* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download 51 - Lab Times
Survey
Document related concepts
Agarose gel electrophoresis wikipedia , lookup
Maurice Wilkins wikipedia , lookup
Molecular evolution wikipedia , lookup
Gel electrophoresis of nucleic acids wikipedia , lookup
Non-coding DNA wikipedia , lookup
Nucleic acid analogue wikipedia , lookup
Evolution of metal ions in biological systems wikipedia , lookup
Genomic library wikipedia , lookup
Artificial gene synthesis wikipedia , lookup
DNA supercoil wikipedia , lookup
Biosynthesis wikipedia , lookup
Community fingerprinting wikipedia , lookup
Molecular cloning wikipedia , lookup
Cre-Lox recombination wikipedia , lookup
Transcript
Products 7-2011 Lab Times page 51 Product survey: Restriction enzymes Precision Cuts Though restriction-free cloning approaches, such as Gateway cloning and In-fusion cloning are gaining ground, classical cloning, based on restriction digestion and ligation of a DNA insert into a vector, is not past its prime and restriction enzymes are still amongst the most popular tools in life science labs. R estriction enzymes (restriction ally methylation) dependent type IV restricendonucleases), which were isolattion enzymes, which also cleave at variaed independently in the mid-1960s ble sites. Hence, only type II restriction enby Werner Arber and Matt Meselson, are zymes, which cleave both DNA strands at part of the bacterifixed positions (either inal restriction-modificaside or outside but close tion system (R-M systo the recognition site), tem) that destroys exare used by molecular ternal DNA, especially biologists for cloning. DNA originating from No wonder then that bacteriophages. The two 645 out of 647 commerkey players of the R-M cially available restricsystem are methyltranstion enzymes belong to ferases and restriction type II and only one to endonucleases. The fortype III and IV, respecmer recognise speciftively (REBASE doesn’t ic DNA sequences and Fast and accurate cuts like the ones list a single type I restrictransfer methyl groups from Edward Scissorhand. That’s tion enzyme under the to particular bases of what molecular biologists expect category “commercialthis site. Restriction en- from restriction enzymes. ly available”). Dependdonucleases that zero in ing on specific features, for the same recognition sequence cleave such as the nature of the recognition mothe DNA – but only if it is not methylated tif, the position of DNA cleavage or the rein either strand. Any external DNA that enquirement for co-factors, type II restriction ters the cell and lacks the methylation patenzymes are further categorised into the tern of the host DNA will thus be destroyed. subtypes A to T. Since the R-M system is vital for the deRelaxed specificity fence of prokaryotes against foreign DNA, Restriction enzymes are epitomes of restriction enzymes are found in almost any specificity. But even restriction enzymes bacteria and archaea. The restriction enwith an almost perfect sequence specificzyme database REBASE, run by restriction ity take it easy under certain conditions enzyme pioneer Richard Roberts and his and may cleave sequences that are simicolleagues, currently lists 246 type I, 3869 lar but not identical to the specific recognitype II, 34 type III and 12 type IV restriction sites. This so called ‘star activity’ may tion enzymes that have been biochemically be triggered by high glycerol contents, low characterised. But that’s only the tip of the ionic strength, high pH, organic solvents iceberg. The reinforced sequencing of bacor high enzyme to DNA ratios. Typical starterial and archaeal genomes has revealed prone restriction enzymes are, for example, thousands of putative restriction enzyme EcoRI, EcoRV, BamHI and KpnI. To avoid genes that need to be further investigated. unpredictable restriction products resultDifferent types ing from star activity, molecular biologists Not all restriction enzyme types are usually pay fastidous attention to restricsuitable for cloning. Type I enzymes, for tion buffers and reaction conditions. If this example, cleave the DNA at random spots doesn’t help, however, genetically engithat may be distant to the recognition site. neered restriction enzymes with reduced Type III enzymes usually cut approximatestar activity may be worth a try. ly 25 bases away from the recognition site Though restriction enzymes are mainand may not cut to completion. The same ly associated with cloning of DNA, some holds basically true for modification (usurecently discovered modification-depend- ent endonucleases grouped into type II and IV restriction enzymes, respectively, may also be used to investigate the epigenomes of higher organisms. Whereas methylation of DNA is employed by prokaryotes to protect their DNA from restriction enzymes, eukaryotes use the methylation of cytosine (5’-methylcytosine) as an epigenetic signal to regulate gene expression. Typical epigenetic markers found in eukaroytic DNA are, e.g., methylated CpG and CHG sites. Mapping methylation One of the major difficulties in epigenomics is to distinguish modified (methylated) from non-modified nucleotides. Modification-dependent restriction enzymes, such as the recently discovered type II M endonuclease MspJI, may be an alternative to existing (suboptimal) methods to identify methylated nucleotides. MspJI recognises 5’ methylated cytosines and cleaves on the 3’ side of the modified cytosine at a fixed distance. That’s a nice feature you might say but where is the connection to epigenetic research? The answer is quite obvious, if you imagine what happens to symmetrical modified sites, such as fully methylated CpG sites during MspJI restriction. Actually, MspJI cleaves on both 3’ sides and cuts out a DNA fragment with a fixed length (a 32-mer) that contains the CpG site in the centre of the fragment. The 32-mer-fragments may be isolated and sequenced to locate methylated cytosines (Cohen-Carni et al., 2011, PNAS 5;108(27):11040-5). Cohen-Carni et al. went even one step further. They digested the DNA with multiple MspJI-like enzymes with different recognition preferences and sequenced the resulting 32-mer pool to map the methylated sites of the genome. Considering the myriad of putative and yet-to-be discovered restriction enzymes, many more applications based on restriction enzymes are on the horizon. And one thing’s certain: the number of commercially available restriction enzymes listed by REBASE will continue to rise. Harald Zähringer page 52 Lab Times 7-2011 Products Restriction Enzymes Company/Distributor Restriction Enzyme Restriction Enzyme Type Miscellaneous, Specialities, Generally PvuRts1I Unique restriction enzyme to study 5’-hydroxymethylcytosine (5-hmC) within the genome — Allows direct discrimination between 5’-hydroxymethyl- Please inquire cytosine and 5’-methylcytosine DNA methylation — Specifically cleaves 5-hmC DNA — No digestion of 5-methylcytosine residues or unmethylated DNA — Recognises the sequence hmCN11-12/N9-10G BamHI Type II — Different buffers for double digestions 20.30 (7,500 units) HindIII Type II — Different buffers for double digestions 18.90 (2,000 units) EcoRI Type II — Different buffers for double digestions 12.60 (5,000 units) EcoRV Type II — Different buffers for double digestions 20.- (3,000 units) XbaI Type II — Different buffers for double digestions 20.- (3,500 units) FastDigest Restriction Enzymes Type II — — — — 176 FastDigest enzymes available 100% activity of all enzymes in one universal buffer Digestion in 5-15 min 100% buffer compatibility with downstream applications — Direct loading of reaction mixture on gels 23.- to 320.- Geneon FaiI YA^TR RT^AYRT^ — 500 units 890.- www.geneon.net Contact: Andreas Steinhoff Phone +49 621-5720864 [email protected] AbsI CC^TCGAGG GGAGCT^CC — 500 units 620.- HindIII A^AGCTT TTCGA^A — 10,000 units 45.- MspI C^CGG GGC^C — 5,000 units 75.- Common restriction enzymes for molecular biology 46 Type IIs — Concentration 10 μ/μl, BSA incl., compatible with other buffer systems 32.50 to 37.50 (S package) 130.- to 150.(L package) EcoRI -- -- 32.50 (15,000 units) 130.- (75,000 units) BamHI -- -- 32.50 (7,500 units) 130.- (37,500 units) NcoI -- -- 37.50 (600 units) 150.- (3,000 units) NotI -- -- 32.50 (300 units) 130.- (1,500 units) TaqI -- -- 32.50 (3,500 units) 130.- (17,500 units) 276 Type II restriction enzymes in total Type II Type IIG Type IIS Type IIB Homing- and Nicking Enzymes — Largest selection of restriction enzymes worldwide — 250 recombinant enzymes for excellent purity and highest stability — 1 buffer – 200 enzymes – 100% activity (NEBuffer 4) — 210 enzymes qualified for quick digestions within 15 min or less — 52 type IIS, 12 type IIG and 4 type IIB restriction enzymes Please request price list from your nearest NEB subsidiary or your local distributor Type II — High fidelity due to dramatically reduced star activity (up to 62,500 x in some cases) — Time-Saver qualified for quick digestion in 5 minutes — Optimised for highest buffer compatibility (NEBuffer 4) — Can be used together with NEB “Standard” enzymes Please request price list from your nearest NEB subsidiary or your local distributor Type II Type IV (McrBC) — Specificity to epigenetically-relevant DNA modifications (5-mC and 5-hmC) — 5 methylation-dependent restriction enzymes (DpnI, FspEI, LpnPI, McrBC, MspJI) / 3 methylationsensitive restriction enzymes (DpnII, HpaI, MspI) Please request price list from your nearest NEB subsidiary or your local distributor Active Motif La Hulpe, Belgium www.activemotif.com Contact: Jörg Plümpe Phone +32 2653-0001 [email protected] Bioron Ludwigshafen, Germany www.bioron.net Contact: Manuel Ammerschläger Phone +49 621 5720 915 [email protected] Thermo Fisher Scientific www.thermoscientific.com/ fermentas Contact: Anja Martin Phone +49 6227 356790 anja.martin@ thermofisher.com Metabion International Martinsried, Germany www.metabion.com Contact: Customer Service Phone +49 89 8993630 [email protected] New England Biolabs Frankfurt, Germany www.neb-online.de Contact: Phone 0800 246-5227 (DE) 00800/246-52277 (AT) [email protected] New England Biolabs France 23 High Fidelity restriction Evry, France enzymes www.neb-online.fr Contact: Phone 0800/100-623 [email protected] New England Biolabs (UK) Hitchin, United Kingdom www.neb.uk.com Contact: Phone 0800/318-486 [email protected] Restriction enzymes for epigenetic analysis Price [EUR] 7-2011 Products Lab Times page 53 Restriction Enzymes Company/Distributor Restriction Enzyme Promega www.promega.com Contact: Local branch or distributor EURx / Roboklon EURx: Gdansk, Poland www.eurx.com.pl Roboklon: Berlin, Germany www.roboklon.com Takara/Clontech Takara Bio Europe Saint-Germain-en-Laye, France www.takara-bio.eu Contact: Francois-Xavier Sicot Phone Europe: +33 1 3904 6880 Toll free phone: 0800 182 5178 (DE) 0808 234 8063 (UK) 0800 563 629 (CH) 0800 296 141 (AT) [email protected] Restriction Enzyme Type Miscellaneous, Specialities, Generally Price [EUR] Over 90 established restriction enzymes; find all restriction enzymes under www.promega.com/ restriction-enzymes -- — Native — Tested for fast digest — Blue/White cloning qualified See catalogue at www.promega.com or contact local branch AccI, AcvI, AluI, ApaI, AvaI, BalI, BamHI, BanII, BglI, BglII, BsiHKCI, BssHII, BstXI, BsuTUI, CviJI, CviJI*, DdeI, DpnI, DraI, EcoRI, EcoRV, FokI, HaeIII, HhaI, HincII, HindIII, HinfI, HpaI, HpaII, KpnI, MboI, MboII, MluI, MmeI, MnlI, MspI, NarI, NcoI, NdeI, NheI, NotI, NruI, NsiI, PinAI, PstI, PvuI, PvuII, RsaI, RsrII, SacI, SacII, SalI, Sau3AI, ScaI, SfiI, SinI, SmaI, SpeI, SphI, SspI, StuI, TaqI, TaqII, TspDTI, TspGWI, Tth111I, XbaI, XhoI 61 type II P (e.g. EcoRI) 7 type II S (e.g. FokI) 4 type II G (e.g. TspDT) 2 type II E (e.g. MboII) 1 type II M (DpnI) — — — — Enzyme dependent/ See webpage for details CviJI, CviJI* Type II P — Extremely short recognition sequences — CviJI and CviJI* cleave single- and double stranded DNA to short fragments (>15 - 20 bp) — Generate quasi-random cleaved DNA for shotgun cloning and generation of randomised libraries — For large-scale mapping or sequencing projects, utilising anonymous primers; high resolution mapping of short DNAs etc. From 0.90 / 1.18 per unit TaqII, TspDTI, TspGWI Type II S, Type II G — Cleave 5’-(11) and 3’-(9) downstream of the recognition sequence — For those cloning strategies that require preserving defined sequence stretches up to 11 bp length downstream of the recognition sequence — Thermostable From 0.65 / 1.30 / 1.30 per unit HpaII, MspI Type II P — MspI cuts both methylated and unmethylated CpG sites, the isoschizomer HpaII does not cut methylated DNA — Comparative restriction of eukaryotic DNA using both HpaII and MspI allows to determine whether a site is methylated From 0.013 / 0.006 per unit DpnI Type II P, Type II M — DpnI cleaves only methylated GATC sites — Digestion of methylated template DNA in site directed mutagenesis reactions From 0.054 per unit Restriction Enzyme Starter Box Set of 13 REs + buffers + rapid, — Supplies 13 of the most commonly used enzymes single solution ligation kit at an attractive price for testing Takara quality <Mighty Mix> + 100 bp DNA ladder in a convenient storage box On request Over 100 restriction endonucleases 19 unique ones Type II — All enzymes available with reaction buffer and loading dye — Most enzymes are available for custom, bulk and OEM supply On request 55 fast cutting restriction enzymes Type II — More than half of Takara REs have been shown to cut On request 1 μg DNA in 5 minutes in standard conditions — We do not charge more for faster digestion — Please request the list of fast cutting enzymes Epigenetic related restricted enzyme 43 CpG methylation-sensitive Type II; — Differential digestion of methylated DNA prior to Sensitive/insensitive pairs of Southern Blot or PCR analyses isoschizomer available (i.e. Hap II/ Msp I; Sau 3A I/Mbo I or Afa I/Rsa I) On request McrBC Specifically cleaves DNA containing methylcytosine preceded by a purine nucleotide (A or G) This enzyme can be used for: — DNA methylation analysis — qAMP: quantitative analysis of DNA methylation using real-time PCR — Enrichment of unmethylated DNA On request Dpn I Dpn I only cleaves when its site is methylated by dam methylase — Cuts DNA prepared from commonly used dam+ E. coli strains but not PCR products On request Highly pure Precise cutting activity Complete with 10x buffer, BSA and dilution buffer Perfectly compatible with enzymes and buffers from third parties — Fast digestion (“5 minutes digest”) at elevated restriction enzyme concentration is possible for most enzymes Six Pack: 6 different restriction enzymes (small sized packages) for 200.- (regardless of the original enzyme price) Double Six Pack: 12 different restriction enzymes (small sized packages) for the price of 10: 360.-