actual sequencing reaction. Global enrichment of epigenetic features or the binding repertoire of DNA binding factors is then assessed in one single experiment. These experiments will enhance our understanding on chromatin dynamics in relation to e.g. development and cancer.


Figure 2: ChIPseq profile
Schematic overview of a ChIPseq profile. Gene a and gene c show enrichment at the 5’ end of genes, whereas gene b is not enriched compared to the genomic background.

Advantages

  • Genomewide DNA-protein network assessment
  • Enhanced detection of protein-bound DNA loci
  • High resolution results
  • Low sample input
  • No background from cytosolic proteins
  • Increased immunoprecipitation efficiencies
  • No variation between experiments
  • No need for additional blocking
  • No contamination of downstream sequencing
  • Fast protocol
  • Convenient DNA isolation with spin columns
Applications
  • Epigenetics
  • DNA binding protein events

Fluidigm Access Array™ System 

Next generation Sequencing Target Enrichment
Next-generation sequencing (NGS) raises the potential for large-scale genomic screens, but sample preparation has been too slow to take full advantage of the high-throughput sequencing capacity. The Fluidigm Access Array System is the first high-throughput, target-enrichment system designed to work with all of the major next-generation sequencing instruments. The Access Array System enables the user to enrich multiple unique targets (such as exons) from a large number of samples, all in one single PCR run. The system combines the throughput benefits of microfluidics with the proven performance and flexibility of PCR. It allows you to obtain quality results while minimizing the time, costs, and labor required for targeted resequencing projects.

Sample Barcoding and  Library preparation for multiplex NGS
The Access Array Integrated Fluidic Circuit (IFC) facilitates parallel amplification of 48 unique samples, against 48 different  target specific primer sets, preparing a total of 48 pooled sequencing libraries, in just a few hours. Every reaction can combine both an amplicon tagging and a barcoding (identification) step that enables all 48 samples to be multiplexed at the sequencing step. This powerful chemistry simplifies the up-front preparation and maximizes the utility of today's next-generation sequencers.

The Access Array™ System
The Fluidigm Access Array System can be used with any PCR-based sample preparation method and with the reagents and primers of your choice. The system includes Access Array IFCs, two IFC Controllers, and the Stand-Alone Thermal Cycler. These components deliver sample throughput and coverage scalable with laboratory needs.

Advantages

  • Target enrichment
  • Sample barcoding for multiplexed sequencing
  • Sequencing library preparation using amplicon tagging
Applications
  • Standard 2 primer PCR
  • FFPE optimized protocol
  • Long range PCR
  • Multiplex PCR
  • 4 primer PCR for NGS sample library preparation
Services
  • Assay design and validation service
  • Barcode libraries available for 454 and Illumina Sequencers

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.

NextGENe 

Next generation Sequencing Target Enrichment
Next generation sequencing (NGS) data analysis is straightforward with NextGENe, the user friendly software package from SoftGenetics. NextGENe's Windows® based operation removes the complexity of scripting and simplifies your analyses by a simple point-and-click wizard (four simple steps from sequencing data to results). The program is used for analyzing sequences derived from all the major sequencing platforms, including the recently launched Ion Torrent sequencer. NextGENe contains a large number of modules for applications like, RNAseq/ChIPseq, mutation discovery and de novo assembly. In addition, NextGENe has experienced significant improvements in data assembly and has acquired highly demanded tools for mutation scoring, variant comparison reports, the transcriptome analysis module and many others. NextGENe is constantly updated by the acquisition (or development) of innovative tools to meet the demands of NGS users. To reveal the customized settings of NextGENe for your NGS project we offer a free demo analysis (100% confidential) with one of your datasets. This exclusive opportunity is the fastest way to fully explore NextGENe as your versatile tool for all your NGS projects.

Advantages

  • Accurate results
  • Easy-to-use graphical user interface
  • No scripting required
  • Free demo analysis
Applications
  • Mutation detection
  • Transcriptome studies
  • ChIP-sequencing
  • De novo assembly

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.

Application Focus

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
  • Convenient formats for easy use with different platforms
  • Functional Validation to guarantee your results on the appropriate sequencing platform (Illumina, 454 or SOLiD)
  • Value Pricing that gives you a competitive advantage
  • Novel Protocol for small RNA suitable for methylated and unmethylated small RNAs
Applications
  • mRNA library preparation for next generation sequencing
  • Small RNA library preparation for sequencing with Illumina and SOLiD
  • Expression library construction

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.

Interview

Nanoscale equipment brings about big changes

interview 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."

Prenatal testing

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."

Special Promotions

Take your chance to win an iPad© 2!

Try our new OneTaq™ DNA Polymerase! Send us your feedback including data and receive a free New England Biolabs lab timer and a chance to win an iPad© 2!

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Experience the Difference

Order antibodies from Cell Signaling Technology before June 30th and get a FREE vial of Cell Lysis Buffer (#9803)!

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  Click here for more special offers