Download Your EasyGuide to DNA Polymerases

Your EasyGuide to DNA Polymerases
Contents.
Page.
Polymerase Chart
4-5
ACCUZYME DNA Polymerase
6
ACCUZYME Mix
AccuSure DNA Polymerase
Bioline
16 The Edge Business Centre
Humber Road
London NW2 6EW
United Kingdom
Bioline USA Inc
PMB 311
28 South Main Street
Randolph MA 02368
USA
Tel: +44 (0)20 8830 5300
Fax: +44 (0)20 8452 2822
Tel: 781 830 0360
Fax: 781 830 0205
7
AccuSure Mix
IMMOLASE™ DNA Polymerase
8
ImmoMix
ImmoMix Red
BIO-X-ACT Short DNA Polymerase
9
BIO-X-ACT Long DNA Polymerase
BIO-X-ACT Short Mix
BIO-X-ACT Long Mix
Bioline GmbH
Im Biotechnologiepark TGZ 2
D-14943 Luckenwalde
Germany
Bioline (Aust) Pty
PO Box 122
Alexandria NSW 1435
Australia
Tel: +49 (0)3371 681 229
Fax: +49 (0)3371 681 244
Tel: +61 (0)2 9209 4180
Fax: +61 (0)2 9209 4763
Diamond DNA Polymerase
10
BIOTAQ DNA Polymerase
11
BIOTAQ Red DNA Polymerase
BIOTAQ Core Kit
BioMix
BioMix Red
MangoTaq DNA Polymerase
12
PolyMate Additive
13
Polymerase Properties
14
Optimised Polymerase/dNTP Mixes
15
Welcome to the Bioline EasyGuide to Polymerases.
This guide aims to simplify your polymerase selection process.
Each polymerase has different characteristics and choosing
the enzyme that suits your individual experiment is crucial.
Bioline polymerases are available separately but are also
supplied as convenient ready-to-use 2x Reaction Mixes
containing the polymerase of choice, dNTPs, MgCl2, and
further additives to achieve optimal reaction conditions.
Note: Bioline polymerase mixes contain our in-house manufactured ultra-pure dNTPs.
Bioline >99% pure nucleotides help you to succeed in your experiments.
BIOLINE DNA Polymerases...
HIGH FIDELITY DNA POLYMERASES
Product
Relative accuracy to Taq
Applications
Advantages
ACCUZYME
+ Up to 47 Fold Greater
+ Assays requiring very high fidelity
+ Extremely high yield coupled with high fidelity
+ Gene cloning
+ Optimised buffer for high fidelity
+ Available heat-activated
ACCUZYME Mix
+ Up to 47 Fold Greater
+ Ready-to-use high fidelity mix
+ Just add template & primers
AccuSure
+ Up to 47 Fold Greater
+ Extremely high fidelity coupled with high specificity
+ Heat-activated for high specificity
+ DHPLC compatible
+ Robust performance
+ Applications requiring minimal handling
+ Ready-to-use master mix
Applications
Advantages
+ Assays requiring a heat-activated assay
+ Robust performance
AccuSure Mix
+ Up to 47 Fold Greater
Heat-activated POLYMERASES
Product
Relative accuracy to Taq
IMMOLASE™
+ Same as Taq
+ Highly suited for use in quantitative assays
ImmoMix/ImmoMix Red
+ Same as Taq
+ Assays requiring ready-to-use mix
+ Fewer handling steps
+ Stable at room temperature for prolonged periods
AccuSure
AccuSure Mix
4
+ Up to 47 Fold Greater
+ Assays requiring high fidelity and high specificity
+ Heat-activated with higher fidelity than Taq
+ Ready-to-use mix
POLYMERASE CHART
OUR GUIDE TO SOURCING THE PERFECT POLYMERASE FOR YOU
POLYMERASES FOR SPECIAL APPLICATIONS
Product
Relative accuracy to Taq
Applications
Advantages
BIO-X-ACT Short
+ Up to 17 Fold Greater
+ Low copy template
+ Suited to difficult templates <3Kb
+ Sensitive templates of <3Kb
+ Can process GC rich templates
+ Higher performance coupled with higher fidelity than Taq
BIO-X-ACT Short Mix
+ Up to 17 Fold Greater
+ Difficult templates requiring minimal handling
+ Ready-to-use mix
BIO-X-ACT Long
+ Up to 17 Fold Greater
+ For long and accurate assays of <20Kb genomic templates
+ Can process ≤20Kb genomic template
BIO-X-ACT Long Mix
+ Up to 17 Fold Greater
+ Assays requiring minimal handling
+ Ready-to-use mix
+ Suited to multiplex reactions
+ Extremely high specificity
+ Extremely high specificity
+ Suited to SNP detection, genotyping
+ Minimal optimisation required
Diamond
+ Same as Taq
+ Ideal for GC-rich templates
TAQ DNA POLYMERASES
Product
Applications
Advantages
BIOTAQ
+ Wide range of assays
+ Very robust, excellent price/performance
+ Can be used in conjunction with PolyMate Additive for enchanced results
BioMix
+ Same as BIOTAQ
+ Ready-to-use mix, Reduced risk of contamination
+ Can be stored at +4˚C for 1 month
MangoTaq
+ Any Taq based reactions
+ Direct loading onto agarose gels
+ Suited to high-throughput
+ Coloured reaction buffer for easy recognition.
5
ACCUZYME DNA Polymerase
ACCUZYME Mix
Features & Applications:
Features & Applications:
•
•
•
•
•
•
•
•
Extremely High fidelity
High yield
Processes fragments up to 5Kb
Produces blunt ends
Compatible with DHPLC columns (detergent free Buffer)
Convenient pre-mixed, pre-optimised solution
Same high fidelity as ACCUZYME
Suitable for high-throughput applications
Description:
Description:
ACCUZYME is a thermostable enzyme possessing 5’ - 3’ DNA
polymerase and 3’ - 5’ proof-reading exonuclease activities, offering
extremely high-fidelity (up to 47-fold higher fidelity than Taq).
ACCUZYME produces blunt-ended amplicons of up to 5Kb in length.
ACCUZYME is also available as a convenient 2x Reaction Mix to
maximise experiment reproducibility. ACCUZYME Mix contains
ACCUZYME DNA Polymerase, ultra-pure dNTPs manufactured by
Bioline and MgCl2. The Mix is optimised and ready-to-use: the user
adds only water, template and primers.
ACCUZYME is supplied with 10x buffer containing MgSO4, which
provides optimal final reaction conditions (2mM Mg2+) for most
experiments. In order to allow optimisation of reaction conditions,
additional MgCl2 is also provided.
ACCUZYME is supplied with an additional 50mM MgCl2 solution for
optional optimisation of reaction conditions.
ACCUZYME is optimised for use in a detergent-free system making it
ideally suited for direct loading of samples on to DHPLC columns.
6
PRODUCT
CONC.
PACK SIZE
CAT NO.
ACCUZYME
2.5u/µl
250 Units
BIO-21051
ACCUZYME
2.5u/µl
500 Units
BIO-21052
ACCUZYME Mix
2x
100 Reactions
BIO-25027
ACCUZYME Mix
2x
500 Reactions
BIO-25028
AccuSure DNA Polymerase
AccuSure Mix
Features & Applications:
Features & Applications:
•
•
•
•
•
•
•
•
•
Heat-activated
High fidelity
Processes fragments up to 5Kb
Produces blunt ends
Compatible with DHPLC columns (detergent free Buffer)
Convenient pre-mixed, pre-optimised solution
Same high-fidelity and specificity as ACCUZYME
Suitable for high-throughput applications
Compatible with DHPLC columns (detergent free Buffer)
Description:
Description:
AccuSure DNA polymerase possesses the same high fidelity as
ACCUZYME (page 5) but has the additional advantage of being
heat-activated, offering enhanced specificity. AccuSure is ideally
suited for difficult templates, such as DNA secondary structures and
microsatellites. AccuSure produces blunt-ended amplicons of up to
5Kb in length.
AccuSure is also available as a convenient 2x Reaction Mix to maximise
experiment reproducibility. AccuSure Mix contains AccuSure DNA
Polymerase, ultra-pure dNTPs manufactured by Bioline and MgCl2. The
Mix is optimised and ready-to-use so that the user adds only water,
template and primers.
AccuSure is optimised for use in a detergent-free system making it
ideally suited for direct loading of samples on to DHPLC columns.
AccuSure is supplied with an additional 50mM MgCl2 solution for
optional optimisation of reaction conditions.
AccuSure is also supplied with 10x buffer containing MgSO4 but, in
order to allow optimisation of reaction conditions, additional MgCl2 is
also provided.
PRODUCT
CONC.
PACK SIZE
CAT NO.
AccuSure
2.5u/µl
250 Units
BIO-21068
AccuSure
2.5u/µl
500 Units
BIO-21069
AccuSure Mix
2x
100 Reactions
BIO-25029
AccuSure Mix
2x
500 Reactions
BIO-25030
7
IMMOLASE™ DNA Polymerase
ImmoMix/ ImmoMix Red
Features & Applications:
ImmoMix/ImmoMix Red are complete ready-to-use heat-activated 2x
Reaction Mixes which only require the user to add water, template and
primers and then pre-heat to 95°C. Both mixes contain IMMOLASE™
DNA Polymerase, dNTPs, MgCl2 and further additives to achieve
optimal reaction conditions.
•
•
•
•
Heat-activated, inactive at room temperature
Highly suited to real-time assays
Ultra high specificity (eliminates non-specific
reaction products)
Leaves an ’A’ overhang
Activity
The illustration compares Taq during its active state
with a potential to extend wrongly annealed primers,
whereas IMMOLASE™ is still inactive.
Temperature (˚C)
IMMOLASE™ DNA Polymerase
Taq DNA Polymerase
Wrongly annealed primers could be
extended in this temperature range
Description:
IMMOLASE™ is a heatactivated thermostable DNA
polymerase isolated from a
novel organism. IMMOLASE™
provides improved specificity
when compared to standard
polymerases and can eliminate
the presence of non-specifics,
such as primer-dimers and misprimed products.
Activity
Activation profile for IMMOLASE™ DNA Polymerase
5
6
7
8
9
10
11
12
Time (minutes)
97˚C
95˚C
93˚C
8
Activation is most effective
at 95˚C for 7-10 minutes
IMMOLASE™ is inactive at
room temperature and must
therefore be activated by
heat treatment before primer
extension.
In addition, ImmoMix Red contains an additional non-toxic, nonhazardous red dye, allowing users to load samples directly onto a gel,
without the need to add loading buffer. Adequate mixing is also ensured
when reactions are set up.
PRODUCT
CONC.
PACK SIZE
CAT NO.
IMMOLASE™
5u/µl
250 Units
BIO-21046
IMMOLASE™
5u/µl
500 Units
BIO-21047
IMMOLASE™
5u/µl
5000 Units
BIO-21048
PRODUCT
CONC.
PACK SIZE
ImmoMix
2x
100 Reactions
BIO-25019
ImmoMix
2x
500 Reactions
BIO-25020
ImmoMix Red
2x
100 Reactions
BIO-25021
ImmoMix Red
2x
500 Reactions
BIO-25022
CAT NO.
BIO-X-ACT DNA Polymerases
BIO-X-ACT Short
Features & Applications:
This polymerase has been optimised to maximise its performance with
difficult templates up to 3Kb (genomic) or 5Kb (lambda DNA).
• Suited to problematic templates
such as GC-rich or dirty DNA
BIO-X-ACT Long
• Possesses 5’ - 3’ polymerase activity
and 3’ - 5’ proof-reading activity,
for enhanced fidelity
This polymerase has been optimised to maximise its performance with
long templates. BIO-X-ACT Long is suitable for genomic fragments
of ≤20Kb or ≤30Kb lambda fragments. BIO-X-ACT Long is our new
version of BIO-X-ACT DNA Polymerase.
Description:
BIO-X-ACT Short & Long Mixes
BIO-X-ACT Short and BIO-X-ACT Long are the polymerases of
choice for difficult applications that otherwise fail. BIO-X-ACT DNA
polymerases contain a proprietary mix of components, which can be
used on almost any template. In addition to spanning difficult regions
successfully, BIO-X-ACT polymerases offer >17-fold fidelity as
compared with Taq. To achieve an optimal pH stability in the reaction,
an additional buffer, OptiBuffer, is supplied. For enhanced specificity,
BIO-X-ACT polymerases may be used with 2x PolyMate Additive (not
supplied).
PRODUCT
Complete ready-to-use 2x Reaction Mixes, which only require the user
to add water, template and primers. The mixes contain BIO-X-ACT
(Short or Long) DNA Polymerase, dNTPs, MgCl2 and further additives to
achieve optimal reaction conditions.
CONC.
PACK SIZE
CAT NO.
PRODUCT
CONC.
PACK SIZE
CAT NO.
BIO-X-ACT Short
4u/µl
250 Units
BIO-21064
BIO-X-ACT Short
4u/µl
500 Units
BIO-21065
BIO-X-ACT Short Mix
2x
100 Reactions
BIO-25025
BIO-X-ACT Short Mix
2x
500 Reactions
BIO-X-ACT Long
4u/µl
250 Units
BIO-25026
BIO-21049
BIO-X-ACT Long Mix
2x
100 Reactions
BIO-X-ACT Long
4u/µl
500 Units
BIO-25023
BIO-21050
BIO-X-ACT Long Mix
2x
500 Reactions
BIO-25024
9
Diamond DNA Polymerase
Features & Applications:
•
•
•
•
•
Extremely high specificity (mis-matched primers not extended)
Ideal for regions such as high GC, inverted repeats,
microsatellites and complex secondary structures
Suited to multiplex, SNP detection, genotyping applications
High recognition for mismatches in allele-specific applications
Suitable for high-throughput and MALDI-TOF spectrometry use
Description:
Diamond DNA Polymerase is designed for difficult templates such as
high GC% regions and microsatellites. When templates are challenging,
the enzyme maintains excellent specificity and minimal background,
even in conditions such as high concentration of Mg2+, primers and
dNTPs. On genomic templates, Diamond can be used with MgCl2
concentrations as high as 10mM.
Diamond also extends through inverted tandem repeats and regions
with high amounts of secondary structure.
Diamond possesses improved nucleotide selection and a lower rate of
mis-match extension, such that only perfectly aligned primers will be
extended (see diagram).
PRODUCT
10
CONC.
PACK SIZE
CAT NO.
Diamond
5u/µl
250 Units
BIO-21058
Diamond
5u/µl
500 Units
BIO-21059
BIOTAQ/ BIOTAQ Red
BIOTAQ Core Kit
BioMix/ BioMix Red
Features & Applications:
Features & Applications:
• Robust performance in a wide
range of applications
• Leaves a ’A’ overhang
• Separate MgCl2 solution supplied
• Highly suited to high-throughput
applications
• Direct loading onto agarose gels
• Ideal for labs setting up new protocols
Both BIOTAQ and BIOTAQ Red are also
available as convenient ready-to-use 2x
Reaction Mixes, BioMix and BioMix Red
respectively. The user only needs to add
water, template and primers.
(BIOTAQ Red only)
• Easy visual recognition
(BIOTAQ Red only)
Description:
BIOTAQ/ BIOTAQ Red are highly purified
thermostable DNA polymerases offering a
very high yield over a wide range of templates
and they are the ideal choice for most assays.
Both BIOTAQ & BIOTAQ Red possess 5’-3’
exonuclease activity and leave an A’ overhang
such that the primer extension product is
suitable for effective integration into TA
cloning vectors.
BIOTAQ Red is an ideal choice for highthroughput applications, since it contains an
inert red dye to facilitate easy recognition.
When the reaction is complete, a sample
of the Reaction Mix can be loaded directly
onto agarose gels without the need for
loading buffer. This enzyme is supplied at a
concentration of 1u/µl.
Description:
The BIOTAQ Core Kit contains all the
individual reagents necessary to perform
polymerase assays on a wide range of
DNA templates. The Core Kit is based
on our widely used BIOTAQ, which offers
dependable high-performance in routine or
high-throughput assays. Additionally, the
kit includes dNTP mix, buffer, MgCl2 and
the special PolyMate Additive for difficult
reactions.
PRODUCT
CONCENTRATION
PACK SIZE
CAT NO.
BIOTAQ DNA Polymerase
5u/ul
500 Units
BIO-21040
BIOTAQ DNA Polymerase
5u/ul
2500 Units
BIO-21060
BIOTAQ Red DNA Polymerase
1u/µl
500 Units
BIO-21041
BIOTAQ Red DNA Polymerase
1u/µl
2500 Units
BIO-21061
BIOTAQ Core Kit
5u/µl
500 Units
BIO-21071
BioMix
2x
100 Reactions
BIO-25011
BioMix
2x
500 Reactions
BIO-25012
BioMix Red
2x
100 Reactions
BIO-25005
BioMix Red
2x
500 Reactions
BIO-25006
11
MangoTaq
Features & Applications:
• Robust performance in a wide range of applications
5,10,15 and 20µl of amplification reactions, in wells prior to electrophoresis
• 10x Reaction Buffer contains inert red and
orange dyes which allow direct gel-loading
• Leaves ‘A’ overhang
• Excellent price/performance
• Highly suited to high-throughput applications
• Separate MgCl2 supplied
Red and orange dyes after electrophoresis.
Above amplification reactions subjected to electroporesis.
Description:
MangoTaq is a new Bioline formulation of Taq DNA Polymerase, and is
supplied with a coloured 10x Reaction Buffer which contains two inert
dyes. The Red and Orange Dyes separate during gel electrophoresis
and provide quick reference points to monitor mobility of DNA samples
in the gel.
The Reaction Mixture containing the inert Red/Orange dyes can be
loaded directly onto an agarose gel for analysis, without the need for
separate gel-loading buffer.
12
MangoTaq offers consistent results across a wide range of DNA
templates and also leaves an ‘A’ overhang such that the primer-extension
product is suitable for effective integration into TA cloning vectors.
MangoTaq is an ideal choice for high-throughput applications, since it
combines a lower Taq concentration (1u/µl) with inert coloured dyes to
facilitate easy recognition.
PRODUCT
CONC.
PACK SIZE
CAT NO.
MangoTaq DNA Polymerase
1u/µl
1000 Units
BIO-21083
MangoTaq DNA Polymerase
1u/µl
2000 Units
BIO-21082
MangoTaq DNA Polymerase
1u/µl
5000 Units
BIO-21078
PolyMate Additive
Features & Applications:
PCR of a 201bp GC-Rich
fragment >66% from Human
TGF - ß gene
• Dramatically increases specificity and yield of any
thermostable DNA polymerase
Lane 1-3: With PolyMate &
1.5mM, 2.0mM & 2.5mM MgCl2
respectively
• Compatible with all commercially available
thermostable DNA polymerases
Lane 4: HyperLadder IV
• Ideal for “difficult” templates
1
2
3
4
5
6
7
Lane 5-7: Without PolyMate
or MgCl2
• Reduces smearing and background
• Developed to enhance the performance and specificity of any
thermostable DNA polymerase in enzyme reactions
Description:
PolyMate is a special 2x additive for use in reactions involving any
thermostable DNA polymerase. PolyMate provides an optimised
composition of reagents, leading to a dramatic improvement of reaction
specificity. By making DNA more accessible for polymerase and
oligonucleotides, PolyMate acts as a melting agent for dirty and/or
difficult templates, which have a high GC or AT content, repetitive
sequences, or difficult melting profiles. PolyMate is recommended for
use with any Bioline thermostable DNA polymerase.
PolyMate assayed with three
different polymerases on a
234bp GC-Rich fragment
>68% from Human ApoE gene
Lane 1: HyperLadder II
Lane 2: Treated with PolyMate
1
2
3
1
PolyMate does not contain magnesium, dNTPs, or buffer components.
In some cases it is necessary to optimise the Magnesium
concentration.
PRODUCT
Note:
PolyMate Additive
2
3
1
2
3
Lane 3: Without PolyMate
PACK SIZE
CAT NO.
2 x 1.2ml
BIO-37041
PolyMate should not be used in combination with any other additives for polymerase reactions.
13
Polymerase Properties
SUITABLE
TEMPLATE
LENGTH
HEAT
ACTIVATED
ACCUZYME
Up to 5Kb
NO
ACCUZYME Mix
Up to 5Kb
NO
AccuSure
Up to 5Kb
YES
AccuSure Mix
Up to 5Kb
YES
IMMOLASE
Up to 5Kb
YES
ImmoMix
Up to 5Kb
YES
BIO-X-ACT Short
Up to 3Kb
NO
BIO-X-ACT Short Mix
Up to 3Kb
NO
BIO-X-ACT Long
3 – 20Kb
NO
BIO-X-ACT Long Mix
3 – 20Kb
NO
Diamond
Up to 5Kb
NO
BIOTAQ
Up to 5Kb
NO
BioMix
Up to 5Kb
NO
MangoTaq
Up to 5Kb
NO
POLYMERASE
14
SENSITIVITY /
LOW COPY
TEMPLATES
SPECIFICITY
*
*
*
*
**
**
**
**
**
**
*
**
**
**
*
*
**
**
***
***
**
**
**
**
***
*
*
*
PROBLEM
TEMPLATES
*
*
*
*
*
*
***
***
***
***
***
FIDELITY
***
***
***
***
**
**
**
**
*
*
*
*
GC-RICH
TEMPLATES
3’ END
MODIFICATION
*
*
*
*
*
*
*
*
**
**
***
*
*
*
Blunt
Blunt
Blunt
Blunt
3’-dA
3’-dA
Mix (3’-dA + Blunt)
Mix (3’-dA + Blunt)
Mix (3’-dA + Blunt)
Mix (3’-dA + Blunt)
3’-dA
3’-dA
3’-dA
3’-dA
YIELD
**
**
**
**
***
***
***
***
**
**
*
***
***
**
Optimised Polymerase/dNTP Mixes
Most Bioline DNA polymerases are supplied as convenient polymerase/
dNTP/MgCl2 mixes. These are complete ready-to-use 2x Reaction
Mixes, which only require the user to add water, template and primers.
Using these optimised mixes, the amount of time needed to set up
reactions and the risk of contamination are dramatically reduced. The
number of pipetting steps is also minimised, which ensures greater
experiment reproducibility.
PRODUCT
CONC.
PACK SIZE
CAT NO.
2x BioMix
2x
100 Reactions
BIO-25011
2x BioMix
2x
500 Reactions
BIO-25012
2x BioMix Red
2x
100 Reactions
BIO-25005
2x BioMix Red
2x
500 Reactions
BIO-25006
2x ImmoMix
2x
100 Reactions
BIO-25019
2x ImmoMix
2x
500 Reactions
BIO-25020
2x ImmoMix Red
2x
100 Reactions
BIO-25021
2x ImmoMix Red
2x
500 Reactions
BIO-25022
2x BIO-X-ACT Short Mix
2x
100 Reactions
BIO-25025
2x BIO-X-ACT Short Mix
2x
500 Reactions
BIO-25026
2x BIO-X-ACT Long Mix
2x
100 Reactions
BIO-25023
2x BIO-X-ACT Long Mix
2x
500 Reactions
BIO-25024
2x ACCUZYME Mix
2x
100 Reactions
BIO-25027
2x ACCUZYME Mix
2x
500 Reactions
BIO-25028
2x AccuSure Mix
2x
100 Reactions
BIO-25029
2x AccuSure Mix
2x
500 Reactions
BIO-25030
•
Convenient, optimised 2x mixes
•
Time saving
•
Highly reproducible results
•
Risk of contamination reduced
15