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Spring 2009, vol. 4.1 NEBexpressions a scientific update from New England Biolabs Welcome to the spring edition The Next Generation of Reagents of NEB expressions. This issue introduces the 2009–10 NEB for Sample Preparation Catalog & Technical Reference, and highlights several components of Fiona Stewart, Ph.D. and Andrew Gardner, New England Biolabs, Inc. our catalog, including new products, reference appendix updates Sample preparation is a critical step in a wide and environmental minireviews. variety of analytical techniques in which a sample The feature article focuses on is biochemically or enzymatically treated for the importance of high quality downstream analysis. In the realm of nucleic acid manipulation, sample preparation relates to the sample preparation for emerging steps that occur following initial purification of DNA. technologies, such as next- Recently, the number of applications that require generation sequencing, microarrays upstream preparation of nucleic acid samples has NEBNext™ DNA Sample Prep Reagent Set 1 facilitates sample preparation and library construction. In addition grown dramatically. Techniques such as next- for downstream analysis. See page 2 for details. to our standalone reagents, NEB generation sequencing, microarrays and library construction all require efficient, uniform and now offers the NEBNext™ Sample unbiased processing of nuclear material for accurate of lowering the cost of sequencing a human Prep Reagent Set 1 to facilitate analysis. As a result, the demand for high quality genome from $3 billion (1) down to $100,000 – sample preparation. sample preparation reagents has grown, as well as and ultimately to $1,000. Since then, several strategies for massively parallel sequencing have As always, we invite your feedback the need for novel reagents, novel formats of existing reagents and more stringent quality controls. been commercialized or are in development. These on our products, services and include pyrosequencing (Roche 454), sequencing by corporate philosophy. Next Generation Sequencing synthesis (Illumina, Intelligent Bio-Systems, Helicos In 2003, the completion of the human genome BioSciences, LaserGen), sequencing by ligation inside: sequence marked the end of a large international (Applied Biosystems/Life Technologies, Complete Genomics, Polonator), real-time sequencing (Pacific Feature Article project, but also the beginning of novel sequencing methods. During the following year, the National Biosciences) and nanopore sequencing (Oxford 1 The Next Generation of Reagents Nanopore Technologies, Sequenom). for Sample Preparation Human Genome Research Institute (NHGRI) began funding sequencing projects with the goal (continued on page 2) New Products 1 HF Restriction Enzyme Sampler 2 NEBNext DNA Sample Prep High Fidelity (HF) Introductory Offer Reagent Set 1 Restriction Enzyme For a limited time, try the 4 Cellular Imaging and HF Restriction Enzyme Sampler for $15 Analysis Technologies Sampler ™ 5 SHuffle Competent E. coli High Fidelity (HF) Restriction Enzymes have been Advantages of HF enzymes Protein Ladder engineered to maximize performance under a wider 5 z Same specificity and price as wild type* 6 High Fidelity (HF) Restriction range of conditions, including reaction volume, z Reduced star activity offers added flexibility Enzymes incubation time and buffer compatibility. For a limited time, purchase the HF Restriction Enzyme z 100% activity in NEBuffer 4, simplifying Technical Tips Sampler, containing sample sizes (20 µl each) of a double digests selection of our most popular restriction enzymes 4 2009–10 NEB Catalog & z Time-Saver qualified – digests DNA in 5 minutes Technical Reference in HF format. The exceptional value of the Sampler makes it the perfect way to try out an HF enzyme. 7 High Fidelity Enzyme FAQs The HF Sampler includes EcoRI-HF from NEB EcoRI from Supplier A z ™ z ™ 7 Restriction Enzyme EcoRI-HF NotI-HF 10 30 50 100 M 10 30 50 100 units Troubleshooting Guide z BamHI-HF™ z BSA Web Tool Focus z SalI-HF™ z NEBuffer 4 PCR Selection Tool 8 High Fidelity (HF) Restriction Enzyme Sampler #R3000S. .$15 See page 6 for additional HF offers. HF enzymes reduce star activity (non-specific cleavage), even at higher enzyme amounts. 50 µl reactions were set up according to manufacturer’s *BamHI-HF is offered at a different $/unit, with a lower cost/vial. recommendations using 1 µl λ DNA and incubated at 37°C overnight. Page 2 Feature Article The Next Generation of Reagents for Sample Preparation (continued from page 1) In recent years, the advent of these technologies customers, NEB offers a broad selection of has revolutionized the world of sequencing and reagents that facilitate sample preparation of NEBNext™ DNA Sample biology. There is an insatiable appetite for DNA DNA (Figure 2). Additionally, NEB has taken a sequencing across diverse scientific disciplines dual approach to developing products for sample Prep Reagent Set 1 including microbiology, evolution, ecology, preparation: The NEBNext™ DNA Sample Prep Reagent forensics, epidemiology, comparative genomics, 1.) Develop novel solutions for multiple Set 1 is a set of reagents that facilitates sample and diagnostic applications. steps in sample preparation processes. preparation for downstream applications Parallel sequencing methods have enabled For example, the PreCR Repair Mix (NEB including next-generation sequencing, library whole genome resequencing of human genomes #M0309) is an optimized enzyme master construction and microarrays. Each of these (2,3), analysis of chromatin binding sites along mix that repairs damaged DNA and also components must pass rigorous quality control the entire genome by ChIP analysis (4,5), offers a simplified workflow. standards and are functionally validated by methylation analysis (6), transcriptome analysis 2.) Offer additional formulations of reagents preparation of a genomic library sequenced (7), metagenomic surveys, and sequencing of for increased ease-of-use in specific on an Illumina Genome Analyzer II, and by ancient DNA (8,9). applications. An example of this construction of an expression library. approach is the NEBNext™ DNA Sample Enzymes included in set: Reagents for Sample Preparation Prep Reagent Set 1, a set of enzymes, z T4 DNA Polymerase r DNA and RNA sample preparation is one of the nucleotides and buffers ideally suited for most important aspects of optimization of the z DNA Polymerase I, sample preparation for next-generation Large (Klenow) Fragment r quality and quantity of nucleic acid material sequencing and expression library for next generation sequencing. The exquisite construction (see side bar for more z T4 Polynucleotide Kinase r sensitivity of these technologies places new information). z Klenow Fragment (3´→ 5´ exo-) r demands on all the reagents involved, including z Quick T4 DNA Ligase r those required for the upstream sample Quality Assurance z 2X Phusion™ High-Fidelity PCR Master Mix* preparation process, which is outlined in Our priority is to provide product formulations Figure 1. During sample preparation, genomic that appropriately address existing and future Buffers and additional components DNA is sheared into 150 – 800 bp fragments. demanding applications, thereby providing included in set: This can be done by nebulization, acoustic maximum convenience and quality to customers. z 10X Phosphorylation Buffer shearing or by treatment with enzymes. DNA As new and more sensitive applications z Deoxynucleotide Solution Mix (10 mM) sheared by nebulization or acoustic shearing develop, quality controls and functional assays z dATP Solution (1 mM) may contain ends that are incompatible (3´ or must evolve to ensure that the more stringent z 5´ overhangs and 3´-phosphates or 5´-OH) demands placed on reagents are met. For 10X NEBuffer 2 with subsequent ligation steps and therefore example, each component of the NEBNext z Quick Ligation Reaction Buffer must be repaired. In some cases, it may be DNA Sample Prep Reagent Set 1 undergoes NEBNext™ DNA Sample Prep Reagent Set 1 necessary to convert a mixture of heterogeneous rigorous quality controls and validation assays. #E6000S 10 reactions. $500 ends to a homogenous population of blunt In addition to the standard quality controls for #E6000L 50 reactions. .$2,000 ended DNA fragments with 5´-phosphates and contaminating endonuclease and exonuclease 3´-OH. Furthermore, if dA tailing is required activities, each component also meets further * Produced by Finnzymes, distributed by New England Biolabs for subsequent ligation, a 3´ dAMP must be Phusion is a trademark of Finnzymes, Oy. added to a blunt ended fragment. Adaptors are † then ligated either to blunt ended or dA tailed Figure 1: Genomic library construction using NEB reagents fragments and the entire library is amplified. PCR to enrich/ Because sample preparation is a series of Ligate Fragmentation Repair dA Tail Purify amplify final coordinated reactions, each step must be as Adapters of Purified Fragment Fragment Ligation adapter modified efficient as possible to maximize yield while at onto DNA Genomic DNA Ends Ends Product fragmented Fragments the same time maintaining convenience and sample cost effectiveness. The efficiency of end repair, dA tailing, ligation and amplification are all dependent on the purity of the enzymes that perform these steps. UÊ£äÊÊ` /*½Ã UÊ/{Ê Ê* For 35 years, New England Biolabs has been UÊ Ê*ÞiÀ>ÃiÊ] UÊiÜÊ­iÝ® UÊ+ÕVÊ}>ÃiÊ UÊ* ÕÃ]ÊÓ8Ê committed to providing enzymes that drive Large (Klenow) UÊ£Ê`/*Ê-

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