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Takara Mirus Bio 10.05 What’s inside: Technical Article: The Future of High Fidelity 2 Application Notes:
Takara Mirus Bio
10.05
What’s inside:
Technical Article:
The Future of High Fidelity
2
Application Notes:
Premix Ex Taq™ (Perfect Real Time)
pCold TF Vector
DICE Thermocycler and Ex Taq™
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10
FAQ Mailbox
Featured Products
New Products
Announcements
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Technical Article

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 thou- sands 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, Taq’s performance is not adequate for other more demanding PCR applications, such as high-fidelity PCR, high-sensi- tivity 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 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, or amplification and rare transcripts or allelic 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 a variety of factors, including template sequence (i.e. GC-rich templates gen- erally increased error rates), cycling parameters, and reaction conditions (i.e. pH, Mg 2+ , dNTP concentration). However, in controlled studies, poly- merases exhibit characteristic rates of base misincorporations, rates of extension from those misincorpora- tions, and 3' 5' exonuclease or proof- reading 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” func- tion. 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 10 6 fold PCR amplification of a 200 bp target, up to 33% of the resulting products may contain errors.

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

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 misincorpo- ration 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 prod- uct yield, reduced product length, and difficulties in optimization.

Mixing a proofreading polymerase with Taq polymerase has been shown to increase amplification performance, and is the basis for several widely- used enzymes, including Takara Ex Taq™ and LA Taq™. These blends provide superior amplification efficien- cy 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 experi- mentally based on indirect phenotypic measurements of mutant frequency, and vary widely. For example, one common method calculates the fre- quency of observed mutants by identi- fying the number of phenotypically altered colonies following bacterial transformation with a PCR-amplified DNA fragment. However, lethal amino acid substitutions derived from misin- corporation of one or more incorrect bases during the PCR reaction will go unnoticed and uncounted, as they result in cell death. Fidelity rates cal- culated via this method are also sub- ject to an additional level of inaccura- cy because some nucleotide changes will not result in clear phenotypic changes 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 the actual results may vary widely from expected numbers.

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Technical Article The Future of High Fidelity Overview of Polymerase Fidelity By Julie Kramer, Marketing Manager,

Technical Article

For information about Takara’s PrimeSTAR HS see page 14

For information about Takara’s PrimeSTAR HS see page 14

For information about Takara’s 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 λ DNA). PrimeSTAR™ is the only cur- rently 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 ther- mostable 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 inacti- vates both the polymerase and exonu- clease functions during reaction assembly. This prevents false initiation events due to mispriming or primer digestion, resulting in lowered back- ground and increased reproducibility.

Takara’s 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 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.

PrimeSTAR™ Fidelity Comparison with Other DNA Polymerases and Taq.

PrimeSTAR™ HS DNA Polymerase Thermococcus kodakaraensis - derived DNA Polymerase Pyrococcus sp. - derived DNA Polymerase
PrimeSTAR™ HS DNA Polymerase
Thermococcus kodakaraensis - derived DNA Polymerase
Pyrococcus sp. - derived DNA Polymerase
Thermus aquaticus - derived DNA Polymerase
0.04
0.03
0.05
0.02
0.01
0

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; 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 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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Application Note

Comparison of Takara’s 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 reac- tion, 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, spe- cially 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 anti- body-mediated Hot Start technology to prevent non-spe- cific amplification, with an optimized real time PCR buffer formulation which provides increased amplification effi- ciency 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 real- time instruments.

Materials and Methods

qPCR amplification of the human ACTB gene was per- formed 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 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 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.

The amplification curves generated with Premix Ex Taqdemonstrate excellent sensitivity and uniformity. With the ABI 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 lower amplification efficiency than the Takara Kit. The curves lack uniformity when compared to Takara’s Kit, indicating lower reproducibility.

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

In summary, Takara Premix Ex Taq™ provides superior efficiency and reproducibility as compared to other leading qPCR kits.

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

» 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 range of 10 orders of magnitude (λ DNA template).

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

Ordering Information

Product No.

Product Name

Quantity

Price

TAK RR039A

Premix Ex Taq™ (Perfect Real Time) DNA Polymerase

200 reactions

$219

TAK RR039B

Premix Ex Taq™ (Perfect Real Time) DNA Polymerase

400 reactions

$437

TAK RR041A

SYBR ® Premix Ex Taq™ (Perfect Real Time)

200 reactions

$273

TAK RR041B

SYBR ® Premix Ex Taq™ (Perfect Real Time)

400 reactions

$534

TAK RR031A

Ex Taq™ R-PCR, Version 2.1

250 U

$189

TAK RR031B

Ex Taq™ R-PCR, Version 2.1

1000 U

$666

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

Application Note

For additional information about Takara’s qPCR products see pages 12-13
For additional information about Takara’s qPCR products see pages 12-13

For additional information about Takara’s qPCR products see pages 12-13

Takara SYBR ® Premix Ex Taq

Takara SYBR Premix Ex Taq ™ ABI’s TaqMan Universal PCR Master Mix Invitrogen’s Platinum Quantitative PCR

ABI’s TaqMan ® Universal PCR Master Mix

Takara SYBR Premix Ex Taq ™ ABI’s TaqMan Universal PCR Master Mix Invitrogen’s Platinum Quantitative PCR

Invitrogen’s Platinum ® Quantitative PCR Supermix -UDG

Takara SYBR Premix Ex Taq ™ ABI’s TaqMan Universal PCR Master Mix Invitrogen’s Platinum Quantitative PCR

Qiagen’s Quantitect Probe PCR Kit

Takara SYBR Premix Ex Taq ™ ABI’s TaqMan Universal PCR Master Mix Invitrogen’s Platinum Quantitative PCR

Figure 1:

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

Cycling conditions:

95°C, 2 min} 1 cycle

94°C, 15 sec. 55°C, 1 min. 72°C,1 min.

Takara SYBR Premix Ex Taq ™ ABI’s TaqMan Universal PCR Master Mix Invitrogen’s Platinum Quantitative PCR

45 cycles

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Application Note For additional information about Takara’s qPCR products see pages 12-13 Takara SYBR Premix Ex

Application Note

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

Application Note The pCold TF Protein Expression System Produces Soluble, Active Protein in Elucidation of protein

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 recombi- nant protein for study. E. coli expression systems are used extensively for production of recombinant proteins, and have two major advantages over other expression sys- tems: (1) ease of use, and (2) low cost. However, some recombinant proteins do not fold correctly during expres- sion in E. coli, resulting in deposits of inactive insoluble protein termed "inclusion bodies".

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 pro- tein expression vectors. The pCold Vectors provide increased in vivo protein yield, purity, and solubility of expressed recombinant proteins using "cold shock" tech- nology. 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 (37°C-15°C). This temperature drop also suppresses expression of other cel- lular 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-expres- sion of one or more chaperone proteins during expression of a heterologous target protein has proven effective for obtaining increased amounts of soluble recombinant pro- tein (see Takara's Chaperone Plasmid Set (TAK 3340)). This procedure, though, lacks the convenience of a single transformation step.

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

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 facili- tate 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 pro- teins.

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 amp r transformants.

3) Inoculate the transformants into medium including 50 µg/ml of ampicillin, and culture with shaking at

37°C.

4) At OD 600 = 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, and continue 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 insoluble fractions or activity assay.

Expression from T7 promoter-driven vectors was per- formed using a standard protocol utilizing IPTG induction and 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.

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Application Note The pCold TF Protein Expression System Produces Soluble, Active Protein in Elucidation of protein

Application Note

Chaperone 2 1 2 1 2 1 2 1 T7 * pCold pCold TF pCold +
Chaperone
2
1
2
1
2
1
2
1
T7
*
pCold
pCold TF
pCold +
22
31
45
66
97
kDa
1. Cell extract solution
2. Soluble fraction
co-expressed
target protein
trigger factor
*
3 3 1 2 3 1 2 3 1 2 2 1 2 3 97 66
3
3
1
2
3
1
2
3
1
2
2
1
2
3
97
66
45
31
22
Chaperone
2
3
kDa
solution
3. Insoluble
fraction
fraction
2. Soluble
1. Cell extract
1
pCold +
pCold
pCold I
Trx
GST
Nus
TF
1

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 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, 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 frac- tion. (Note: The molecular weight of the target protein is larger than its actual size and varies due to fused expres- sion 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.

Product Name

Quantity

Price

TAK 3365

pCold TF Vector

25 µg

$727

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Application Note Chaperone 2 1 2 1 2 1 2 1 T7 * pCold pCold TF

» FAQ Mailbox «

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 T m 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 single monoclonal antibody which blocks both PrimeSTAR™’s polymerase and nuclease activities.

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

Simple comparison of the fidelity rates available for dif- ferent PCR enzymes is not possible due to the multitude of different fidelity measurement techniques employed by different manufacturers. Takara Bio has determined PrimeSTAR™’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 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™ HS’s extremely high fidelity, with a calculated error frequency of only 0.0048%.

What is the composition of PrimeSTAR™ HS Buffer (Mg 2+ )?

The PrimeSTAR™ HS Buffer composition is proprietary.

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 effi- ciency.

What is the source of PrimeSTAR™ HS DNA poly- merase? 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).

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' exonucle- ase 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 enzy- matically phosphorylated or made using PCR primers possessing phosphoric acid residues at their 5' termini.

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» FAQ Mailbox « FAQ Mailbox PrimeSTAR™ HS DNA Polymerase FAQ Can PrimeSTAR™ HS reactions use

FAQ Mailbox

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 dH 2 O 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 dH 2 O for negative con- trol). 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 back- ground levels (i.e. threshold line), and is set in the expo- nential amplification phase to allow the most accurate reading.

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. com- posed 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 reac- tions are required for 3 genes. One package of SYBR ® Premix Ex Taq™ (Perfect Real Time) contains sufficient reagent for 200 reactions (50 µl 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 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 -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 with your hands, followed by gentle mixing. Do not vor- tex!! We have verified that this product shows good performance after the precipitate is dissolved complete- ly.

What is the composition of SYBR ® Premix Ex

Taq™?

The Premix contains Ex Taq™ Hot Start DNA Polymerase, buffer, dNTP mix, Mg 2+ and SYBR ® Green I. The Mg 2+ and SYBR ® Green I concentrations are pro- prietary.

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 inter- nal 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.

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 µl of ROX to 1 ml of SYBR ® Premix Ex Taq™. Store this solution at 4°C (protected from light), and use within one month for best performance.

ROX Reference Dye II should not be premixed prior to reaction assembly.

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FAQ Mailbox SYBR Premix Ex Taq (Perfect Real Time) FAQ How many reactions (points) are recommended

Application Note

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 mix which offers high amplification efficiency (up to 100X Taq), superior reproducibility, and extended product length (to 30 kb on λ DNA).

The Takara Thermocycler Dice provides convenience, relia- bility, multifunctionality, and a gradient feature to select temperature conditions at each step, and program a tem- perature gradient up to 20°C 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 µL amplification reactions using 1.25 U of Ex TaqDNA Polymerase and 0.5 µg of λ DNA were assembled according to manufacturer’s instructions. Half of the reac- tions were amplified using shuttle PCR under the following conditions:

In both protocols, robust, reproducible amplification was apparent.

These results demonstrate that Takara’s 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 52°C 72°C M
M 52°C
72°C M

Figure 1: Evaluation of annealing/extension tempera-

tures in two-step gradient PCR. Higher annealing/exten- sion temperatures reduced amplification of low molecular weight non-specific products, and increased target yield.

98°C

10 sec

52

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

 
98°C 10 sec 52 to 72°C 5 min 30 cycles (Gradient temperature range: 20°C) The remainder

45

to 65°C

30 sec

30 cycles

98°C 10 sec 52 to 72°C 5 min 30 cycles (Gradient temperature range: 20°C) The remainder

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 of the target product.

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

M 45°C 65°C M
M 45°C
65°C M

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.

Product Name

Quantity

Price

TAK RR001A

Ex Taq™ DNA Polymerase

250U

$158

TAK RR001B

Ex Taq™ DNA Polymerase

1000U

$567

TAK RR001C

Ex Taq™ DNA Polymerase

3000U

$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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Application Note Highly Efficient Gradient PCR of an 8 kb DNA Fragment using Takara Ex Taq

Treasure Map

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.

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2. 1. How many Resource Centers are on the website? What is the length of genomic
2.
1.
How many Resource Centers are on the website?
What is the length of genomic
DNA that LA Taq™ DNA
Polymerase can amplify?
3. How many reactions can you run with
the DNA Ligation
Kit, Mighty Mix?
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. What technology do the pCold
Vectors utilize?
8.
What is the cell transduction
efficiency of cell line
K-562 on Retronectin ® ?
6. 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
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Treasure Map Takara Mirus Bio announces the re-launching of our website at www.takaramirusbio.com. The upgraded site
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888-251-6618 • www.takaramirusbio.com

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

Featured Products

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.

Wide dynamic range: Possesses a dynamic range of 7-8 orders of magnitude (λ 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 conven- ient (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.

Amplification Curve (upper panel) and Melting Curve (lower panel) for SYBR ® Premix Ex Taq™ (Perfect
Amplification Curve
(upper panel) and
Melting Curve (lower
panel) for SYBR ®
Premix Ex Taq™
(Perfect Real Time)
on the SmartCycler ®
(Cepheid).
Ordering Information
Catalog No.
Product Name
SYBR ® Premix Ex Taq™
SYBR ® Premix Ex Taq™
Quantity
Price
TAK RR041A
200 reactions
$273
TAK RR041B
400 reactions
$534
Featured Products Featured Products SYBR Premix Ex Taq ™ (Perfect Real Time) For superior real time

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 (λ 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 Takara’s high sensitivity, high efficiency Ex TaqHot Start DNA Polymerase, and an optimized real time buffer which provides superior specificity and increased amplifica- tion efficiency for real time PCR. Antibody-mediated hot start technology prevents nonspecific amplification due to misprim- ing and/or formation of primer dimers during the reaction assembly. The Taq antibody-polymerase complex is dena- tured in the first cycling step, releasing the polymerase and allowing DNA synthesis to proceed.

Featured Products Featured Products SYBR Premix Ex Taq ™ (Perfect Real Time) For superior real time

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.

Product Name

Quantity

Price

TAK RR039A

Premix Ex Taq

200 reactions

$219

TAK RR039B

Premix Ex Taq

400 reactions

$437

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Takara Mirus Bio

888-251-6618 • www.takaramirusbio.com

Featured Products Featured Products SYBR Premix Ex Taq ™ (Perfect Real Time) For superior real time

Featured Products

Ex Taq TM R-PCR, Version 2.1

For 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 of magnitude using probe detection and 7-8 orders of mag- nitude 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 sys- tem developed specifically for real time PCR on the Cepheid 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 polymerase until the temperature is elevated. The binding of this anti- body prevents nonspecific amplification due to mispriming and/or formation of primer dimers during reaction assembly. An optimized buffer formulation provides increased speci- ficity for real time PCR, further enhancing performance.

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 For high specificity qPCR Features • High sensitivity: Detect
Featured Products Ex Taq R-PCR, Version 2.1 For high specificity qPCR Features • High sensitivity: Detect

Ordering Information

Catalog No.

Product Name

Quantity

Price

TAK RR031A

Ex Taq™ R-PCR, Ver 2.1

250 Units

$189

TAK RR031B

Ex Taq™ R-PCR, Ver 2.1

1000 Units

$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 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.

The Real Time One Step RNA PCR Kit is designed to per- form 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

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

Featured Products Ex Taq R-PCR, Version 2.1 For high specificity qPCR Features • High sensitivity: Detect

Amplification curve.

Featured Products Ex Taq R-PCR, Version 2.1 For high specificity qPCR Features • High sensitivity: Detect

Melting curve.

Detection of Rat Atp5fl with the SmartCycler ® II System. A real time One-Step RT-PCR reaction was performed using total RNA (1 pg–100 ng) prepared from rat liver 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.

Ordering Information

Catalog No.

Product Name

Quantity

Price

TAK RR026

Real Time One Step

100 reactions

$309

RNA PCR Kit

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Takara Mirus Bio

888-251-6618 • www.takaramirusbio.com

Featured Products Ex Taq R-PCR, Version 2.1 For high specificity qPCR Features • High sensitivity: Detect

New Products

New Products

pCold TF Vector

New Products Ne w Pr oducts pCold TF Vector For increased soluble protein expression Features •

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.

Takara's pCold TF DNA Vector is a fusion cold shock expres- sion vector that expresses Trigger Factor (TF) chaperone as a 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 of its E. coli origin, TF is highly expressed in E. coli expression systems. 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 cspA promoter to ensure strict regulation of expression.

cspA 3’UTR Multiple cloning site Factor Xa site Thrombin site HRV 3C Protease site Trigger Factor
cspA 3’UTR
Multiple cloning site
Factor Xa site
Thrombin site
HRV 3C Protease site
Trigger Factor (TF)
His • Tag
TEE
cspA 5’UTR
lac operator
cspA promoter
C
p
pCold TF DNA
o
(5,769 bp)
m
l
E
A
1
Ordering Information
G
Catalog No.
Product Name
Quantity
Price
TAK 3365
pCold TF
25 ug
$767
o
I
r
i
3
1
M
l
a
c
I

SPP System™ (Single Protein Production System)

New Products Ne w Pr oducts pCold TF Vector For increased soluble protein expression Features •

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 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 syn- thesis. 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 vir tu-

al 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 par ticularly suitable for structural analy- sis 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 Price TAK 3367 SPP System™ I 1 set $720
Ordering Information
Catalog No.
Product Name
Quantity
Price
TAK 3367
SPP System™ I
1 set
$720
Please visit our website for additional products.
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T akara Mirus Bio

888-251-6618 • www .takaramirusbio.com

New Products Ne w Pr oducts pCold TF Vector For increased soluble protein expression Features •

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

Try it today and prove it to yourself. Call or visit our web site to request

New Products

PrimeSTAR™ HS DNA Polymerase

Try it today and prove it to yourself. Call or visit our web site to request

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 impor- tant for amplification of cDNA libraries.

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 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, the antibody-mediated hot start formulation prevents false initi- ation events during reaction assembly due to mispriming or primer digestion, thus, lowering background.

PrimeSTAR TM Company N Company B Company I 1 2 3 4 5 1 2 3
PrimeSTAR TM
Company N
Company B
Company I
1
2
3
4
5
1
2
3
4
5
1
2
3
4
5
1
2
3
4
5

- 2kb

Comparison of PrimeSTAR TM HS Amplification Efficiency with Competitors on a 2 kb Human Genomic DNA Fragment.

Superior amplification efficiency was apparent

using PrimeSTAR TM HS on a human genomic (DCLRE1A) 2 kb template. Human genomic DNA was used in the following quantities:

Lane1:

0 ng (dH 2 O),

Lane 2: 100 pg, Lane 3:

1 ng, Lane 4: 10 ng, Lane 5: 100 ng.

M1 M2 1 2 3 4 5 6 7 8 9 10 M2 M1
M1
M2
1
2
3
4
5
6
7
8
9
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: λ-Hind III-digest

M2: pHY Marker

A Single PCR Cycling Protocol Produces Optimal Amplifcation of

Fragments of Varying Sizes.

A single protocol allows efficient amplifica-

tion of target fragments from 0.5 - 8.5 kb, making PrimeSTAR™ HS an

excellent choice for a cDNA librar y cloning.

Try it today and prove it to yourself. Call or visit our web site to request

Ordering Information

Catalog No.

Product Name

Quantity

Price

TAK R010A

PrimeSTAR™ HS

250 Units

$218

TAK R010B

PrimeSTAR™ HS

1000 Units

$785

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 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 Bio’s 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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T akara Mirus Bio

888-251-6618 • www .takaramirusbio.com

Try it today and prove it to yourself. Call or visit our web site to request

Coming Soon!

We’ve relaunched our website - check it out today!

www.takaramirusbio.com

While you browse the website, fill out our “Treasure Map” (pg. 11), to win a FREE hat.

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Order Your Copy of Takara’s 2006 Life Science Calendar!

(Available 11/05)

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Try a free sample of

PrimeSTAR™ HS DNA Polymerase!

(see pg. 15)

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

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