Takara Mirus Bio

10.05

Whats inside:
Technical Article: The Future of High Fidelity Application Notes: Premix Ex Taq (Perfect Real Time) pCold TF Vector DICE Thermocycler and Ex Taq FAQ Mailbox Featured Products New Products Announcements
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The Future of High Fidelity

Overview of Polymerase Fidelity
By Julie Kramer, Marketing Manager, Takara Mirus Bio

PCR has become a basic laboratory procedure, being performed thousands of time each day in laboratories worldwide. Taq Polymerase was the first thermostable polymerase to be made available to researchers, and is still the most widely-used PCR enzyme. It is a highly processive enzyme, suitable for many routine PCR applications. However, Taqs performance is not adequate for other more demanding PCR applications, such as high-fidelity PCR, high-sensitivity PCR, or the synthesis of long or complex DNA targets. Importance of High Fidelity High polymerase fidelity (i.e. a low rate of base misincorporations, or errors) is most important in PCR applications where downstream sequencing or gene expression of the amplified product is desired. It is also significant in applications requiring amplification of low-copy-number templates (requiring many rounds of amplification), longer target sequences, or amplification and rare transcripts or allelic mutants. cDNA library construction, site directed mutagenesis, and mutation detection are also particularly sensitive to error rate. Enzyme fidelity can by influenced by a variety of factors, including template sequence (i.e. GC-rich templates generally increased error rates), cycling parameters, and reaction conditions (i.e. pH, Mg2+, dNTP concentration). However, in controlled studies, polymerases exhibit characteristic rates of base misincorporations, rates of extension from those misincorporations, and 3' 5' exonuclease or proofreading activity. These factors together result in an intrinsic error rate for each polymerase.

Polymerase Fidelity

Taq polymerase and related Thermus family polymerases generally possess a high rate of base misincorporations, a low rate of extension from these misincorporations, and lack a 3' 5' exonuclease or proofreading function. Their error rates are the highest among the most widely-studied viral and bacterial polymerases. Additionally, the low extension rate actually acts somewhat as a de facto proofreading function, as incorrect templates fall out of the amplifiable pool. However, this results in lower yield and sensitivity, particularly on longer products.
Using conventional mutant-based fidelity assays, the recorded error rates of about 10-4 are common for Taq. This number may seem low, but this means that after one fairly typical 106 fold PCR amplification of a 200 bp target, up to 33% of the resulting products may contain errors.
The expected fraction of PCRinduced mutants can be calculated according to the following formula:

Technical Article

Mixing a proofreading polymerase with Taq polymerase has been shown to increase amplification performance, and is the basis for several widelyused enzymes, including Takara Ex Taq and LA Taq. These blends provide superior amplification efficiency and product length as compared to Taq or the proofreader alone. Fidelity is also much improved over Taq Polymerase alone, but may still cause problems in some applications. Calculated Error Rate Error rate and fidelity are calculated via the following formulas: Error Rate= # misincorporated bases/# bases synthesized Fidelity= 1/error rate Most quoted error rates are experimentally based on indirect phenotypic measurements of mutant frequency, and vary widely. For example, one common method calculates the frequency of observed mutants by identifying the number of phenotypically altered colonies following bacterial transformation with a PCR-amplified DNA fragment. However, lethal amino acid substitutions derived from misincorporation of one or more incorrect bases during the PCR reaction will go unnoticed and uncounted, as they result in cell death. Fidelity rates calculated via this method are also subject to an additional level of inaccuracy because some nucleotide changes will not result in clear phenotypic changes of the expressed protein (usually beta-galactosidase). Therefore, these conventional methods of calculating error rates can provide useful comparisons within a single set of reaction conditions, but the actual results may vary widely from expected numbers.

F(>1) = 1- e-bfd b= length of target sequence f= error rate d=number of doublings

Pyrococcus sp. polymerases (also called proofreading polymerases) have an even higher initial misincorporation rate than Taq, but because they contain a 3' to 5' exonuclease activity, they generally possess much lower error rates than Taq Polymerase or other Thermus-family polymerases. However, these enzymes often display low processivity, resulting in low product yield, reduced product length, and difficulties in optimization.

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For information about Takaras PrimeSTAR HS see page 14

Takara Bio recently introduced PrimeSTAR HS DNA Polymerase, a novel new DNA polymerase which offers very high fidelity as well as excellent amplification efficiency and extended product length (8.5 kb for human genomic DNA; 22 kb for DNA). PrimeSTAR is the only currently available DNA polymerase whose error rate (only 12 errors per 250,000 bases) is determined by DNA sequencing. PrimeSTAR HS PrimeSTAR HS is a recombinant enzyme expressed in E. coli. It was derived from a proprietary thermostable bacterial strain, and was chosen by Takara after studying a panel of bacterial strains which had been identified as potential producers of high fidelity polymerases. It has a very strong 3' 5' exonuclease activity, high replication accuracy, and extremely high priming efficiency. It also contains an antibody which inactivates both the polymerase and exonuclease functions during reaction assembly. This prevents false initiation events due to mispriming or primer digestion, resulting in lowered background and increased reproducibility. Takaras fidelity assay for PrimeSTAR HS is as follows: Eight arbitrarily selected GC-rich regions were amplified with PrimeSTAR HS and other enzymes, using Thermus thermophilus HB8 genomic DNA as a template. Each product (approx. 500 bp each) was then cloned into a suitable plasmid. Multiple clones were selected and subjected to sequence analysis. Sequence analysis of DNA fragments amplified using PrimeSTAR HS demonstrated only 12 mismatched bases per 249,941 total bases. This is higher fidelity than Thermococcus kodakaraensis DNA Polymerase (KOD), Pfu, and 10X higher fidelity than Taq DNA polymerase. Additionally, when compared with other commercially available high fidelity enzymes, PrimeSTAR HS demonstrates superior efficiency on both a 500 bp Thermus and 2 kb human genomic DNA fragments (see page 14). Thus, PrimeSTAR HS answers the need for convenient, robust, easy-to-optimize high-fidelity DNA polymerases, and offers the method of choice for superior highfidelity PCR results.

References:
(1) Cha, R.; Thilly, W. in PCR Primer, A Laboratory Manual, 1995, 34-51. (2) Keohavong, P.; and Thilly, W. Proc. Natl. Acad. Sci. USA, 1989, 86:9253-9257. (3) Pavlov, R.; et. al. TRENDS in Biotechnology, 2004, 22:254-261. (4) Barnes, W. Proc. Natl. Acad. USA, 1994, 91:2216-220.

Technical Article

PrimeSTAR Fidelity Comparison with Other DNA Polymerases and Taq.

PrimeSTAR HS DNA Polymerase

Thermococcus kodakaraensis - derived DNA Polymerase Pyrococcus sp. - derived DNA Polymerase

Thermus aquaticus - derived DNA Polymerase

0 0.01 0.02 0.03 0.04 0.05

Fidelity comparison with competitors sequencing results showed only 12/249,941 mismatched bases in DNA fragments amplified using PrimeSTAR HS.

PrimeSTAR HS Offers: High Accuracy: A strong exonuclease activity, resulting in an extremely low error rate of only 12 errors per 250,000 bp. High Efficiency: Amplification efficiency higher than Taq Polymerase; excellent performance even on GC-rich templates. Robust Amplification: Tolerance to varying reaction conditions means a single PCR cycling protocol can be used to amplify products of varying sizes. Extended Product Length: Amplify targets of up to 8.5 kb on human genomic DNA; 10 kb on E. coli genomic DNA; and 22 kb on lambda DNA. Fast Reaction Times: Increased priming efficiency results in rapid annealing times and improved specificity. High Specificity: Antibody-mediated hot-start prevents false initiation events during reaction assembly due to mispriming or primer digestion.

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Comparison of Takaras New Premix Ex Taq (Perfect Real Time) versus Competing qPCR Kits
Introduction Real Time PCR offers a quantitative method to study product amounts during the early stages of a PCR reaction, when the amount of product corresponds to the amount of initial template present in the reaction. Premix Ex Taq (Perfect Real Time) is a 2X premix, specially designed for high-speed, high sensitivity real time PCR using either detection probes (i.e. TaqMan and other molecular probe technologies) or SYBR Green I (not included). This premix combines high-performance Takara Ex Taq Hot Start DNA Polymerase, which uses antibody-mediated Hot Start technology to prevent non-specific amplification, with an optimized real time PCR buffer formulation which provides increased amplification efficiency and further improved specificity for high speed real time PCR. Premix Ex Taq reactions are fast and easy, and generate exceptional PCR results on all major realtime instruments. The amplification curves generated with Premix Ex Taq demonstrate excellent sensitivity and uniformity. With the ABI Kit, only one curve appears, indicating sensitivity several logs lower than Takara s. Both the Invitrogen and Qiagen Kits resulted in amplification curves which are shifted to the right, indicating lower amplification efficiency than the Takara Kit. The curves lack uniformity when compared to Takaras Kit, indicating lower reproducibility. Additionally, reactions with Takara Premix Ex Taq can be completed in as little as 50 minutes, making it compatible with fast PCR systems. In summary, Takara Premix Ex Taq provides superior efficiency and reproducibility as compared to other leading qPCR kits.

Application Note

Features of Premix Ex Taq (Perfect Real Time) Include: Materials and Methods qPCR amplification of the human ACTB gene was performed using cDNA templates corresponding to 100 ng-1 pg of total RNA using TaqMan probes (ABI) and Premix Ex Taq (Perfect Real Time). These results were compared against results generated using three different leading competitor qPCR kits on the Roche LightCycler. Reactions were performed according to the manufacturers protocols.
Fast: Reaction can be completed in as little as 50

minutes.
High Sensitivity: Detects as few as 10 template

copies.
Versatility: Compatible with Smart Cycler,

LightCycler, ABI PRISM 7000/7700/7900 HT, Applied Biosystems 7500 Real-Time PCR Systems, and other real time PCR instruments.
Wide Dynamic Range: Possesses a dynamic

Results and Discussion Amplification efficiency was determined using Premix Ex Taq (Perfect Real Time) and three leading competitor qPCR enzymes on the Roche LightCycler. The results can be seen in the figure on the next page.

range of 10 orders of magnitude ( DNA template).

Convenient: Supplied as a premix formulation; two

ROX reference dyes are supplied separately.

Ordering Information
Product No. TAK RR039A TAK RR039B TAK RR041A TAK RR041B TAK RR031A TAK RR031B Product Name Premix Ex Taq (Perfect Real Time) DNA Polymerase Premix Ex Taq (Perfect Real Time) DNA Polymerase SYBR Premix Ex Taq (Perfect Real Time) SYBR Premix Ex Taq (Perfect Real Time) Ex Taq R-PCR, Version 2.1 Ex Taq R-PCR, Version 2.1 Quantity 200 reactions 400 reactions 200 reactions 400 reactions 250 U 1000 U Price $219 $437 $273 $534 $189 $666

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For additional information about Takaras qPCR products see pages 12-13

Takara SYBR Premix Ex Taq

ABIs TaqMan Universal PCR Master Mix

Application Note

Invitrogens Platinum Quantitative PCR Supermix -UDG

Qiagens Quantitect Probe PCR Kit

Figure 1:

Performance of SYBR Premix Ex Taq (Perfect Real Time) vs. ABIs TaqMan Universal PCR Master Mix using a Roche LightCycler. Cycling conditions: 95C, 2 min} 1 cycle 94C, 15 sec. 55C, 1 min. 72C,1 min. 45 cycles

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The pCold TF Protein Expression System Produces Soluble, Active Protein in E. coli
Elucidation of protein structure and function maintains an important role in post-genomic sequencing and analysis studies. An efficient protein production system is critical for obtaining large amounts of correctly folded recombinant protein for study. E. coli expression systems are used extensively for production of recombinant proteins, and have two major advantages over other expression systems: (1) ease of use, and (2) low cost. However, some recombinant proteins do not fold correctly during expression in E. coli, resulting in deposits of inactive insoluble protein termed "inclusion bodies". a 5' untranslated region (5' UTR), a translation enhancing element (TEE), a His-Tag sequence, and a multicloning site (MCS). A lac operator is inserted downstream of the cspA promoter to ensure strict regulation of expression. Additionally, recognition sites for HRV 3C Protease, Thrombin, and Factor Xa are located between the TF-Tag and the multiple cloning site (MCS) and function to facilitate tag removal from the expressed fusion protein. Most E. coli strains can serve as expression hosts. pCold TF DNA Vector combines high-yield cold shock expression technology with Trigger Factor (chaperone) expression in a single vector to facilitate correct protein folding, thus enabling efficient soluble protein production for otherwise intractable target proteins. The following experiment compares results generated using pCold I; pCold I co-expressed with a Chaperone Plasmid; pCold TF; and T7 promoter constructs to express various proteins.

Application Note

Series of pCold Vectors In collaboration with Prof. Masayori Inouye (University of Medicine and Dentistry of New Jersey), Takara Bio has developed the pCold DNA Vectors, a series of novel protein expression vectors. The pCold Vectors provide increased in vivo protein yield, purity, and solubility of expressed recombinant proteins using "cold shock" technology. More specifically, the cspA (cold shock protein A) promoter and related elements have been incorporated into these vectors to upregulate target protein production at lowered incubation temperatures (37C-15C). This temperature drop also suppresses expression of other cellular proteins, represses protease activity, and temporarily halts overall cell growth. This process allows expression of target proteins at high yield, high purity (up to 60% of cellular protein), and increased solubility as compared with conventional E. coli expression systems. Co-expression of one or more chaperone proteins during expression of a heterologous target protein has proven effective for obtaining increased amounts of soluble recombinant protein (see Takara's Chaperone Plasmid Set (TAK 3340)). This procedure, though, lacks the convenience of a single transformation step.

Materials and Methods pCold DNA I and pCold TF DNA cloning and expression procedures* were conducted as follows: 1) Insert the target gene to the multicloning site of the pCold DNA vector for expression. 2) Transform the E.coli host strain (e.g. BL21) with the expression plasmid, and select for ampr transformants. 3) Inoculate the transformants into medium including 50 g/ml of ampicillin, and culture with shaking at 37C. 4) At OD600= 0.4 - 0.5, refrigerate the culture at 15C (without shaking) for 30 minutes. 5) Add IPTG to a final concentration of 0.1- 1.0 mM, and continue the culture with shaking at 15C for 24 hours. 6) Collect the cells, and confirm the expression of the target protein with SDS-PAGE in soluble and insoluble fractions or activity assay. Expression from T7 promoter-driven vectors was performed using a standard protocol utilizing IPTG induction and subsequent culturing at 37C. *Cultivation/induction conditions (culture medium, aeration, timing of induction, concentration of inducer, cultivation time after induction) should be optimized for each target protein. 6

pCold TF Vectors Takara's pCold TF DNA Vector is a fusion cold shock expression vector that expresses a molecular chaperone (Trigger Factor (TF)) as a soluble tag. Trigger Factor is a prokaryotic ribosome-associated chaperone protein (48 kDa) which facilitates co-translational folding of newly expressed polypeptides. Because of its E. coli origin, TF is highly expressed in E. coli expression systems. The pCold TF DNA Vector consists of the cspA (cold shock) promoter plus additional downstream sequences including

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kDa

pCold TF 1 2

pCold 1 2

pCold + Chaperone 1 2

T7 1 2 1. Cell extract solution 2. Soluble fraction target protein co-expressed trigger factor
kDa 97 66 45 31 22

Trx

GST

Nus

1 2 3 1 2 3 1 2 3

pCold pCold I pCold + Chaperone TF 1 2 3 1 2 3 1 2 3

Application Note

97 66 45 31 22

1. Cell extract solution 2. Soluble fraction 3. Insoluble fraction

Figure 1: Expression of Soluble Protein A Using the pCold TF Expression System

Figure 2: Increased Expression of Soluble Protein B Using the pCold Expression System

Results and Discussion Protein expression using the pCold TF Expression Vector was compared with protein expression using (1) the pCold DNA I Vector alone, (2) co-expression using the pCold DNA I Vector with Takara's Chaperone Plasmid pTf16, and (3) a T7 promoter expression system which included other tags for solubilization. Figure 1 demonstrates the successful production of enzyme protein A using the pCold TF system. Expression of this protein, with an estimated molecular weight of 29 kDa, was not seen as an exact band with either the T7 expression system or even with pCold I (either individual expression or chaperone co-expression). However, the expression of the target protein and target plus tag (29 kDa and 52 kDa) was observed using pCold TF, and most of the obtained protein was in soluble form. Subsequent assays confirmed that the expressed enzyme A retains activity even as a fusion protein. Figure 2 demonstrates improved levels of soluble protein B using pCold TF. Expression of soluble enzyme protein B (M.W: ~63 kDa) was not observed using either pCold DNA I alone or pCold I co-expressed with chaperone proteins, nor with a T7 expression vector that included other tags for solubilization (Trx Tag [~12 kDa], Nus Tag [~55 kDa], and GST Tag [~26 kDa]). However, when the pCold TF DNA Vector was used, the target protein was present at an expression level much higher than with other systems and tags, and most of the expressed target protein was observed in the soluble fraction. (Note: The molecular weight of the target protein is larger than its actual size and varies due to fused expression with different tags). In summary, the pCold TF expression system offers a convenient high yield, high purity alternative for efficient soluble protein expression of otherwise intractable target proteins.

Ordering Information
Catalog No. TAK 3365 Product Name pCold TF Vector Quantity 25 g Price $727

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 FAQ Mailbox
PrimeSTAR HS DNA Polymerase FAQ
Can PrimeSTAR HS reactions use the same PCR cycling conditions that are used with Taq Polymerase?
PrimeSTAR HS cannot use the same PCR cycling conditions that are used with Taq Polymerase. Takara strongly recommends following the conditions described in the PrimeSTAR HS product protocol, since the characteristics of this enzyme are very different from those of Taq Polymerase. Takara recommends the following initial cycle protocol for primers with a Tm of >55C: denaturing step, 98C, 10 sec; annealing step 55C, 5 sec.; extension step, 72C, 1 min./kb; for 30 cycles.

What is the composition of PrimeSTAR HS Buffer (Mg2+)?

The PrimeSTAR HS Buffer composition is proprietary.

FAQ Mailbox

Why does PrimeSTAR HS use a 5X concentration buffer?

A 5X concentration buffer was determined to provide the best optimization for this reaction system.

How does PrimeSTAR HS differ from KOD Hot Start?

PrimeSTAR HS provides higher fidelity than KOD Hot Start while offering the same level of amplification efficiency.

What is the basis of PrimeSTAR HSs antibodymediated Hot Start Technology?

PrimeSTAR HSs Hot Start Technology uses a single monoclonal antibody which blocks both PrimeSTARs polymerase and nuclease activities.

What is the source of PrimeSTAR HS DNA polymerase? Is it a cloned enzyme?

PrimeSTAR HS is a recombinant enzyme that is expressed in E. coli. It was derived from a proprietary thermostable bacterial strain chosen by Takara after studying various strains that were identified as producing high fidelity enzymes. PrimeSTAR HS was not obtained from the same bacterial strain that was used to produce KOD (Pfx).

What advantage is offered by Takaras measurement of PrimeSTAR HSs fidelity using sequence analysis?
Simple comparison of the fidelity rates available for different PCR enzymes is not possible due to the multitude of different fidelity measurement techniques employed by different manufacturers. Takara Bio has determined PrimeSTARs error rate based upon genotype, that is, the error rate as determined by actual sequence analysis. The method Takara Bio used to obtain their fidelity data is presented below: Eight arbitrarily selected GC-rich regions were amplified with PrimeSTAR HS and other enzymes, using the Thermus thermophilus HB8 genomic DNA as a template. Each PCR product (approx. 500 bp each) was cloned into a suitable plasmid. For each different DNA region cloned, multiple clones were picked, and subjected to sequence analysis. Sequence analysis results of DNA fragments amplified using PrimeSTAR HS demonstrated only 12 mismatched bases per 249,941 total bases. This data confirms PrimeSTAR HSs extremely high fidelity, with a calculated error frequency of only 0.0048%.

Are PrimeSTAR HS PCR products suitable for TA cloning?

PCR products cannot be used directly for TA cloning. The termini are blunt-ended due to the 3' 5' exonuclease activity of this enzyme. PrimeSTAR HS PCR products should be used for blunt-end cloning. Takara recommends use of a dephosphorylated vector and phosphorylated PCR products. Products can be enzymatically phosphorylated or made using PCR primers possessing phosphoric acid residues at their 5' termini.

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SYBR Premix Ex Taq (Perfect Real Time) FAQ

How many reactions (points) are recommended for a typical standard curve?
Generally 5 or 6 reactions (5 or 6 points) are used to establish the standard curve, plus dH2O for a negative control. Takara has used cDNAs which corresponded to 1 pg, 10 pg, 100 pg, 1 ng, 10 ng and 100 ng of mouse liver total RNA respectively (and dH2O for negative control). If possible, establish the standard curve within a Ct range of ~ 15-35. The Ct(cycle threshold) is the number of cycles at which fluorescence intensity is measureable above background levels (i.e. threshold line), and is set in the exponential amplification phase to allow the most accurate reading. eral minutes, protected from light, or by warming with your hands, followed by gentle mixing. Do not vortex!! We have verified that this product shows good performance after the precipitate is dissolved completely.

What is the composition of SYBR Premix Ex Taq?

The Premix contains Ex Taq Hot Start DNA Polymerase, buffer, dNTP mix, Mg2+ and SYBR Green I. The Mg2+ and SYBR Green I concentrations are proprietary.

FAQ Mailbox

What is the purpose of the ROX reference dye included with the SYBR Premix Ex Taq?
ROX (i.e. Carboxy-X-Rhodamine) is a convenient internal reference standard for use in normalizing signals due to non-PCR related fluorescence fluctuations that occur either between wells or over time. Please note that two types of ROX Reference Dye (Original Version ROX and ROX II) are supplied with this product. Use the Original Version ROX for normalization with the ABI PRISM 7000/7700/7900HT and Applied Biosystems 7300 Real-Time PCR Systems. Use ROX II reference dye for normalization whith Applied biosystems 7500 Real-Time PCR System.

How do I determine the number of qPCR reactions for my experiments? For example, if I have two different cell lines and want to characterize three different genes in each?
For each of the 3 genes, a standard curve (e.g. composed of 7 data points) plus 2 experimental samples that are run in triplicate are performed. Therefore, 3(triplicate) x (7 pts + 2 samples) x 3(genes) = 81 reactions are required for 3 genes. One package of SYBR Premix Ex Taq (Perfect Real Time) contains sufficient reagent for 200 reactions (50 l reaction volume).

What PCR product size is optimal for real time PCR?

A size range of 80-150 bp is generally recommended for qPCR amplification, although sizes up to 300 bp are possible.

Can you make a master mix of the ROX reference dyes and SYBR Premix Ex Taq to help avoid pipetting errors?
Original Version ROX Reference Dye can be premixed. Add 40 l of ROX to 1 ml of SYBR Premix Ex Taq. Store this solution at 4C (protected from light), and use within one month for best performance. ROX Reference Dye II should not be premixed prior to reaction assembly.

Can SYBR Green I dye precipitate? Is there a good way to resuspend SYBR Green I?
A greenish-yellow precipitate can sometimes be observed in SYBR Premix Ex Taq when stored at -20C. If this occurs, dissolve the precipitate completely by letting the tube stand at room temperature for sev-

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Highly Efficient Gradient PCR of an 8 kb DNA Fragment using Takara Ex Taq DNA Polymerase and the Takara PCR Dice Thermocycler
Introduction:
Takaras Ex Taq DNA Polymerase is a polymerase mix which offers high amplification efficiency (up to 100X Taq), superior reproducibility, and extended product length (to 30 kb on DNA). In both protocols, robust, reproducible amplification was apparent. These results demonstrate that Takaras Ex Taq DNA Polymerase and Dice Thermocycler can be combined to produce easy optimization and superior reproducibility and yield even with challenging DNA fragment lengths.
M 52C 72C M

Application Note

The Takara Thermocycler Dice provides convenience, reliability, multifunctionality, and a gradient feature to select temperature conditions at each step, and program a temperature gradient up to 20C across the rows of a sample block. This allows optimization of reaction conditions in a single experiment. The following experiment demonstrates optimization of the PCR conditions for an 8 kb DNA fragment amplified with Ex Taq DNA Polymerase on the PCR Thermocycler Dice.

Materials and Methods:

50 L amplification reactions using 1.25 U of Ex Taq DNA Polymerase and 0.5 g of DNA were assembled according to manufacturers instructions. Half of the reactions were amplified using shuttle PCR under the following conditions: 98C 10 sec 52 to 72C 5 min 30 cycles (Gradient temperature range: 20C) The remainder were amplified using the following threestep protocol: 98C 10 sec 30 cycles
Figure 1: Evaluation of annealing/extension temperatures in two-step gradient PCR. Higher annealing/extension temperatures reduced amplification of low molecular weight non-specific products, and increased target yield. M 45C 65C M

45 to 65C 30 sec 72C 5 min

(Gradient temperature range for annealing step: 20C)

Results and Discussion:

In the two-step protocol (Figure 1), higher annealing/extension temperatures reduced the amplification of low molecular weight non-specific products, and increased the yield of the target product. In the three-step protocol (Figure 2), higher annealing temperatures reduced the amplification of low molecular weight non-specific products.

Figure 2: Evaluation of annealing temperatures in three-step gradient PCR. Higher annealing temperatures reduced the amplification of non-specific products.

Ordering Information
Product No. TAK RR001A TAK RR001B TAK RR001C Product Name Ex Taq DNA Polymerase Ex Taq DNA Polymerase Ex Taq DNA Polymerase Quantity 250U 1000U 3000U Price $158 $567 $1483

To order the Takara Thermocycler Dice (TP600) please contact Sanyo Biomedical at 905-760-4049 (Canada) or Fisher Scientific at 800-766-7000 (USA).

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Takara Mirus Bio announces the re-launching of our website at www.takaramirusbio.com. The
upgraded site features increased technical content (FAQs, reference lists, product manuals, articles, etc.), easier navigation, and two Resource Centers providing convenient, centralized pages from which to access technical information on our PCR and Protein Expression and Folding product lines.

Please check out our site and as you browse, fill out the Treasure Map below to win a FREE Takara baseball cap!
Offer good while supplies last.

Treasure Map
1.
What is the length of genomic DNA that LA Taq DNA Polymerase can amplify? How many reactions can you run with the DNA Ligation Kit, Mighty Mix?

Treasure Map

2.

How many Resource Centers are on the website?

3.

4.

What is the fidelity of Ex Taq DNA Polymerase?

7.

Can SYBR Premix Ex Taq (Perfect Real Time) be used on the MJ Opticon instrument?

5. 6.

What technology do the pCold Vectors utilize?

8.

What is the cell transduction efficiency of cell line K-562 on Retronectin?

How many plasmids are included in the Chaperone Plasmid Set?

9.

What is the assay range of the Procollagen Type 1 C-Peptide EIA Kit?

10.

What does CA stand for in the Refolding CA Kit?

Fax your answers to 608-441-2845 Fill in the above Name________________________________________ Treasure Map and Sorry! This promotion has ended. win a FREE baseball cap! E-mail_______________________________________
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Featured Products
SYBR Premix Ex Taq (Perfect Real Time)
For superior real time PCR using SYBR Green I
Features
High sensitivity: Detects as few as 100 copies. Versatility: Compatible with most qPCR instruments.
Amplification Curve (upper panel) and Melting Curve (lower panel) for SYBR Premix Ex Taq (Perfect Real Time) on the SmartCycler (Cepheid).

Featured Products

Wide dynamic range: Possesses a dynamic range of 7-8 orders of magnitude ( DNA template). High speed capability: Optimized buffer system allows quick reaction times. Convenient: Separate tubes of ROX reference dye are supplied. SYBR Premix Ex Taq (Perfect Real Time) is a convenient (2X) premix consisting of Takara's high sensitivity, high efficiency Ex Taq Hot Start DNA Polymerase, SYBR Green I, dNTPs, and an optimized real time buffer which provides superior specificity and increased amplification efficiency for real time PCR, plus separate tubes of ROX Reference Dye. Antibody-mediated hot start technology is used for reduced background.

Ordering Information
Catalog No. TAK RR041A TAK RR041B Product Name SYBR Premix Ex Taq SYBR Premix Ex Taq Quantity 200 reactions 400 reactions Price $273 $534

Premix Ex Taq (Perfect Real Time)

For excellent real time PCR using probe detection
Features
High sensitivity: Detects as few as 10 template copies (probe detection). Versatility: Compatible with most qPCR instruments. Wide dynamic range: Possesses a dynamic range of 10 orders of magnitude ( DNA template). High speed capability: Optimized buffer system allows quick reaction times. Premix Ex Taq (Perfect Real Time) is a 2X premix specially designed for high speed, high sensitivity real time PCR using either detection probes or SYBR Green I (not included). It consists of Takaras high sensitivity, high efficiency Ex Taq Hot Start DNA Polymerase, and an optimized real time buffer which provides superior specificity and increased amplification efficiency for real time PCR. Antibody-mediated hot start technology prevents nonspecific amplification due to mispriming and/or formation of primer dimers during the reaction assembly. The Taq antibody-polymerase complex is denatured in the first cycling step, releasing the polymerase and allowing DNA synthesis to proceed.

Amplification curve for Premix Ex Taq (Perfect Real Time) using the TaqMan Gene Expression Assay on the Applied Biosystems 7500 Real-Time PCR System.

Ordering Information
Catalog No. TAK RR039A TAK RR039B Product Name Premix Ex Taq Premix Ex Taq Quantity 200 reactions 400 reactions Price $219 $437

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Ex TaqTM R-PCR, Version 2.1

For high specificity qPCR
Features
High sensitivity: Detect as few as 10 copies. Superior specificity: Hot-Start formulation prevents mispriming and lowers nonspecific amplification products. Wide dynamic range: Dynamic range of 10 orders of magnitude using probe detection and 7-8 orders of magnitude for SYBR Green I. Flexibility: Use with both SYBR Green I and detection probes such as TaqMan. The Ex Taq R-PCR Version 2.1 is an enzyme-buffer system developed specifically for real time PCR on the Cepheid SmartCycler. This product combines the high performance of Ex Taq Hot-Start Polymerase with an optimized R-PCR buffer for high specificity.

Ex Taq R-PCR Version 2.1 offers the excellent specificity, sensitivity, and low background required for superior real time PCR.

Featured Products

Ex Taq R-PCR Version 2.1 contains a monoclonal antibody to Taq Polymerase, which binds to the polymerase until the temperature is elevated. The binding of this antibody prevents nonspecific amplification due to mispriming and/or formation of primer dimers during reaction assembly. An optimized buffer formulation provides increased specificity for real time PCR, further enhancing performance.

Ordering Information
Catalog No. TAK RR031A TAK RR031B Product Name Ex Taq R-PCR, Ver 2.1 Ex Taq R-PCR, Ver 2.1 Quantity 250 Units 1000 Units Price $189 $666

Real Time One Step RNA PCR Kit

For sensitive real time RT-PCR
Features
Single tube reaction: Optimized to perform both RT-PCR and PCR using sequential reaction conditions limiting contamination. High specificity: Uses Ex Taq Hot Start Technology. Large fragments amplified: Up to 800 bp. Flexible: Use either SYBR Green I or probe detection. The Real Time One Step RNA PCR Kit is designed to perform cDNA synthesis using AMV RT-XL followed by PCR amplification using Takara Ex Taq HS in a single tube. Real time monitoring of the amplification process takes place using either SYBR Green I or various detection probes. This kit is compatible with the Smart Cycler (Cepheid) and other real time instruments. This kit is suitable for detection of small amounts of RNA (i.e. RNA virus). It is supplied with ROX Reference Dye to
Amplification curve.
SmartCycler

nomalize the fluorescent signal between reactions (for instruments that are equipped with this option).

Melting curve.

Detection of Rat Atp5fl with the II System. A real time One-Step RT-PCR reaction was performed using total RNA (1 pg100 ng) prepared from rat liver as a template. The target gene was detected in the range of 10 pg100 ng. The melting curve shows that a single product was amplified at all template concentrations.

Ordering Information
Catalog No. TAK RR026 Product Name Real Time One Step RNA PCR Kit Quantity 100 reactions Price $309

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New Products
pCold TF Vector
For increased soluble protein expression
Features
High yield recombinant protein: Up to 60%, of expressed intracellular protein is the target protein. Soluble expression level is increased: Due to fused expression with Trigger Factor, proteins that are insoluble in conventional expression systems may be expressed in soluble form. Wide range of E. coli hosts: Compatible with most E. coli strains. More protease are available for tag release: Cut sites for 3 proteases are included.
Amp
M
cspA 3UTR Multiple cloning site Factor Xa site Thrombin site HRV 3C Protease site Trigger Factor (TF) His Tag TEE cspA 5UTR lac operator cspA promoter

New Products

IG 13

pCold TF DNA
lac I
Quantity 25 ug

(5,769 bp)

Takara's pCold TF DNA Vector is a fusion cold shock expression vector that expresses Trigger Factor (TF) chaperone as a soluble tag. Trigger Factor is a prokaryotic ribosome-associated chaperone protein (48 kDa) which facilitates co-translational folding of newly expressed polypeptides. Because of its E. coli origin, TF is highly expressed in E. coli expression systems. The pCold TF DNA Vector consists of the cspA promoter plus additional downstream sequences including a 5' untranslated region (5' UTR), a translation enhancing element (TEE), a His-Tag sequence, and a multicloning site (MCS). A lac operator is inserted downstream of the cspA promoter to ensure strict regulation of expression.

ColE1 ori

Ordering Information
Catalog No. TAK 3365 Product Name pCold TF Price $767

SPP System (Single Protein Production System)

For high-purity protein expression in E. coli
Features
Yields signal to noise ratios unparalleled by any in vivo expression system Enables expression of proteins which are toxic or easily aggregate Cold-temperature expression increases protein stability and solubility Well suited for labeling applications such as NMR Takaras new SPP System yields signal to noise ratios unparalleled by any in vivo protein expression system. It uses MazF, a bacterial toxin that acts as an mRNA interferase to efficiently and selectively degrade cellular mRNAs in vivo. This results in a precipitous drop in total cellular protein synthesis. Concomitant expression of MazF and a target gene engineered to encode an ACA-less mRNA results in sustained and high-level (up to 90%) target gene expression in the virtual absence of background protein synthesis. In addition, cspA promoter-based cold temperature expression improves both the stability and solubility of expressed proteins. Nearly exclusive labeling of the target protein can be achieved, making SPP technology particularly suitable for structural analysis of proteins by NMR. The SPP System is also particularly appropriate for expression of proteins that are toxic or tend to easily aggregate when expressed at high levels. Reference:
Suzuki, M.; et. al. Molecular Cell, 2005, 18, 253-261.

Ordering Information
Catalog No. Product Name Quantity 1 set TAK 3367 SPP System I Please visit our website for additional products. Price $720

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Try it today and prove it to yourself. Call or visit our web site to request a sample of this product.

PrimeSTAR HS DNA Polymerase

For accurate and efficient high fidelity PCR
Features
Superior accuracy: A strong exonuclease activity results in an extremely low error rate, with only 12 of 250,000 bp containing errors as determined by DNA sequence analysis. Excellent efficiency: High efficiency amplification-even higher than Taq Polymerase. Robust amplification: Tolerance to varying reaction conditions means a single PCR cycling protocol can be used to amplify products of varying sizes. GC-rich targets: Robust performance even with GC-rich templates. Excellent for blunt-end cloning: Low error rate allows for excellent cloning results, which is particularly important for amplification of cDNA libraries.
PrimeSTARTM 1 2 3 4 5 Company N 2 3 4 5 Company B 1 2 3 4 5 Company I 1 2 3 4 5

PrimeSTAR HS DNA Polymerase is a novel new high fidelity PCR enzyme which provides maximum fidelity as well as extended product length (8.5 kb for human genomic DNA; 22 kb for DNA). Targeted for demanding cloning (i.e. amplification of cDNA libraries) and sequencing applications, it offers extremely high accuracy, and fidelity calculated by sequence analysis. It also offers excellent amplification efficiency and shortened reaction times. Finally, the antibody-mediated hot start formulation prevents false initiation events during reaction assembly due to mispriming or primer digestion, thus, lowering background.

New Products

- 2kb

Comparison of PrimeSTARTM HS Amplification Efficiency with Competitors on a 2 kb Human Genomic DNA Fragment. Superior amplification efficiency was apparent using PrimeSTARTM HS on a human genomic (DCLRE1A) 2 kb template. Human genomic DNA was used in the following quantities: Lane1: 0 ng (dH2O), Lane 2: 100 pg, Lane 3: 1 ng, Lane 4: 10 ng, Lane 5: 100 ng.

M1 M2 1

10 M2 M1

1. DCLRE1A 4 Kb 2. beta-globin 8.5 Kb 3. beta-globin 6 Kb 4. DCLRE1A 2 Kb 5. p53 0.5 Kb 6. p53 4 Kb 7. beta-globin 7.5 Kb 8. DCLRE1A 8 Kb 9. DCLRE1A 1 Kb 10. p53 6 Kb M1: -Hind III-digest M2: pHY Marker

Ordering Information
Catalog No. TAK R010A TAK R010B Product Name PrimeSTAR HS PrimeSTAR HS Quantity 250 Units 1000 Units Price $218 $785

A Single PCR Cycling Protocol Produces Optimal Amplifcation of Fragments of Varying Sizes. A single protocol allows efficient amplification of target fragments from 0.5 - 8.5 kb, making PrimeSTAR HS an excellent choice for a cDNA library cloning.

Ex Taq, LA Taq, SPP System and PrimeSTAR are trademarks of, and RetroNectin is a registered trademark of Takara Bio Inc. SYBR is a registered trademark of Molecular Probes, Inc. TaqMan is registered trademark of Roche Molecular Systems. LightCycler is a trademark of the Roche group. Platinum is registered trademark of Invitrogen. Smart Cycler is a registered trademark of Cepheid. PRISM is a registered trademark of Applied Biosystems. MJ Opticon is a registered trademark of BioRad Laboratories, Inc. Takara PCR Related Products are sold under a licensing arrangement with Roche Molecular Systems and F. Hoffman La Roche Ltd.and Applied Biosystems. Takara Bios Hot-Start PCR-Related products are licensed under U.S. Patent 5,338,671 and 5,587,287 and corresponding patents in other countries. His Tag sequences in pCold I, II, and TF are licensed from Hoffman-LaRoche, Inc. pCold DNA and the SPP System are covered by U.S. patents and pending patents owned and issued by the University of Medicine and Dentistry of New Jersey.

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Weve relaunched our website - check it out today!

www.takaramirusbio.com

n! oo S ing m Co
Order Your Copy of

While you browse the website, fill out our Treasure Map (pg. 11), Sorry! This promotion has to win a FREE expired. hat.

Takaras 2006 Life Science Calendar!

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(Available 11/05)

PrimeSTAR HS DNA Polymerase!

(see pg. 15)

Takara Mirus Bio 505 S. Rosa Road - Suite 101 Madison WI 53719 USA