RNA Depletion Kits for NGS Library Prep

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FEATURE ARTICLE The effect of nucleic acid modifications on digestion by DNA exonucleases

APPLICATION NOTE Eliminate residual gDNA from low copy plasmid purifications

NEW PRODUCTS New globin & rRNA depletion kits for NGS library prep

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CONTENTS

02 30% Off Luna qPCR and RT-qPCR 30% Off All Luna qPCR Kits Kits: Make a simpler choice and save on Luna kits until the end of December. Find the right Luna product for your application 03 RNA → cDNA in 13 minutes: The LunaScript RT SuperMix Kit offers the shortest available protocol for first-strand Select your cDNA synthesis. 2 detection method Dye-based Probe-based 04 Feature Article: The effect of nucleic acid modifications on digestion by DNA exonucleases. Genomic DNA Luna Universal Luna Universal Probe qPCR Master Mix qPCR Master Mix or cDNA (NEB #M3003) (NEB #M3004) 07 Something to chew on: Common Select applications for exonucleases and 1your target endonucleases. Luna Universal Luna Universal Probe RNA One-Step RT-qPCR Kit One-Step RT-qPCR Kit 08 Application Note: Using Exonuclease (NEB #E3005) (NEB #E3006) V (RecBCD) to Eliminate Residual Genomic DNA When Purifying Low Copy Plasmids with the Monarch Plasmid Miniprep Kit. Make a simpler choice

09 Featured New Products: Template • One product per application simplifies Switching RT Enzyme Mix for cDNA selection with Blue Tracking Dye amplification, 5´ RACE and nd2 strand cDNA synthesis and Q5U Hot Start High- • Convenient master mix and supermix Fidelity DNA Polymerase for amplification formats with user-friendly protocols of bisulfite-converted, deamninated, or simplify reaction setup damaged DNA. • Non-interfering, visible tracking dye 10 New globin and rRNA depletion kits helps to eliminate pipetting errors for NGS library prep: These new kits employ the NEBNext RNase H-based depletion workflow. ORDERING INFORMATION: LIST OFFER PRODUCT NEB # SIZE PRICE PRICE Download the NEB AR App to your device and M3003S 200 rxns (2 x 1 ml) £95 £66.50 look out for the AR icon in the 2019-20 NEB Luna Universal qPCR M3003L 500 rxns (5 x 1 ml) £226 £158.20 TERMS & CONDITIONS: Master Mix M3003X 1000 rxns (10 x 1 ml) £383 £268.10 Catalogue & Technical Reference to discover Offer valid in the UK M3003E 2500 rxns (1 x 25 ml) £853 £597.10 and Ireland only from videos, tutorials and immersive experiences. M3004S 200 rxns (2 x 1 ml) £95 £66.50 1/10/19 - 31/12/19. No Request your copy at Luna Universal Probe M3004L 500 rxns (5 x 1 ml) £190 £133.00 cash or cash equivalent. qPCR Master Mix M3004X 1000 rxns (10 x 1 ml) £330 £231.00 No substitution. Offer www.neb.uk.com/request M3004E 2500 rxns (1 x 25 ml) £734 £513.80 valid on new orders for Luna products E3005S 200 rxns £205 £143.50 only (#M3003S/L/X/E, Luna Universal One- E3005L 500 rxns £502 £351.40 M3004S/L/X/E, #E3005S/ Step RT-qPCR Kit E3005X 1000 rxns £780 £546.00 L/X/E, #E3006S/L/X/E, E3005E 2500 rxns £1734 £1213.80 #E3010S/L). Offer may not be applied to existing, NEB E3006S 200 rxns £180 £126.00 pending or prior orders. Luna Universal Probe E3006L 500 rxns £405 £283.50 Cannot be combined with One-Step RT-qPCR Kit £497.00 AR E3006X 1000 rxns £710 any other promotions. No E3006E 2500 rxns £1564 £1094.80 other discounts apply. Void LunaScript RT E3010S 25 rxns £124 £86.80 if copied or transferred and SuperMix Kit E3010L 100 rxns £386 £270.20 where prohibited by law. be INSPIRED drive DISCOVERY stay GENUINE One or more of the NEB products in this publication are covered by patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc. For more information, please email us at [email protected]. The use of these products may require you to obtain additional third party intellectual property rights for certain applications. Your purchase, acceptance, and/ New England Biolabs (UK) Ltd or payment of and for NEB’s products is pursuant to NEB’s Terms of Sale at www.neb.uk.com/terms-conditions. NEB does not agree to and is not bound by any other terms or conditions, Tel: 0800 318486 | Email: [email protected] | www.neb.uk.com unless those terms and conditions have been expressly agreed to in writing by a duly authorized officer of NEB. SUPERSCRIPT and VILO are trademarks of Thermo Fisher Scientific, Inc. iSCRIPT is a trademark of Bio-Rad Laboratories, Inc. BIOSCRIPT is a trademark Bioline LLC. qSCRIPT is a Cover photo: Viacheslav Rubel/Shutterstock.com registered trademark of Quanta BioSciences. ILLUMINA®, RIBO-ZERO®, GLOBIN-ZERO® and TruSeq® are registered trademarks of Illumina, Inc. AGENCOURT® and RNACLEAN® are registered trademarks of Beckman Coulter, Inc. SMART-SEQ® is a registered trademark of Clontech Laboratories. © 2019 New England Biolabs (UK) Ltd. All rights reserved. 30% Off All Luna qPCR Kits RNA → cDNA in 13 minutes with Advantages

LunaScript RT SuperMix Kit! • Simplify reaction setup with convenient supermix format Our latest addition to the Luna qPCR/RT-qPCR portfolio, the LunaScript RT SuperMix Kit is optimized for first strand cDNA synthesis in the context of a One-Step RNA cDNA DNA • Eliminate pipetting errors with two-stepRT-qPCR RT-qPCR workflow. It delivers best-in-class performance, user-friendly non-interfering, visible tracking dye

protocols, and includes a convenientReverse blue dye to track your sample. qPCR The cDNA products generatedtranscription by LunaScript have been extensively evaluated • Synthesize cDNA in less than in qPCR using the Luna qPCR LunaMaster® Universal Mixes. One-Step In combination, these products 15 minutes RT-qPCR Kit (NEB #E3005) provide a two-step RT-qPCR workflow with excellent sensitivity and accurate, Luna Universal Probe One-Step • Experience best-in-class linear quantitation. RT-qPCR Kit (NEB #E3006) performance, as all Luna products have undergone rigorous testing to optimize specificity, sensitivity, Two-Step accuracy and reproducibility RT-qPCR RNA cDNA cDNA DNA

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LunaScript Temp. (°C) Random primer cDNA RT 95 annealing & synthesis inactivation partial extension 85 Request a FREE 2 min., 25° 10 min., 55° 1 min., 95° 55 LunaScript 50 LunaScript RT ® SuperScript IV VILO™ 46 SuperMix sample at Maxima H Minus 42 25 LUNAqPCR.com iScript™ BioScript™ GrandScript qScript® qScript XLT SuperScript III VILO

0 5 10 15 20 25 30 35 40 45 50 50 60 65 70 75 Time (min)

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Luna Universal qPCR & RT-qPCR Products 03 FEATURE ARTICLE ~ The effect of nucleic acid modifications on digestion by DNA exonucleases

by Greg Lohman, Ph.D., New England Biolabs, Inc.

New England Biolabs offers a wide variety of exonucleases with a range of nucleotide structure specificity. Exonucleases can be active on ssDNA and/or dsDNA, initiate from the 5´ end and/or the 3´ end of polynucleotides, and can also act on RNA. Exonucleases have many applications in molecular biology, including removal of PCR primers, cleanup of plasmid DNA and production of ssDNA from dsDNA. In this article, we explore the interaction between commercially available exonucleases on oligonucleotides that have chemical modifications added during phosphoramidite synthesis, including phosphorothioate diester bonds, 2´-modified riboses, modified bases, and 5´ and 3´ end modifications. We discuss how modifications can be used to selectively protect some polynucleotides from digestion in vitro, and which modifications will be cleaved like natural DNA. This information can be helpful for designing primers that are stable to exonucleases, protecting specific strands of DNA, and preparing oligonucleotides with modifications that will be resistant to rapid cleavage by common exonuclease activities.

The ability of nucleases to hydrolyze phosphodi- chart, Common Applications of Exonucleases and Figure 1: ester bonds in nucleic acids is among the earliest Non-specific Endonucleases, at go.neb.com/Exos- Examples of exonuclease directionality nucleic acid enzyme activities to be characterized Endos, (also see page 7). 3→5 exonuclease (1-6). Endonucleases cleave internal phosphodiester What about cases where you only want to degrade bonds, while exonucleases, the focus of this article, some of the ssDNA in a reaction? Or, when you 5´ 3´ must begin at the 5´ or 3´ end of a nucleic acid want to make ssDNA from a dsDNA substrate, but 3´ 5´ strand and cleave the bonds sequentially (Figure 1). which strand is degraded matters greatly? What Exonucleases may be DNA or RNA specific, and about cases where the ends of your nucleic acids can act on single-stranded or double-stranded are modified—will exonucleases still digest the sub- 5→3 exonuclease nucleic acids, or both. Double-strand specific exo- strate, or cleave the modification? Several methods 5´ 3´ nucleases may initiate at blunt ends, nicks, or short depend on selective protection of polynucleotides, 3´ 5´ single-stranded 5´ or 3´ overhangs, though most such as protection of primers from degradation by exonucleases are active on a subset of these struc- polymerase exonuclease domains (17), selective tures. For a summary of the substrate specificity of protection of one strand of a DNA duplex for the Bidirectional exonuclease exonucleases available from NEB, view our newly- production of ssDNA (14-16), and the protection updated selection chart, Properties of Exonucleases of polynucleotides from degradation by serum 5´ 3´ and Non-specific Endonucleases, at go.neb.com/ nucleases, as in the case of RNA interference 3´ 5´ ExosEndos. drugs (18, 19). In each of these cases, it is critical A variety of DNA exonucleases have been charac- to understand the influence of modifications on Pictured are double stranded exonucleases with a 5´ to 3´ terized from many different organisms; in vivo, these exonuclease activity—which modifications inhibit polarity (top), a 3´ to 5´ polarity (middle), and a bidirec- enzymes play critical roles in polynucleotide repair, nucleotide cleavage and which do not. tional nuclease (bottom). recycling, error correction, and protection from ex- Recently, researchers at NEB have worked to ogenous DNA (6-8). In vitro, exonucleases are used characterize the interaction between exonucleases in many applications where it is desirable to remove and modified polynucleotides, as part of a broader modifications to inhibit nuclease activity, as well certain nucleic acids. For example, Exonuclease V effort to gain deeper insight into the sequence and as which modifications have little to no effect on (RecBCD) (Exo V, NEB #M0345) is often used to structural determinants of nuclease activity and exonuclease digestion. remove contaminating linear ssDNA and dsDNA specificity. In an effort to catalog the modifica- from plasmid preparations (4,9); T7 Exonuclease tions that inhibit exonuclease digestion, we treated (T7 Exo, NEB #M0263) can be used to generate polynucleotides containing a range of modifications Phosphorothioate linkages 3´ overhangs in DNA (4, 10, 11); Exonuclease I (including non-standard bases, sugars and backbone A phosphorothioate (pt) bond is a phosphodiester (Exo I, NEB #M0293), Thermolabile Exonuclease I chemistries) with exonucleases under the recom- linkage where one of the two non-bridging oxy- (NEB #M0568) or Exonuclease VII (Exo VII, NEB mended in vitro reaction conditions. This article will gens has been replaced by a sulfur (Figure 2). This #M0379) can be used to eliminate ssDNA PCR summarize data from the literature, as well as the modification has been used for decades to inhibit primers, leaving double-stranded products undigest- key results from NEB’s work related to understand- nuclease phosphodiesterase and phosphoryl trans- ed (12, 13), and Lambda Exonuclease (Lambda Exo, ing the activity of exonucleases on chemically ferase activities, as well as for gaining mechanistic NEB #M0262) can be used to convert dsDNA to modified polynucleotides. We will focus on the insights into these enzymes (20, 23). Chemically, ssDNA for a variety of applications (14-16). More most widely used—and most successful—method the substitution of oxygen for sulfur does not information on common applications of exonucleas- for blocking nuclease activity, the phosphorothioate dramatically change the reactivity of the bond, and es available from NEB can be found in our selection bond (20-23), but will also discuss the use of other pt-containing polynucleotides can still function in many enzymatic reactions. In a typical phospho-

04 Figure 2: at the first phosphodiester (5, 26). Importantly, any Examples of common nucleotide modifications and their effect on exonuclease activity enzyme with endonuclease activity, like DNase I, will simply ignore the ends and degrade the poly- Modi ed phosphodiesters (pt bonds) Modi ed bases do not inhibit nucleotides from the inside out (unless every phos- block exonuclease activity exonuclease activity -O O O phodiester bond is replaced by a phosphorothio- 5´ – 5´ O OH O– ate). Keeping these important exceptions in mind, - O P O Base O O O P O - Base O O O O O T phosphorothioate bonds remain the most generally O– O H O N O O P O HN N Base 5´ H applicable (and relatively inexpensive) way to pro- O O S P O O– O Base O O N O O P O– O– O tect oligonucleotides from digestion by exonucle- O O O P – O O O 5-Fluorescein dT ases. For a complete list of DNA exonucleases and O P – O O O 3´ O T OH 3´ their interaction with pt bonds, view our selection 3´ HO O NH2 O chart, Activity of Exonucleases and Non-Specific

Phosphorothioate 3-inverted dT 5-inverted dT N N 5´ HN (pt) bond 5´ 5´ N Endonucleases, at go.neb.com/ExosEndos. N O NH2 O O N N O O N O O H O

O O O 2´-modified nucleosides

5´ and 3´ end modi cation 3´ 3´ 3´ does not block exonuclease activity Generally, DNA exonucleases do not digest RNA IsoC IsoG Super T

O portions of oligonucleotides, though RNA is itself

HN NH susceptible to RNases and nonspecific hydrolysis. O H H Sugar modi cations inhibit H P We have further found that hybridizing RNA to N O O Base O– S O most exonuclease activity O DNA strands does not block the activity of dsDNA

5´ 5´ 5´ 5 Biotin O exonucleases on the DNA strand. Hybridiza- O P O– O Base O Base O Base O O O O tion of ssDNA to RNA will block the activity of

3´ O ssDNA exonucleases as effectively as hybridization 5´ P O OH O OMe O O O O – Base O O O 3´ 3´ 3´ to dsDNA. Additionally, certain 2´-O-modified OMe HN NH riboses, are both stable to spontaneous hydrolysis O 3 Biotin H H – Ribo 2-Methoxy 2-Methoxyethyl O P O H and offer strong resistance to exonuclease activity N (MOE) O S O (27). 2´-O-methyl and 2´-O-methoxyethyl (MOE) nucleosides, which contain bulky substituents off the sugar ring, have been shown to grant strong resistance to nucleases and additionally increase the diester bond, the two non-bridging oxygens are a single pt bond is insufficient to fully protect an strength of annealing to complementary DNA and chemically equivalent. When one of these oxygens oligonucleotide from exonuclease digestion. When RNA. These features have found utility in antisense is replaced by sulfur, however, the phosphorus is the pt bond is installed via an oxidation step during nuclease strategies, to make oligonucleotides that now connected to four distinct groups, rendering phosphoramidite synthesis, a nearly equal amount are both resistant to degradation and able to bind tightly to target RNAs. it a chiral center with two possible configurations of each isomer (SP and RP) is formed at each pt referred to as “SP” and “RP” (Figure 3). It is this key linkage (20). Since most enzymes can cleave one of These sugar modifications also work in vitro to feature that confers resistance for the majority of these isomers, a single chemically installed pt will block exonuclease activity quite strongly. Our stud- nuclease enzymes studied; one configuration will protect only half the molecules from digestion by a ies have found that, while a single terminal MOE react at rates similar to a phosphodiester, while given exonuclease. Thus, it is typically recommend- nucleoside only weakly inhibits exonuclease activ- the other is significantly inhibitory or completely ed that 3–6 pt bonds be used to block exonuclease ity, three successive MOE modifications provide unreactive. Isomer reactivity varies from enzyme digestion, to prevent this read-through. One might enhanced resistance to many exonucle to enzyme, and different pt isomers can inhibit expect that because each bond is a 50:50 mixture enzymes that catalyze the same reaction (e.g., phos- of isomers, when presented with 5 consecutive phoryl transfer). For example, DNA Polymerase I isomers, a given enzyme could cleave the first bond (DNA Pol I, NEB #M0209) can incorporate on half the molecules, then half of the molecules deoxynucleotide triphosphates with a pt ester at that had the first bond hydrolyzed would have the α phosphate (dNTPαS), allowing formation the second hydrolyzed, and so on, such that there Figure 3: of pt-bonded polynucleotides. However, it can would be a range of partially degraded products. Chirality of phosphorothioate bonds only react with SP configured dNTPαS molecules, In practice, it has been reported (and confirmed by and does so with inversion of the stereocenter to recent results at NEB) that five consecutive pt bonds 5´ O– O form exclusively RP-configured pt bonds in the completely block all exonuclease activity at all pt product. Conversely, the 3´→ 5´exo activity of this bond positions (16). The exact reasons for this are S P O Base S P O Base O O O O– polymerase cleaves RP, but not SP configured bonds not currently known, but it is likely that exonucle- → ases engage multiple bases at once, and the net ef- 5´ (20). Alternatively, the 3´ 5´ exo activity of E. coli O O O O Exonuclease III (Exo III, NEB #M0206) cleaves fect of the isomeric mixture somehow prevents the P O– P O– S , but not R configured pt bonds (24). Therefore, active site from properly organizing around bonds O O P P 3´ 3´ DNA created from the incorporation of dNTPαS that are the normally cleavable pt isomer. RP SP by DNA Pol I is highly resistant to exonuclease There are several commonly used exonucleases that cleavage by Exo III (25). are not blocked even by 5 consecutive pt bonds; Phosphorothioates can block many, but not all, for example, Exo V, Exo VII and T5 Exonuclease exonucleases. To block exonuclease cleavage, the pt (T5 Exo, NEB #M0363) all can cleave, leaving bonds must be placed at the end(s) where the en- short oligos instead of cutting at every bond in a zyme initiates, e.g., the 5´ end for Lambda Exo and series, and thus can digest DNA by skipping over the 3´ end for Exo III. It is important to note that termini blocked by multiple pt bonds and cleaving continued on page 4...

05 ases, including Exo I, Exo III, Lambda Exo, T3 degradation by serum exonucleases for aptamers match the polarity of the exonucleases used, and be Exonuclease (T3 Exo) and polymerase exonucleases. and other modified oligonucleotides. In our hands, aware that several exonucleases can read-through or Similar to pt bonds, several exonucleases can digest 3´-inverted dT blocked only the relatively weak bypass terminal pt bonds; your choice of nucleases through these regions, notably T5 Exo, T7 Exo, 3´→ 5´ exonuclease activity of DNA Polymerase is as important as the modifications used. Aside Exo V, Exo VII and Exo VIII. Overall, exonuclease I, Large (Klenow) Fragment (NEB #M0210) and from pt bonds, MOE nucleotides are the next best inhibition by MOE is quite strong, but pt bonds are Exonuclease T (Exo T, NEB #M0265), but did choice for providing nuclease resistance in vitro, more effective and are typically cheaper to prepare not block more active exonucleases such as in T7 with similar caveats to pt bonds. The vast majority and incorporate. However, if for some reason the DNA Polymerase (NEB #M0274), Exo I or Exo III. of end modifications, including affinity tags and pt chemistry is not desired, 2´-O-modified ribose Similarly, 5´-inverted dT partially inhibited only fluorophores, as well as internal non-standard bases, moieties are a viable alternative. Lambda Exo activity, which is known to require a provide little, if any, nuclease resistance, and will be 5´-phosphate for efficient initiation. Other 5´→ 3´ cleaved completely in vitro. Other 5´/3´ end modifications exonucleases were not significantly inhibited by this Several other modifications, such as the inverted de- modification, showing complete digestion after a References oxythymidine bases and dideoxynucleotides (Figure one-hour incubation under the recommended usage 1. Lehman, I. R., and Nussbaum, A. L. (1964) J. Biol. Chem. conditions. 239, 2628-2636. 2) have been reported to suppress serum nuclease 2. Nichols, N. M. (2011) Curr. Protoc. Mol. Biol. Chapter 3, activity when appended to the end of synthetic We do not recommend 5´/3´ end modification as Unit3.12. oligonucleotides (27). Many other modifications a good strategy for producing nucleotides resistant 3. Richardson, C. C., Lehman, I. R., and Kornberg, A. (1964) to exonuclease degradation in vitro. Researchers J. Biol. Chem. 239, 251-258. may be attached through “linkers” at either the 5´ 4. McReynolds, L. A., and Nichols, N. M. (2011) Curr. Protoc. or 3´ end, including fluorescent tags, biotin or other should be aware that these modifications will be Mol. Biol. Chapter 3, Unit 3.11. affinity labels, or reactive groups for attachment to cleaved by the majority of exonucleases, potentially 5. Lovett, S. T. (2011) EcoSal Plus 4. beads or surfaces. These linkers are typically con- leading to the loss of fluorescent labels and affinity 6. Yang, W. (2011) Q. Rev. Biophys. 44, 1-93. 7. Bebenek, A., and Ziuzia-Graczyk, I. (2018) Curr. Genet. 64, nected to the 5´ or 3´ end via a phosphodiester, but tags. If a modification stable to exonuclease activity 985-996. what is the interaction of these modified ends with is needed, a better strategy is to use internal labels 8. Tsutakawa, S. E., Lafrance-Vanasse, J., and Tainer, J. A. exonucleases? connected to the 5-methyl position of dT (e.g., (2014) DNA Repair (Amst). 19, 95-107. 9. Karu, A. E., MacKay, V., Goldmark, P. J., and Linn, S. Fluorescein dT, Figure 2). If these modified dT We have surveyed a range of these modifications in (1973) J. Biol. Chem. 248, 4874-4884. typical in vitro exonuclease assays. In general, while bases are used near the end of an oligo, they can 10. Kerr, C., and Sadowski, P. D. (1972) J. Biol. Chem. 247, 305-310. many provide modest inhibition as compared to a be protected with surrounding pt bonds (Figure 4). The linkage to the base is not susceptible to enzy- 11. Straus, N. A., and Zagursky, R. J. (1991) Biotechniques. 10, 5´-phosphate, all exonucleases tested could cleave 376-384. all modifications connected through phospho- matic cleavage, and the pt bonds will protect the 12. Li, H. H., Cui, X. F., and Arnheim, N. (1991) Nucleic. Acids. diester bonds. Interestingly, this poor inhibition backbone from digestion, as described above. Res. 19, 3139-3141. 13. Enzymatic PCR Cleanup using Exonuclease I and Shrimp held true for the inverted dT modifications, which Alkaline Phosphatase. New England Biolabs, Ipswich, MA. have been reported to grant extra stability versus Base modifications 14. Civit, L., Fragoso, A., and O’Sullivan, C. K. (2012) Anal. None of the exonucleases available from NEB were Biochem. 431, 132-138. 15. Murgha, Y. E., Rouillard, J. M., and Gulari, E. (2014) PLoS significantly inhibited by modified bases under the One 9, e94752. conditions we tested. Modifications tested included 16. Nikiforov, T. T., Rendle, R. B., Kotewicz, M. L., and Rog- Figure 4: Designing oligonucleotides 5-methyl-substituted dT (e.g., Fluorescein dT), ers, Y. H. (1994) PCR Methods Appl. 3, 285-291. with nuclease-resistant modifications 17. Skerra, A. (1992) Nucleic Acids Res. 20, 3551-3554. deoxyuridine, the Tm-enhancing “super T,” and the 18. Evers, M. M., Toonen, L. J., and van Roon-Mom, W. M. non-natural base pair isoG:isoC (Figure 2) (28). (2015) Adv. Drug Deliv. Rev. 87, 90-103. A. End uorescein (FAM)-labeled DNA All modified substrates were digested completely 19. Lundin, K. E., Gissberg, O., and Smith, C. I. (2015) Hum. Gene Ther. 26, 475-485. 5´ FAM 3´ by all the exonucleases tested. Some modifications 20. Eckstein, F. (1985) Annu. Rev. Biochem. 54, 367-402. 3´ 5´ showed weak blockage, pausing at the modifica- 21. Spitzer, S., and Eckstein, F. (1988) Nucleic Acids Res. 16, tion site before completely degrading the substrate. 11691-11704. B. pt bonds For several exonucleases tested, modified dT bases 22. Eckstein, F., and Gish, G. (1989) Trends Biochem Sci 14, 97-100. X with large modifications off the 5-methyl position 23. Eckstein, F. (2014) Nucleic Acid Ther. 24, 374-387. 5´ FAM 3´ (Figure 2) showed a buildup of partially-digested 24. Putney, S. D., Benkovic, S. J., and Schimmel, P. R. (1981) 3´ 5´ intermediates, apparently stalling just before the Proc. Natl. Acad. Sci. U S A, 78, 7350-7354. modification; in no case did this resistance approach 25. Yang, Z., Sismour, A. M., and Benner, S. A. (2007) Nucleic Acids Res. 35, 3118-3127. the inhibition seen for 2´ MOE sugars or pt link- 26. Sayers, J. R., and Eckstein, F. (1990) J. Biol. Chem. 265, C. Internal FAMdT surrounded by pt bonds ages. 18311-18317. FAM 27. Kratschmer, C., and Levy, M. (2017) Nucleic Acid Ther. 27, X 335-344. 5´ T 3´ Conclusion 28. Piccirilli, J. A., Krauch, T., Moroney, S. E., and Benner, S. A. (1990) Nature, 343, 33-37. 3´ 5´ We have evaluated a variety of chemical modifications for their inhibition of exonuclease activity at (A) End fluorescein (FAM) labeled-DNA is rapidly the 5´ and 3´ ends of oligonucleotides. Broadly, the degraded by exonucleases. (B) pt bonds between nucleotides phosphorothioate modification, one of the more prevent the DNA strand from being degraded, but the well-known used modifications to block nuclease end label can still be cleaved. (C) An internal FAMdT cleavage, remains the most effective choice to pro- surrounded by pt bonds will prevent the exonuclease from tect oligonucleotides from degradation. However, removing the label. one must be careful to use multiple pt bonds, place them at the correct end of the oligonucleotide to

Featured Resources: Request your copy of our endonuclease magnet or exonuclease poster at

06 www.neb.com/ExosEndosRequest Something to chew on. Common Applications for Exonucleases and Endonucleases

Did you know that NEB offers the largest supply of these important tools, and has a team of experts studying the function and optimization of these enzymes? We also offer several helpful tools to help you find the best enzyme to facilitate your work, including selection charts, recommended applications, usage guidelines and more.

Not sure which exonuclease or endonuclease to choose? Find the right enzyme for your application using the table below.

Application Recommended Enzyme(s) NEB # PRICE Removal of 3´ overhangs Quick Blunting Kit E1201S/L £74 / £299

5´ overhang fill-in treatment Quick Blunting Kit E1201S/L £74 / £299

Removal of ss primers for nested PCR reactions Thermolabile Exonuclease I M0568S/L £69 / £276 Exonuclease I M0293S/L £62 / £247 Removal of primers post PCR prior to DNA sequencing or SNP detection Thermolabile Exonuclease I (1) M0568S/L £69 / £276 Exonuclease VII (2) M0379S/L £142 / £566 Mapping positions of introns in genomic DNA Exonuclease VII M0379S/L £142 / £566

Removal of primers with or without 3´ or 5´ terminal phosphorothioate bonds Exonuclease VII M0379S/L £142 / £566 Generating ssDNA from linear dsDNA: If 5´ → 3´ polarity required Lambda Exonuclease (3) M0262S/L £63 / £251 If 3´ → 5´ polarity required Exonuclease III (E. coli) (4) M0206S/L £57 / £225 Exonuclease III (E. coli) plus M0206S/L £57 / £225 Preparation of nested deletions in double-stranded DNA Exonuclease VII M0379S/L £142 / £566 Exonuclease III ( ) (5) M0206S/L £57 / £225 Site-directed mutagenesis E. coli T7 Exonuclease (6) M0263S/L £53 / £216 Nick-site extension T7 Exonuclease M0263S/L £53 / £216

Degradation of denatured DNA from alkaline-based plasmid purification methods for T5 Exonuclease M0363S/L £59 / £234 improving DNA cloning T5 Exonuclease (7) M0363S/L £59 / £234 Removal of chromosomal/linear DNA in plasmid preparations Exonuclease V (RecBCD) (8) M0345S/L £74 / £299 T5 Exonuclease (9) M0363S/L £59 / £234 Removal of unligated products (linear dsDNA) from ligated circular double-stranded DNA Exonuclease V (RecBCD) (10) M0345S/L £74 / £299 Removal of residual gDNA after purification of low copy plasmid Exonuclease V (RecBCD) M0345S/L £74 / £299

Removal of contaminating DNA from RNA samples DNase I M0303S/L £66 / £264

Conversion of single-stranded DNA or RNA to 5´-mononucleotides Nuclease P1 M0660S £46

Analysis of base composition, potential damage and modification of nucleic acids Nuclease P1 M0660S £46

Progressive shortening of both ends of double-stranded DNA Nuclease BAL-31 M0213S £57

Preparation of double-stranded DNA fragments with 5´-OH and 3´-phosphate Micrococcal Nuclease M0247S £60

Degradation of nucleic acids (both DNA and RNA) in crude cell-free extracts Micrococcal Nuclease M0247S £60

Preparation of rabbit reticulocyte Micrococcal Nuclease M0247S £60

Chromatin Immunoprecipitation (ChIP) analysis Micrococcal Nuclease M0247S £60

Notes: 6. Removes nicked-strand DNA from 5´ to 3´ 1. Rapid heat inactivation versus Exonuclease I 7. Degrades linear ss + dsDNA, nicked DNA 2. For 3´ chemically modified primers 8. Degrades linear ss + dsDNA: preferred as Exo V will save nicked plasmids resulting in 3. Strand targeted for removal requires one 5´ end with phosphate higher yields especially for low-copy number plasmid prep 4. Strand targeted for removal requires a 5´ overhang, a blunt end, or a 3´ overhang 9. Only the unnicked form of ligated circular double-stranded DNA remains (with less than 4 bases) 10. Both nicked and unnicked form of ligated circular double-stranded DNA remains 5. Removes nicked-strand DNA from 3´ to 5´ 07 APPLICATION NOTE Using Exonuclease V (RecBCD) to Eliminate Residual Genomic DNA When Purifying Low Copy Plasmids with the Monarch Plasmid Miniprep Kit Peichung Hsieh, Ph.D., New England Biolabs, Inc.

Materials 3. Check OD600 nm (usually it will be around Results: • Endonuclease V (RecBCD) (NEB #M0345) 4 O.D./ml of cells). Three milliliters of an overnight culture of • NEB 10-beta Competent E coli (High Efficiency) 4. Harvest 3 ml of the overnight culture and purify NEB-10 beta competent E. coli cells transformed (NEB #C3019) the plasmid DNA using the Monarch Plasmid with pBAC were processed using the Monarch Plas- • Antibiotic, typically Chloramphenicol Miniprep Kit (NEB #T1010) following the mid DNA Kit and an equivalent Miniprep kit from recommended protocol. another vendor. After isolating the DNA, samples • LB Media were treated with Exonuclease V (RecBCD) and 5. In the final elution step, elute the DNA • Monarch Plasmid Miniprep Kit (NEB #T1010) then digested with EcoRI. Samples were run on with 30 μl of Monarch DNA Elution Buffer (pre- an agarose gel to assess the quality of the isolated heated to 50°C). DNA, and whether or not the Exonuclease V- Introduction 6. To the eluted DNA, add 4 μl of NEBuffer 4 treated DNA could be digested to completion. The (10X), 4 μl of 10 mM ATP, and 2 μl of Exo- The use of low and/or single-copy plasmids to Exonuclease V-treated samples showed no gDNA nuclease V (RecBCD). Mix reaction and incubate clone large pieces of DNA (up to 200 kb) or to contamination (#3-6) while the untreated samples at 37°C for 1 hr. drive expression of slow folding or toxic proteins in exhibited a significant amount of gDNA as seen by E.coli is a commonly used strategy. Purification of 7. Heat-inactivate the Exonuclease V by incubating the smear observed in those samples (#1,2,7,8). low-copy plasmids or bacterial artificial chromo- at 70°C for 30 min. The plasmid DNA is now These results indicate that Endonuclease V can be somes (BACs) presents some challenges that are not ready for restriction enzyme digestion, PCR or used to efficiently degrade contaminating gDNA evident when working with higher copy number transformation. from plasmid purification steps, including those of plasmids, such as pUC19. The ratio between bacte- Note: Typically, 30-60 ng of single-copy plas- low copy number. rial gDNA and plasmid DNA is higher, thereby mid can be purified from 3 ml of an overnight reducing yield of the desired plasmid produced by E.coli culture with (O.D. 600 nm = 4 O.D/ml) typical plasmid miniprep protocols. Additionally, elevated levels of host gDNA are often co-purified, reducing the accuracy of quantitation by UV ab- sorbance or dsDNA specific dyes. Neither method pBAC samples exhibit no bacterial gDNA contamination after can distinguish the contribution from gDNA to the treatment with Exonuclease V (RecBCD) overall nucleic acid content. Co-purification of host gDNA also affects the appearance of the sample Removal of gDNA contamination from low-copy number plasmid purification when resolving by gel electrophoresis and adds un- Column Q N Q N Q N Q N wanted contaminating DNA for any amplification- Exo V – – + + + + – – N0552 based application. EcoRI – – – – + + + + Exonuclease V (RecBCD, NEB #M0345) is an exonuclease that degrades both linear ss- and dsDNA, while keeping the circular DNA intact. Treatment of miniprep DNA samples of low copy plasmids with this exonuclease degrades the contaminating gDNA, restoring purity and ease of use in downstream applications.

Protocol 1. Transform an endA- strain (e.g. NEB 10-beta, NEB #C3019) with the BAC plasmid DNA and plate outgrowth onto a media plate with ap- propriate antibiotic. Incubate overnight at 30°C. BACs with CamR require reduced stringency selection. Chloramphenicol levels should be maintained between 10-15 μg/ml on the selective plate. Note: strains with an F’ plasmid are not compat- Miniprep plasmid DNA samples isolated with the Monarch Plasmid Miniprep kit (N) and a ible with BACs or miniF plasmids. similar kit from a competitor (Q) were either treated (+) or not treated (-) with Exonucle- ase V, and then digested with EcoRI. The samples treated with Exonuclease V showed no 2. Pick a colony, inoculate 10 ml LB + antibiotic, contaminating gDNA and they were correctly cut with EcoRI. and incubate overnight at 30°C (200-250 RPM). Learn more and request a sample at NEBmonarch.com 08 NEW PRODUCTS Template Switching RT Enzyme Mix Advantages Template switching overview

5´ 3´ • Prepare RNA-seq libraries from extremely low RT primers Cap or ppp mRNA 3´ template RT primer 5´ TTTTTT input: single cells/nuclei or 2 pg total RNA Oligo (dT) Reverse NNNNNN transcription • Low background for RNA-seq or 5´ RACE Random • Use with various TSOs, RT primers and DNA CCACGA 5´ 3´ Gene-speci c 3´ 5´ polymerases for full-length cDNA amplification cDNA Non-templated nucleotides Optional adaptor • Enjoy faster protocols as compared to at 5´ end Template alternative RNA-seq methods (i.e., Smart-Seq®) Template-switching switching oligo (TSO)

5´ 3´ Applications 3´ 5´

• cDNA amplification 3´ 5´ cDNA with a sequence-of-choice (adaptor, etc.) • 5´ RACE at the 3´ end (5´ end of transcript)

nd • 2 strand cDNA synthesis (full coverage of the Upon reaching the 5´ end of the RNA template, the reverse transcriptase adds a few non- 5´ end of the transcript) templated nucleotides to the 3´ end of the cDNA. These non-templated nucleotides can anneal to a TSO with a known sequence handle of choice, prompting the reverse transcriptase to switch from the RNA template to the TSO. The resulting cDNA contains a universal sequence (complementary to the TSO sequence) at the 3´ end. PRODUCT NEB # SIZE PRICE Template Switching RT Enzyme Mix M0466S/L 20/100 rxns £81 / £324

Q5U Hot Start High-Fidelity DNA Polymerase

Advantages Common applications enabled by • Enable room termperature setup with Q5U Hot Start High-Fidelity Polymerase aptamer-based hot start formulation Bisulfite-treated DNA enymatically-deaminated DNA • Superior amplification of bisulfite-converted, m deamninated, or damaged DNA (e.g. FFPE) U C U U U

• Generate higher assembly efficiency and Damaged DNA (e.g., FFPE) improve accuracy in USER cloning U I • Utilize optimized protocols for recommended U I applications dU incorporation/carryover prevention dUTP ...U...U...

USER cloning U PRODUCT NEB # SIZE PRICE Q5U Hot Start High-Fidelity M0515S/L 100/500 units £145 / £570 U DNA Polymerase

Archaeal family B-type polymerases can incorporate/tolerate a variety of modified nucleotides but will stall upon encountering uracil and inosine residues. Q5U Hot Start High-Fidelity DNA Polymerase is a modified Q5 High-Fidelity DNA polymerase, which efficiently incorporates dUTP and amplifies uracil-containing templates.

09 Get more of what you want.

NEBNext RNA Depletion Kits Advantages

Abundant RNAs can conceal the biological significance of less abundant transcripts, • Suitable for low-quality (e.g., FFPE) and so their efficient and specific removal is desirable. NEBNext RNA Depletion and high-quality RNA kits facilitate this removal, while ensuring retention of RNAs of interest. These kits • Compatible with a broad range of employ the efficient RNase H method (1,2), as well as close probe tiling of abundant input amounts: 10ng–1µg RNAs, thereby ensuring that even degraded RNA is hybridized and subsequently removed. • Superior depletion of abundant RNAs, with retention of RNAs of interest

Globin & rRNA Depletion Kits (Human/Mouse/Rat) • Fast workflow: 2 hours, with less than 10 minutes hands-on time The NEBNext RNaseH-based depletion method can be applied to abundant RNAs beyond rRNA. In blood samples, the great majority of RNA is comprised of rRNA • Depleted RNA is suitable for and globin mRNA, and the removal of both is desirable. The NEBNext Globin & RNA-seq, random-primed cDNA rRNA Depletion Kit (Human/Mouse/Rat) depletes globin mRNA (HBA1/2, HBB, synthesis, or other downstream RNA analysis applications HBD, HBM, HBG1/2, HBE1, HBQ1 and HBZ), cytoplasmic rRNA (5S, 5.8S, 18S, 28S, ITS and ETS) and mitochondrial rRNA (12S, 16S). • Available with optional Agencourt® The kit is effective with human, mouse and rat total RNA preparations, both intact RNAClean® XP Beads for RNA and degraded. Purification When only mRNA (and not non-coding RNA) is of interest, the Globin & rRNA Depletion Kits can be used following poly(A) mRNA enrichment (e.g. using the 1. Adiconis, X. et al. (2013). Nature Methods 10; 623-629. NEBNext poly(A) mRNA Magnetic Isolation Module NEB #E7490). 2. Morlan, J.D. et al. (2012). PLoS One 7, e42882.

Depletion of globin mRNA and ribosomal RNA enriches for RNAs of interest across species

rRNA Globin RNAs of interest

uman Whole Blood Mouse Whole Blood Rat Whole Blood Human, mouse and rat whole blood total RNA (1 µg)

100 was depleted of rRNA alone, or rRNA and globin mRNA transcripts, using the NEBNext Globin & rRNA Depletion 80 Kit. RNA-seq libraries were prepared from untreated and 60 depleted RNA using the NEBNext Ultra II RNA Library 40 Prep Kit for Illumina followed by paired-end sequencing (2

Percent of Reads 20 x 75 bp). Reads were identified as rRNA or globin mRNA

0 using mirabait (6 or more, 25-mers), and levels of rRNA and No rRNA rRNA & globin No rRNA rRNA & globin No rRNA rRNA & globin globin mRNA remaining were calculated by dividing matched depletion depletion depletion depletion depletion depletion depletion depletion depletion reads by the total number of reads passing instrument quality filtering.

10 Transcript expression correlation is maintained after depletion Consistent depletion of globin mRNA and rRNA across of Globin mRNA and rRNA species and across inputs

10,000 NEBNext® Illumina® NEBNext Illumina A. uman Whole Blood 1,000 100 rRNA 5 Globin * <0.9% † ≤0.01% 80 4 ‡ ≤0.07% 100 60 3

d (TPM, log 10) 40 2

e 10 t e

l 20 1 rRNA Reads (%) p * Globin Reads (%) † † ‡ † 1 0 * * 0 1 µg 100 ng 10 ng 1 µg 100 ng 10 ng Und e R2=0.98 R2=0.80 0.1 0.1 1 10 100 1,000 0.1 1 10 100 1,000 10,000 B. Mouse Whole Blood Depleted (TPM, log 10) 60 rRNA 5 Globin § <0.08% 50 4 40 Human whole blood total RNA (1 µg) was depleted of rRNA and globin mRNA using 3 30 the NEBNext Globin & rRNA Depletion Kit or Globin-Zero Gold rRNA Depletion Kit 2 (Illumina). RNA-seq libraries were prepared from untreated and depleted RNA using the 20 10 1 rRNA Reads (%) NEBNext Ultra II RNA Library Prep Kit for Illumina followed by paired-end sequencing Globin Reads (%) § § 0 0 (2 x 75 bp). GENCODE v27 transcript abundances were estimated using Salmon. TPM 1 µg 100 ng 10 ng 1 µg 100 ng 10 ng (Transcript Per Million mapped reads) of protein coding transcripts, and R2 values for the linear fit are shown. Correlation analysis of the transcripts indicates better transcript C. Rat Whole Blood expression correlation between depleted and undepleted samples for the NEBNext Globin & 50 rRNA 5 Globin rRNA Depletion Kit. Treatment does not alter the abundances of non-targeted transcripts. 40 4 30 3

20 2

10 1 rRNA Reads (%) Globin Reads (%) 0 0 Ordering Information 1 µg 100 ng 10 ng 1 µg 100 ng 10 ng

PRODUCTS NEB # SIZE PRICE Human, mouse and rat whole blood total RNA (1 µg, 100 ng and 10 ng) was depleted NEBNext rRNA Depletion Kit (Human/Mouse/ E6310S 6 rxns £312 of rRNA and globin mRNA using the NEBNext Globin & rRNA Depletion Kit or Rat) E6310L 24 rxns £1134 Globin-Zero® Gold rRNA Depletion Kit (Illumina). RNA-seq libraries were prepared E6310X 96 rxns £4082 from untreated and depleted RNA using the NEBNext Ultra II RNA Library Prep Kit for NEBNext rRNA Depletion Kit (Human/Mouse/ E6350S 6 rxns £322 Illumina followed by paired-end sequencing (2 x 75 bp). Reads were identified as ribosomal Rat) with RNA Sample Purification Beads E6350L 24 rxns £1182 or globin using mirabait (6 or more, 25-mers), and levels of rRNA and globin mRNA E6350X 96 rxns £4253 remaining were calculated by dividing matched reads by the total number of reads passing New NEBNext Globin & rRNA Depletion Kit E7750S 6 rxns £321 instrument quality filtering. The data represents an average of 3 replicates and error bars (Human/Mouse/Rat) E7750L 24 rxns £1167 indicate standard error. The NEBNext Globin Depletion Kit is superior at depleting rRNA E7750X 96 rxns £4205 across species, and at depleting over 99% of globin mRNA. New NEBNext Globin & rRNA Depletion Kit E7755S 6 rxns £332 (Human/Mouse/Rat) with RNA Sample E7755L 24 rxns £1217 Purification Beads E7755X 96 rxns £4381 COMPANION PRODUCTS NEBNext Poly(A) mRNA Magnetic Isolation E7490S 24 rxns £69 Module E7490L 96 rxns £247 It’s time to celebrate! NEBNext Ultra II Directional RNA Library Prep E7760S 24 rxns £851 For 10 years, NEB has helped advance next generation Kit for Illumina E7760L 96 rxns £2751 sequencing (NGS) by streamlining sample prep workflows, NEBNext Ultra II Directional RNA Library Prep E7765S 24 rxns £896 minimizing inputs, and improving library yield and quality. with Sample Purification Beads E7765L 96 rxns £3054 NEBNext Ultra II RNA Library Prep Kit for E7770S 24 rxns £851 Illumina E7770L 96 rxns £2751 NEBNext Ultra II RNA Library Prep with E7775S 24 rxns £896 Sample Purification Beads E7775L 96 rxns £3054 NEBNext Library Quant Kit for Illumina E7630S 100 rxns £101 E7630L 500 rxns £422 NEBNext Magnetic Separation Rack S1515S 24 tubes £437

New to NEBNext? Get started with a free sample at NEBNext.com.

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