BioView_TAKARA

What’s inside:Technical Article:

The Future of High Fidelity 2Application Notes:

Premix Ex Taq™ (Perfect Real Time) 4pCold TF Vector 6DICE Thermocycler and Ex Taq™ 10

FAQ Mailbox 8Featured Products 12New Products 14Announcements 16

Takara Mirus Bio 10.05

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PCR has become a basic laboratoryprocedure, being performed thou-sands of time each day in laboratoriesworldwide. Taq Polymerase was thefirst thermostable polymerase to bemade available to researchers, and isstill the most widely-used PCRenzyme. It is a highly processiveenzyme, suitable for many routinePCR applications. However, Taq’sperformance is not adequate for othermore demanding PCR applications,such as high-fidelity PCR, high-sensi-tivity PCR, or the synthesis of long orcomplex DNA targets.

Importance of High Fidelity

High polymerase fidelity (i.e. a lowrate of base misincorporations, orerrors) is most important in PCR appli-cations where downstream sequenc-ing or gene expression of the ampli-fied product is desired. It is also sig-nificant in applications requiring ampli-fication of low-copy-number templates(requiring many rounds of amplifica-tion), longer target sequences, oramplification and rare transcripts orallelic mutants. cDNA library con-struction, site directed mutagenesis,and mutation detection are also partic-ularly sensitive to error rate.

Enzyme fidelity can by influenced by avariety of factors, including templatesequence (i.e. GC-rich templates gen-erally increased error rates), cyclingparameters, and reaction conditions(i.e. pH, Mg2+, dNTP concentration).However, in controlled studies, poly-merases exhibit characteristic rates ofbase misincorporations, rates ofextension from those misincorpora-tions, and 3'�5' exonuclease or proof-reading activity. These factors togetherresult in an intrinsic “error rate” foreach polymerase.

Polymerase Fidelity

Taq polymerase and related Thermusfamily polymerases generally possessa high rate of base misincorporations,a low rate of extension from thesemisincorporations, and lack a 3'�5'exonuclease or “proofreading” func-tion. Their error rates are the highestamong the most widely-studied viraland bacterial polymerases.Additionally, the low extension rateactually acts somewhat as a de factoproofreading function, as incorrecttemplates fall out of the amplifiablepool. However, this results in loweryield and sensitivity, particularly onlonger products.

Using conventional mutant-basedfidelity assays, the recorded errorrates of about 10-4 are common forTaq. This number may seem low, butthis means that after one fairly typical106 fold PCR amplification of a 200 bptarget, up to 33% of the resultingproducts may contain errors.

Pyrococcus sp. polymerases (alsocalled “proofreading” polymerases)have an even higher initial misincorpo-ration rate than Taq, but because theycontain a 3' to 5' exonuclease activity,they generally possess much lowererror rates than Taq Polymerase orother Thermus-family polymerases.However, these enzymes often displaylow processivity, resulting in low prod-uct yield, reduced product length, anddifficulties in optimization.

Mixing a proofreading polymerasewith Taq polymerase has been shownto increase amplification performance,and is the basis for several widely-used enzymes, including Takara Ex Taq™ and LA Taq™. These blendsprovide superior amplification efficien-cy and product length as compared toTaq or the proofreader alone. Fidelityis also much improved over TaqPolymerase alone, but may still causeproblems in some applications.

Calculated Error Rate

Error rate and fidelity are calculatedvia the following formulas:

Most quoted error rates are experi-mentally based on indirect phenotypicmeasurements of mutant frequency,and vary widely. For example, onecommon method calculates the fre-quency of observed mutants by identi-fying the number of phenotypicallyaltered colonies following bacterialtransformation with a PCR-amplifiedDNA fragment. However, lethal aminoacid substitutions derived from misin-corporation of one or more incorrectbases during the PCR reaction will gounnoticed and uncounted, as theyresult in cell death. Fidelity rates cal-culated via this method are also sub-ject to an additional level of inaccura-cy because some nucleotide changeswill not result in clear phenotypicchanges of the expressed protein(usually beta-galactosidase).Therefore, these conventional meth-ods of calculating error rates can pro-vide useful comparisons within a sin-gle set of reaction conditions, but theactual results may vary widely fromexpected numbers.

2Takara Mirus Bio • 888-251-6618 • www.takaramirusbio.comBIOVIEW

The Future of High FidelityOverview of Polymerase FidelityBy Julie Kramer, Marketing Manager, Takara Mirus Bio

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The expected fraction of PCR-induced mutants can be calculatedaccording to the following formula:

F(>1) = 1- e-bfd

b= length of target sequence

f= error rate

d=number of doublings

Error Rate= # misincorporated bases/# bases synthesized

Fidelity= 1/error rate

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Takara Bio recently introducedPrimeSTAR™ HS DNA Polymerase, anovel new DNA polymerase whichoffers very high fidelity as well asexcellent amplification efficiency andextended product length (8.5 kb forhuman genomic DNA; 22 kb for λDNA). PrimeSTAR™ is the only cur-rently available DNA polymerasewhose error rate (only 12 errors per250,000 bases) is determined by DNAsequencing.

PrimeSTAR™ HS

PrimeSTAR™ HS is a recombinantenzyme expressed in E. coli. It wasderived from a proprietary ther-mostable bacterial strain, and waschosen by Takara after studying apanel of bacterial strains which hadbeen identified as potential producersof high fidelity polymerases. It has avery strong 3'�5' exonuclease activity,high replication accuracy, andextremely high priming efficiency. Italso contains an antibody which inacti-vates both the polymerase and exonu-clease functions during reactionassembly. This prevents false initiationevents due to mispriming or primerdigestion, resulting in lowered back-ground and increased reproducibility.

Takara’s fidelity assay forPrimeSTAR™ HS is as follows:

Eight arbitrarily selected GC-richregions were amplified withPrimeSTAR™ HS and other enzymes,using Thermus thermophilus HB8genomic DNA as a template. Eachproduct (approx. 500 bp each) wasthen cloned into a suitable plasmid.Multiple clones were selected andsubjected to sequence analysis.

Sequence analysis of DNA fragmentsamplified using PrimeSTAR™ HSdemonstrated only 12 mismatched

bases per 249,941 total bases. This ishigher fidelity than Thermococcuskodakaraensis DNA Polymerase(KOD), Pfu, and 10X higher fidelitythan Taq DNA polymerase.

Additionally, when compared withother commercially available highfidelity enzymes, PrimeSTAR™ HSdemonstrates superior efficiency onboth a 500 bp Thermus and 2 kbhuman genomic DNA fragments (seepage 14). Thus, PrimeSTAR™ HSanswers the need for convenient,robust, easy-to-optimize high-fidelity

DNA polymerases, and offers themethod of choice for superior high-fidelity 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.

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For information about Takara’s PrimeSTAR™ HSsee page 14

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PrimeSTAR™ HS DNA Polymerase

Thermococcus kodakaraensis - derived DNA Polymerase

Pyrococcus sp. - derived DNA Polymerase

Thermus aquaticus - derived DNA Polymerase

PrimeSTAR™ Fidelity Comparison with Other DNA Polymerases and Taq.

PrimeSTAR™ HS Offers:

» High Accuracy: A strong exonuclease activity, resulting in an extremely lowerror rate of only 12 errors per 250,000 bp.

» High Efficiency: Amplification efficiency higher than Taq Polymerase; excel-lent performance even on GC-rich templates.

» Robust Amplification: Tolerance to varying reaction conditions means a sin-gle PCR cycling protocol can be used to amplify products of varying sizes.

» Extended Product Length: Amplify targets of up to 8.5 kb on humangenomic DNA; 10 kb on E. coli genomic DNA; and 22 kb on lambda DNA.

» Fast Reaction Times: Increased priming efficiency results in rapid annealingtimes and improved specificity.

» High Specificity: Antibody-mediated hot-start prevents false initiation eventsduring reaction assembly due to mispriming or primer digestion.

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

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Comparison of Takara’s New Premix Ex Taq™(Perfect Real Time) versus Competing qPCR Kits Introduction

Real Time PCR offers a quantitative method to studyproduct amounts during the early stages of a PCR reac-tion, when the amount of product corresponds to theamount of initial template present in the reaction.

Premix Ex Taq™ (Perfect Real Time) is a 2X premix, spe-cially designed for high-speed, high sensitivity real timePCR using either detection probes (i.e. TaqMan® and othermolecular probe technologies) or SYBR® Green I (notincluded). This premix combines high-performance TakaraEx Taq™ Hot Start DNA Polymerase, which uses anti-body-mediated Hot Start technology to prevent non-spe-cific amplification, with an optimized real time PCR bufferformulation which provides increased amplification effi-ciency and further improved specificity for high speed realtime PCR. Premix Ex Taq™ reactions are fast and easy,and generate exceptional PCR results on all major real-time instruments.

Materials and Methods

qPCR amplification of the human ACTB gene was per-formed using cDNA templates corresponding to 100 ng-1pg of total RNA using TaqMan® probes (ABI) and PremixEx Taq™ (Perfect Real Time). These results were com-pared against results generated using three different lead-ing competitor qPCR kits on the Roche LightCycler®.Reactions were performed according to the manufactur-er’s protocols.

Results and Discussion

Amplification efficiency was determined using Premix ExTaq™ (Perfect Real Time) and three leading competitorqPCR enzymes on the Roche LightCycler®. The resultscan be seen in the figure on the next page.

The amplification curves generated with Premix Ex Taq™demonstrate excellent sensitivity and uniformity. With theABI Kit, only one curve appears, indicating sensitivity sev-eral logs lower than Takara’ s.

Both the Invitrogen and Qiagen Kits resulted in amplifica-tion curves which are shifted to the right, indicating loweramplification efficiency than the Takara Kit. The curveslack uniformity when compared to Takara’s Kit, indicatinglower reproducibility.

Additionally, reactions with Takara Premix Ex Taq™ canbe completed in as little as 50 minutes, making it compati-ble with fast PCR systems.

In summary, Takara Premix Ex Taq™ provides superiorefficiency and reproducibility as compared to other leadingqPCR kits.

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

Features of Premix Ex Taq™ (Perfect Real Time)Include:

» Fast: Reaction can be completed in as little as 50minutes.

» High Sensitivity: Detects as few as 10 templatecopies.

» Versatility: Compatible with Smart Cycler®,LightCycler®, ABI PRISM® 7000/7700/7900 HT,Applied Biosystems 7500 Real-Time PCRSystems, and other real time PCR instruments.

» Wide Dynamic Range: Possesses a dynamicrange of 10 orders of magnitude (λ DNA template).

» Convenient: Supplied as a premix formulation; twoROX reference dyes are supplied separately.

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Qiagen’s Quantitect ProbePCR Kit

Invitrogen’s Platinum® Quantitative PCRSupermix -UDG

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For additional informationabout Takara’s qPCR

products see pages 12-13

Takara SYBR® Premix Ex Taq™ ABI’s TaqMan® Universal PCR Master Mix

Figure 1: Performance of SYBR® Premix Ex Taq™ (Perfect Real Time) vs.ABI’s TaqMan® Universal PCR Master Mix using a RocheLightCycler®.

Cycling conditions: 95°C, 2 min} 1 cycle

94°C, 15 sec.

55°C, 1 min. 45 cycles

72°C,1 min.

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Elucidation of protein structure and function maintains animportant role in post-genomic sequencing and analysisstudies. An efficient protein production system is criticalfor obtaining large amounts of correctly folded recombi-nant protein for study. E. coli expression systems are usedextensively for production of recombinant proteins, andhave two major advantages over other expression sys-tems: (1) ease of use, and (2) low cost. However, somerecombinant proteins do not fold correctly during expres-sion in E. coli, resulting in deposits of inactive insolubleprotein termed "inclusion bodies".

Series of pCold Vectors

In collaboration with Prof. Masayori Inouye (University ofMedicine and Dentistry of New Jersey), Takara Bio hasdeveloped the pCold DNA Vectors, a series of novel pro-tein expression vectors. The pCold Vectors provideincreased in vivo protein yield, purity, and solubility ofexpressed recombinant proteins using "cold shock" tech-nology. More specifically, the cspA (cold shock protein A)promoter and related elements have been incorporatedinto these vectors to upregulate target protein productionat lowered incubation temperatures (37°C-15°C). Thistemperature drop also suppresses expression of other cel-lular proteins, represses protease activity, and temporarilyhalts overall cell growth. This process allows expressionof target proteins at high yield, high purity (up to 60% ofcellular protein), and increased solubility as comparedwith conventional E. coli expression systems. Co-expres-sion of one or more chaperone proteins during expressionof a heterologous target protein has proven effective forobtaining increased amounts of soluble recombinant pro-tein (see Takara's Chaperone Plasmid Set (TAK 3340)).This procedure, though, lacks the convenience of a singletransformation step.

pCold TF Vectors

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

a 5' untranslated region (5' UTR), a translation enhancingelement (TEE), a His-Tag sequence, and a multicloningsite (MCS). A lac operator is inserted downstream of thecspA promoter to ensure strict regulation of expression.Additionally, recognition sites for HRV 3C Protease,Thrombin, and Factor Xa are located between the TF-Tagand the multiple cloning site (MCS) and function to facili-tate tag removal from the expressed fusion protein. MostE. coli strains can serve as expression hosts.

pCold TF DNA Vector combines high-yield cold shockexpression technology with Trigger Factor (chaperone)expression in a single vector to facilitate correct proteinfolding, thus enabling efficient soluble protein productionfor otherwise intractable target proteins. The followingexperiment compares results generated using pCold I;pCold I co-expressed with a Chaperone Plasmid; pColdTF; and T7 promoter constructs to express various pro-teins.

Materials and Methods

pCold DNA I and pCold TF DNA cloning and expressionprocedures* were conducted as follows:

1) Insert the target gene to the multicloning site of thepCold DNA vector for expression.

2) Transform the E.coli host strain (e.g. BL21) with theexpression plasmid, and select for ampr transformants.

3) Inoculate the transformants into medium including 50 µg/ml of ampicillin, and culture with shaking at37°C.

4) At OD600= 0.4 - 0.5, refrigerate the culture at 15°C(without shaking) for 30 minutes.

5) Add IPTG to a final concentration of 0.1- 1.0 mM, andcontinue the culture with shaking at 15°C for 24 hours.

6) Collect the cells, and confirm the expression of the tar-get protein with SDS-PAGE in soluble and insolublefractions or activity assay.

Expression from T7 promoter-driven vectors was per-formed using a standard protocol utilizing IPTG inductionand subsequent culturing at 37°C.

*Cultivation/induction conditions (culture medium, aera-tion, timing of induction, concentration of inducer, cultiva-tion time after induction) should be optimized for each tar-get protein.

The pCold TF Protein Expression SystemProduces Soluble, Active Protein in E. coli

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Results and Discussion

Protein expression using the pCold TF Expression Vectorwas compared with protein expression using (1) the pColdDNA I Vector alone, (2) co-expression using the pColdDNA I Vector with Takara's Chaperone Plasmid pTf16, and(3) a T7 promoter expression system which included othertags for solubilization.

Figure 1 demonstrates the successful production ofenzyme protein A using the pCold TF system. Expressionof this protein, with an estimated molecular weight of 29 kDa, was not seen as an exact band with either the T7expression system or even with pCold I (either individualexpression or chaperone co-expression).

However, the expression of the target protein and targetplus tag (29 kDa and 52 kDa) was observed using pColdTF, and most of the obtained protein was in soluble form.Subsequent assays confirmed that the expressed enzymeA retains activity even as a fusion protein.

Figure 2 demonstrates improved levels of soluble proteinB using pCold TF. Expression of soluble enzyme proteinB (M.W: ~63 kDa) was not observed using either pColdDNA I alone or pCold I co-expressed with chaperone pro-teins, 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, thetarget protein was present at an expression level muchhigher than with other systems and tags, and most of theexpressed target protein was observed in the soluble frac-tion. (Note: The molecular weight of the target protein islarger than its actual size and varies due to fused expres-sion with different tags).

In summary, the pCold TF expression system offers aconvenient high yield, high purity alternative for efficientsoluble protein expression of otherwise intractable targetproteins.


Ordering InformationCatalog No. Product Name Quantity PriceTAK 3365 pCold TF Vector 25 µg $727

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Figure 1: Expression of Soluble Protein A Using thepCold TF Expression System

Figure 2: Increased Expression of Soluble Protein BUsing the pCold Expression System

pCold TF pColdpCold +

Chaperone T71 2 1 2 1 2 1 2

kDa

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1. Cell extract solution

2. Soluble fraction

target protein

co-expressed

trigger factor**

Trx GST Nus

1 2 3 1 2 3 1 2 3

kDa

9766

45

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pColdTF

pCold I pCold +Chaperone

1 2 3 1 2 3 1 2 3

1. Cell extract solution

2. Soluble fraction

3. Insoluble fraction

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» FAQ Mailbox «

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PrimeSTAR™ HS DNA Polymerase FAQCan PrimeSTAR™ HS reactions use the same PCRcycling conditions that are used with Taq Polymerase?

PrimeSTAR™ HS cannot use the same PCR cyclingconditions that are used with Taq Polymerase. Takarastrongly recommends following the conditions describedin the PrimeSTAR™ HS product protocol, since thecharacteristics of this enzyme are very different fromthose of Taq Polymerase.

Takara recommends the following initial cycle protocolfor primers with a Tm of >55°C: denaturing step, 98°C, 10 sec; annealing step 55°C, 5 sec.; extension step,72°C, 1 min./kb; for 30 cycles.

What is the basis of PrimeSTAR™ HS’s antibody-mediated Hot Start Technology?

PrimeSTAR™ HS’s Hot Start Technology uses a singlemonoclonal antibody which blocks both PrimeSTAR™’spolymerase and nuclease activities.

What advantage is offered by Takara’s measure-ment of PrimeSTAR™ HS’s fidelity using sequenceanalysis?

Simple comparison of the fidelity rates available for dif-ferent PCR enzymes is not possible due to the multitudeof different fidelity measurement techniques employedby different manufacturers. Takara Bio has determinedPrimeSTAR™’s error rate based upon genotype, that is,the error rate as determined by actual sequence analy-sis. The method Takara Bio used to obtain their fidelitydata is presented below:

Eight arbitrarily selected GC-rich regions were amplifiedwith PrimeSTAR™ HS and other enzymes, using theThermus thermophilus HB8 genomic DNA as a template.Each PCR product (approx. 500 bp each) was cloned intoa suitable plasmid. For each different DNA region cloned,multiple clones were picked, and subjected to sequenceanalysis. Sequence analysis results of DNA fragmentsamplified using PrimeSTAR™ HS demonstrated only 12mismatched bases per 249,941 total bases. This dataconfirms PrimeSTAR™ HS’s extremely high fidelity, with acalculated error frequency of only 0.0048%.

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

The PrimeSTAR™ HS Buffer composition is proprietary.

Why does PrimeSTAR™ HS use a 5X concentrationbuffer?

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

How does PrimeSTAR™ HS differ from KOD HotStart?

PrimeSTAR™ HS provides higher fidelity than KOD HotStart while offering the same level of amplification effi-ciency.

What is the source of PrimeSTAR™ HS DNA poly-merase? Is it a cloned enzyme?

PrimeSTAR™ HS is a recombinant enzyme that isexpressed in E. coli. It was derived from a proprietarythermostable bacterial strain chosen by Takara afterstudying various strains that were identified as producinghigh fidelity enzymes. PrimeSTAR™ HS was notobtained from the same bacterial strain that was used toproduce KOD (Pfx).

Are PrimeSTAR™ HS PCR products suitable for TAcloning?

PCR products cannot be used directly for TA cloning.The termini are blunt-ended due to the 3'�5' exonucle-ase activity of this enzyme. PrimeSTAR™ HS PCRproducts should be used for blunt-end cloning. Takararecommends use of a dephosphorylated vector andphosphorylated PCR products. Products can be enzy-matically phosphorylated or made using PCR primerspossessing phosphoric acid residues at their 5' termini.

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SYBR® Premix Ex Taq™ (Perfect Real Time) FAQ

How many reactions (points) are recommendedfor a typical standard curve?

Generally 5 or 6 reactions (5 or 6 points) are used toestablish the standard curve, plus dH2O for a negativecontrol. Takara has used cDNAs which corresponded to1 pg, 10 pg, 100 pg, 1 ng, 10 ng and 100 ng of mouseliver total RNA respectively (and dH2O for negative con-trol). If possible, establish the standard curve within aCt range of ~ 15-35.

The Ct(cycle threshold) is the number of cycles at whichfluorescence intensity is measureable above back-ground levels (i.e. threshold line), and is set in the expo-nential amplification phase to allow the most accuratereading.

How do I determine the number of qPCR reactionsfor my experiments? For example, if I have twodifferent cell lines and want to characterize threedifferent genes in each?

For each of the 3 genes, a standard curve (e.g. com-posed of 7 data points) plus 2 experimental samplesthat are run in triplicate are performed. Therefore,3(triplicate) x (7 pts + 2 samples) x 3(genes) = 81 reac-tions are required for 3 genes. One package of SYBR®

Premix Ex Taq™ (Perfect Real Time) contains sufficientreagent for 200 reactions (50 µl reaction volume).

What PCR product size is optimal for real timePCR?

A size range of 80-150 bp is generally recommendedfor qPCR amplification, although sizes up to 300 bp arepossible.

Can SYBR® Green I dye precipitate? Is there agood way to resuspend SYBR® Green I?

A greenish-yellow precipitate can sometimes beobserved in SYBR® Premix Ex Taq™ when stored at -20°C. If this occurs, dissolve the precipitate complete-ly by letting the tube stand at room temperature for sev-

eral minutes, protected from light, or by warming withyour hands, followed by gentle mixing. Do not vor-tex!! We have verified that this product shows goodperformance after the precipitate is dissolved complete-ly.

What is the composition of SYBR® Premix ExTaq™?

The Premix contains Ex Taq™ Hot Start DNAPolymerase, buffer, dNTP mix, Mg2+ and SYBR® GreenI. The Mg2+ and SYBR® Green I concentrations are pro-prietary.

What is the purpose of the ROX reference dyeincluded with the SYBR® Premix Ex Taq™?

ROX (i.e. Carboxy-X-Rhodamine) is a convenient inter-nal reference standard for use in normalizing signalsdue to non-PCR related fluorescence fluctuations thatoccur either between wells or over time. Please notethat two types of ROX Reference Dye (Original VersionROX and ROX II) are supplied with this product. Usethe Original Version ROX for normalization with the ABIPRISM® 7000/7700/7900HT and Applied Biosystems7300 Real-Time PCR Systems. Use ROX II referencedye for normalization whith Applied biosystems 7500Real-Time PCR System.

Can you make a master mix of the ROX referencedyes and SYBR® Premix Ex Taq™ to help avoidpipetting errors?

Original Version ROX Reference Dye can be premixed.Add 40 µl of ROX to 1 ml of SYBR® Premix Ex Taq™.Store this solution at 4°C (protected from light), anduse within one month for best performance.

ROX Reference Dye II should not be premixed prior toreaction assembly.

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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:

Takara’s Ex Taq™ DNA Polymerase is a polymerase mixwhich offers high amplification efficiency (up to 100X Taq),superior reproducibility, and extended product length (to 30kb on λ DNA).

The Takara Thermocycler Dice provides convenience, relia-bility, multifunctionality, and a gradient feature to selecttemperature conditions at each step, and program a tem-perature gradient up to 20°C across the rows of a sampleblock. This allows optimization of reaction conditions in asingle experiment.

The following experiment demonstrates optimization of thePCR conditions for an 8 kb DNA fragment amplified with ExTaq™ DNA Polymerase on the PCR Thermocycler Dice.

Materials and Methods:

50 µL amplification reactions using 1.25 U of Ex Taq™DNA Polymerase and 0.5 µg of λ DNA were assembledaccording to manufacturer’s instructions. Half of the reac-tions were amplified using shuttle PCR under the followingconditions:

98°C 10 sec52 to 72°C 5 min 30 cycles

(Gradient temperature range: 20°C)

The remainder were amplified using the following three-step protocol:

98°C 10 sec

45 to 65°C 30 sec 30 cycles

72°C 5 min

(Gradient temperature range for annealing step: 20°C)

Results and Discussion:

In the two-step protocol (Figure 1), higher annealing/exten-sion temperatures reduced the amplification of low molecu-lar weight non-specific products, and increased the yield ofthe target product.

In the three-step protocol (Figure 2), higher annealing tem-peratures reduced the amplification of low molecularweight non-specific products.

In both protocols, robust, reproducible amplification wasapparent.

These results demonstrate that Takara’s Ex Taq™ DNAPolymerase and Dice Thermocycler can be combined toproduce easy optimization and superior reproducibility andyield even with challenging DNA fragment lengths.

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

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

Figure 1: Evaluation of annealing/extension tempera-tures in two-step gradient PCR. Higher annealing/exten-sion temperatures reduced amplification of low molecularweight non-specific products, and increased target yield.

Figure 2: Evaluation of annealing temperatures inthree-step gradient PCR. Higher annealing temperaturesreduced the amplification of non-specific products.

M 52°C 72°C M

M 45°C 65°C M

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2. How many Resource Centers are on the website?1. What is the length of genomicDNA that LA Taq™ DNAPolymerase can amplify?

3. How many reactions can you run withthe DNA LigationKit, Mighty Mix?

4. What is the fidelity of Ex Taq™DNA Polymerase?

5. What technology do the pColdVectors utilize?

6. How many plasmids are includedin the Chaperone PlasmidSet?

7. Can SYBR® Premix Ex Taq™(Perfect Real Time) beused on the MJOpticon® instrument?

8. What is the cell transductionefficiency of cell line K-562 on Retronectin®?

9. What is the assay range of theProcollagen Type 1 C-Peptide EIA Kit? 10. What does CA stand for

in the Refolding CA Kit?

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Takara Mirus Bio announces the re-launching of our website at www.takaramirusbio.com. The

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Featured Products

Ordering InformationCatalog No. Product Name Quantity PriceTAK RR041A SYBR® Premix Ex Taq™ 200 reactions $273TAK RR041B SYBR® Premix Ex Taq™ 400 reactions $534

Features• High sensitivity: Detects as few as 100 copies.

• Versatility: Compatible with most qPCR instruments.

• Wide dynamic range: Possesses a dynamic range of 7-8orders of magnitude (λ DNA template).

• High speed capability: Optimized buffer system allowsquick reaction times.

• Convenient: Separate tubes of ROX reference dye aresupplied.

SYBR® Premix Ex Taq™ (Perfect Real Time) is a conven-ient (2X) premix consisting of Takara's high sensitivity, highefficiency Ex Taq™ Hot Start DNA Polymerase, SYBR®

Green I, dNTPs, and an optimized real time buffer whichprovides superior specificity and increased amplificationefficiency for real time PCR, plus separate tubes of ROXReference Dye. Antibody-mediated hot start technology isused for reduced background.

Amplification Curve(upper panel) andMelting Curve (lowerpanel) for SYBR®

Premix Ex Taq™(Perfect Real Time)on the SmartCycler®

(Cepheid).

SYBR® Premix Ex Taq™ (Perfect Real Time)For superior real time PCR using SYBR® Green I

Premix Ex Taq™ (Perfect Real Time)For excellent real time PCR using probe detection

Premix Ex Taq™ (Perfect Real Time) is a 2X premix speciallydesigned for high speed, high sensitivity real time PCR usingeither detection probes or SYBR® Green I (not included). Itconsists of Takara’s high sensitivity, high efficiency Ex Taq™Hot Start DNA Polymerase, and an optimized real time bufferwhich provides superior specificity and increased amplifica-tion efficiency for real time PCR. Antibody-mediated hot starttechnology prevents nonspecific amplification due to misprim-ing and/or formation of primer dimers during the reactionassembly. The Taq antibody-polymerase complex is dena-tured in the first cycling step, releasing the polymerase andallowing DNA synthesis to proceed.

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

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 10orders of magnitude (λ DNA template).

• High speed capability: Optimized buffer system allowsquick reaction times.

Ordering InformationCatalog No. Product Name Quantity PriceTAK RR039A Premix Ex Taq™ 200 reactions $219TAK RR039B Premix Ex Taq™ 400 reactions $437

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Ex TaqTM R-PCR, Version 2.1For high specificity qPCR

Features• High sensitivity: Detect as few as 10 copies.

• Superior specificity: Hot-Start formulation prevents mis-priming and lowers nonspecific amplification products.

• Wide dynamic range: Dynamic range of 10 orders ofmagnitude using probe detection and 7-8 orders of mag-nitude for SYBR® Green I.

• Flexibility: Use with both SYBR® Green I and detectionprobes such as TaqMan®.

The Ex Taq™ R-PCR Version 2.1 is an enzyme-buffer sys-tem developed specifically for real time PCR on theCepheid SmartCycler®. This product combines the high per-formance of Ex Taq™ Hot-Start Polymerase with an opti-mized R-PCR buffer for high specificity.

Ex Taq™ R-PCR Version 2.1 contains a monoclonal anti-body to Taq Polymerase, which binds to the polymeraseuntil the temperature is elevated. The binding of this anti-body prevents nonspecific amplification due to misprimingand/or formation of primer dimers during reaction assembly.An optimized buffer formulation provides increased speci-ficity for real time PCR, further enhancing performance.

Real Time One Step RNA PCR KitFor sensitive real time RT-PCR

The Real Time One Step RNA PCR Kit is designed to per-form cDNA synthesis using AMV RT-XL followed by PCRamplification using Takara Ex Taq™ HS in a single tube.Real time monitoring of the amplification process takesplace using either SYBR® Green I or various detectionprobes. 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. Melting curve.

Detection of Rat Atp5fl with the SmartCycler® II System. A real time One-StepRT-PCR reaction was performed using total RNA (1 pg–100 ng) prepared from ratliver as a template. The target gene was detected in the range of 10 pg–100 ng.The melting curve shows that a single product was amplified at all template con-centrations.

Features

• Single tube reaction: Optimized to perform both RT-PCRand PCR using sequential reaction conditions limiting con-tamination.

• High specificity: Uses Ex Taq™ Hot Start Technology.

• Large fragments amplified: Up to 800 bp.

• Flexible: Use either SYBR® Green I or probe detection.

Ordering InformationCatalog No. Product Name Quantity PriceTAK RR031A Ex Taq™ R-PCR, Ver 2.1 250 Units $189TAK RR031B Ex Taq™ R-PCR, Ver 2.1 1000 Units $666

Ordering InformationCatalog No. Product Name Quantity PriceTAK RR026 Real Time One Step 100 reactions $309

RNA PCR Kit

Featu

red P

rod

ucts

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

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

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New

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Features• High yield recombinant protein: Up to 60%, of

expressed intracellular protein is the target protein.

• Soluble expression level is increased: Due to fusedexpression with Trigger Factor, proteins that are insolublein conventional expression systems may be expressed insoluble form.

• Wide range of E. coli hosts: Compatible with mostE. coli strains.

• More protease are available for tag release: Cut sitesfor 3 proteases are included.

Takara's pCold TF DNA Vector is a fusion cold shock expres-sion vector that expresses Trigger Factor (TF) chaperone asa soluble tag. Trigger Factor is a prokaryotic ribosome-asso-ciated chaperone protein (48 kDa) which facilitates co-trans-lational folding of newly expressed polypeptides. Because ofits E. coli origin, TF is highly expressed in E. coli expressionsystems. The pCold TF DNA Vector consists of the cspA pro-moter plus additional downstream sequences including a 5'untranslated region (5' UTR), a translation enhancing ele-ment (TEE), a His-Tag sequence, and a multicloning site(MCS). A lac operator is inserted downstream of the cspApromoter to ensure strict regulation of expression.

Ordering InformationCatalog No. Product Name Quantity PriceTAK 3365 pCold TF 25 ug $767

New ProductspCold TF VectorFor increased soluble protein expression

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

Takara’s new SPP System™ yields signal to noise ratiosunparalleled by any in vivo protein expression system. It usesMazF, a bacterial toxin that acts as an “mRNA interferase” toefficiently and selectively degrade cellular mRNAs in vivo.This results in a precipitous drop in total cellular protein syn-thesis. Concomitant expression of MazF and a target geneengineered to encode an ACA-less mRNA results in sustainedand high-level (up to 90%) target gene expression in the virtu-

M13 IG

pCold TF DNA(5,769 bp)

ColE1 ori

Am

pcspA 5’UTRlac operatorcspA promoter

lac

I

cspA 3’UTRMultiple cloning siteFactor Xa siteThrombin siteHRV 3C Protease siteTrigger Factor (TF)His • TagTEE

al absence of background protein synthesis. In addition, cspApromoter-based cold temperature expression improves both thestability and solubility of expressed proteins.Nearly exclusive labeling of the target protein can be achieved,making SPP technology particularly suitable for structural analy-sis of proteins by NMR. The SPP System™ is also particularlyappropriate for expression of proteins that are toxic or tend toeasily aggregate when expressed at high levels.

Reference:Suzuki, M.; et. al. Molecular Cell, 2005, 18, 253-261.

Ordering InformationCatalog No. Product Name Quantity PriceTAK 3367 SPP System™ I 1 set $720Please visit our website for additional products.

newsletterOct05vfinal_2.qxp 10/31/05 9:02 AM Page 14

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PrimeSTAR™ HS DNA PolymeraseFor accurate and efficient high fidelity PCR

PrimeSTAR™ HS DNA Polymerase is a novel new highfidelity PCR enzyme which provides maximum fidelity aswell as extended product length (8.5 kb for human genom-ic DNA; 22 kb for λ DNA). Targeted for demanding cloning(i.e. amplification of cDNA libraries) and sequencing appli-cations, it offers extremely high accuracy, and fidelity calcu-lated by sequence analysis. It also offers excellent amplifi-cation efficiency and shortened reaction times. Finally, theantibody-mediated hot start formulation prevents false initi-ation events during reaction assembly due to mispriming orprimer digestion, thus, lowering background.

Features• Superior accuracy: A strong exonuclease activity

results in an extremely low error rate, with only 12 of250,000 bp containing errors as determined by DNAsequence analysis.

• Excellent efficiency: High efficiency amplification-evenhigher than Taq Polymerase.

• Robust amplification: Tolerance to varying reactionconditions means a single PCR cycling protocol can beused to amplify products of varying sizes.

• GC-rich targets: Robust performance even with GC-richtemplates.

• Excellent for blunt-end cloning: Low error rate allowsfor excellent cloning results, which is particularly impor-tant for amplification of cDNA libraries.

A Single PCR Cycling Protocol Produces Optimal Amplifcation ofFragments of Varying Sizes. A single protocol allows efficient amplifica-tion of target fragments from 0.5 - 8.5 kb, making PrimeSTAR™ HS anexcellent choice for a cDNA library cloning.

Ordering InformationCatalog No. Product Name Quantity PriceTAK R010A PrimeSTAR™ HS 250 Units $218TAK R010B PrimeSTAR™ HS 1000 Units $785

PrimeSTARTM Company N Company B Company I1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5

- 2kb

Comparison of PrimeSTARTM HSAmplification Efficiency with Competitorson a 2 kb Human Genomic DNA Fragment.Superior amplification efficiency was apparentusing PrimeSTARTM HS on a human genomic(DCLRE1A) 2 kb template. Human genomicDNA 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.

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

1. DCLRE1A 4 Kb2. beta-globin 8.5 Kb3. beta-globin 6 Kb4. DCLRE1A 2 Kb5. p53 0.5 Kb6. p53 4 Kb7. beta-globin 7.5 Kb8. DCLRE1A 8 Kb9. DCLRE1A 1 Kb10. p53 6 KbM1: λ-Hind III-digestM2: pHY Marker

M1 M2 1 2 3 4 5 6 7 8 9 10 M2 M1

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 trade-mark of Molecular Probes, Inc. TaqMan® is registered trademark of Roche Molecular Systems. LightCycler® is a trademark of the Roche group. Platinum® is registeredtrademark of Invitrogen. Smart Cycler® is a registered trademark of Cepheid. PRISM® is a registered trademark of Applied Biosystems. MJ Opticon® is a registeredtrademark of BioRad Laboratories, Inc. Takara PCR Related Products are sold under a licensing arrangement with Roche Molecular Systems and F. Hoffman LaRoche Ltd.and Applied Biosystems. Takara Bio’s Hot-Start PCR-Related products are licensed under U.S. Patent 5,338,671 and 5,587,287 and correspondingpatents 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 byU.S. patents and pending patents owned and issued by the University of Medicine and Dentistry of New Jersey.

newsletterOct05vfinal_2.qxp 10/31/05 9:02 AM Page 15

While youbrowse thewebsite, fill outour “TreasureMap” (pg. 11),to win a FREEhat.

We’ve relaunched our website - check it out today!www.takaramirusbio.com

Try a free sample of

PrimeSTAR™ HS DNA Polymerase!(see pg. 15)

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

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noise ratios unparalleled

ensure strict regulation

translation enhancing element

perfect real time

arbitrarily selected gc

quick reaction times

newly expressed polypeptides

rox reference dyes