Download 51 - Lab Times

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.-