dNTP Guide Web

8/8/2019 dNTP Guide Web

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dNTP Guide

Primary manufacturer with facilities in Germany

ISO 9001:2000 certified Quality Management System

From micro-litres to multi-litres to lyophilisates

Free lot-specific qualification samples

Single lot reservations for up to 12 months

Individual delivery schedulesCustom testing and documentation

Custom formulations, packaging & labeling

Deoxynucleotides


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Jena Biosciences products include nucleosides, nu-

cleotides and their non-natural analogs, recombinant

proteins & protein production systems, reagents or

the crystallization o biological macromolecules and

tailor-made solutions or molecular biology and bio-

chemistry.

In our chemistry division, we have hundreds o naturaland modifed nucleotides available on stock. In addi-

tion, with our pre-made building blocks and in-house

expertise we manuacture even the most exotic nucle-

otide analog rom the mg to kg scale.

In the feld o recombinant protein production, Jena

Bioscience has developed its proprietary LEXSY tech-

nology. LEXSY (Leishmania Expression System) is based

on a S1-classifed unicellular organism that combines

easy handling with a ull eukaryotic protein olding

and modifcation machinery including mammalian-like

glycosylation. LEXSY is primarily used or the expres-

sion o proteins that are expressed at low yields or are

inactive in the established systems, and expression

levels o up to 300 mg/L o culture were achieved.

Jena BioscienceCompany profi le

For the crystallization o biological macromolecules

which is the bottleneck in determining the 3D-structure o

any protein we oer specialized reagents or crystal

screening, crystal optimization and phasing that can re-

duce the time or obtaining high quality crystals suitable

or X-ray diraction rom several years to a ew days.

Our specialized reagents are complemented with alarge selection o products or any molecular biology &

biochemistry laboratory such as kits or Standard PCR

and Real-Time PCR, oligonucleotides, cloning enzymes,

mutagenesis technologies, and many more

We combine highest quality standards or all our prod-

ucts with individualized customer support. We establish

direct lines o communication rom clients to our in-house

scientists, resulting in productive interactions among

people with similar and research interests who speak the

same language. Furthermore, we oer support programs

and attractive discount schemes or young scientists es-

tablishing their own labs. I you wish to receive more

inormation on Jena Bioscience, just send us an E-mail to

[email protected].

Jena Bioscience GmbH was founded by a team of scientists from the Max-Planck-Institute for MolecularPhysiology in Dortmund. 25+ years of academic know how were condensed into the company in orderto develop innovative reagents and technologies for the life science market.

Since the start up in 1998, the company has evolved into an established global reagent supplier withmore than 3000 products on stock and a customer base in 50+ countries. Jena Bioscience serves threemajor client groups: Research laboratories at universities, industry, government, hospitals and medical schools

Pharmaceutical industry in the process from lead discovery through to pre-clinical stages Laboratory & diagnostic reagent kit producers and re-sellers

Our company premises are located in the city of Jena / Germany with a subsidiary in Teltow, in the vicin-ity of the German capital Berlin.


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dNTP Guide

Specifications 4

Production Technology 5

Quality 5

Purity 5

(I) Nucleosidic Contaminants 6

(II) Inorganic Species 7

(III) Macromolecular Contaminants 7

Functionality 8

Sales Terms and Conditions 10

Fax Order Form 11


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Deoxynucleotides(dNTPs)

dATP; 100 mMSodium SaltSolution

dCTP; 100 mMSodium SaltSolution

dGTP; 100 mMSodium SaltSolution

dTTP; 100 mMSodium SaltSolution

dUTP; 100 mMSodium SaltSolution

Nomenclature2-Deoxyadenosine5-triphosphate

2-Deoxycytidine5-triphosphate

2-Deoxyguanosine5-triphosphate

2-Deoxythymidine5-triphosphate

2-Deoxyuridine5-triphosphate

CAS Number 1927-31-7 102783-51-7 93919-41-6 18423-43-3 102814-08-4

Catalog Number NU-1001 NU-1002 NU-1003 NU-1004 NU-1008

Formula (Anion) C10

H13

N5O

12P

3C

9H

13N

3O

13P

3C

10H

13N

5O

13P

3C

10H

14N

2O

14P

3C

9H

12N

2O

14P

3

Formula Weight(g mol-1)

488.16 464.13 504.16 479.14 465.12

Storage at -20 C at -20 C at -20 C at -20 C at -20 C

Stability24 months romcertifcation date

24 months romcertifcation date




Appearanceclear colorlesssolution

clear colorlesssolution




Concentration (1)(22C, pH 7.0)

100-110 mM( = 15.1 l mmol -1

cm-1

, 259 nm)

100-110 mM( = 8.9 l mmol -1

cm-1

, 271 nm)

100-110 mM( = 14.2 l mmol -1

cm-1

, 252 nm)

100-110 mM( = 9.5 l mmol-1

cm-1

, 267 nm)

100-110 mM( = 9.8 l mmol-1

cm-1

, 262 nm)A

250 nm/ A

260 nm

(22 C, pH 7.0)0.78 0.02 0.82 0.02 1.15 0.03 0.64 0.02 0.74 0.02

A280 nm

/ A260 nm

(22 C, pH 7.0)0.15 0.01 0.97 0.02 0.67 0.02 0.74 0.02 0.38 0.02

A290 nm

/ A260 nm

(22 C, pH 7.0)

NA 0.30 0.02 0.28 0.02 0.24 0.02 0.04 0.01

pH (4C) 8.5 0.1 8.5 0.1 8.5 0.1 8.5 0.1 8.5 0.1

dNTP (HPLC area) 99.0w% 99.0% 99.0% 99.0% 99.0%

dNDP (HPLC area) 0.9% 0.9% 0.9% 0.9% 0.9%

dNMP (HPLC area) 0.5% 0.5% 0.5% 0.5% 0.5%

Low Copy LongRange PCR(18 kb, lambda DNA,template dilutionseries)(2)

PCR ragment with100 pg o templateor less




PCR ragment with100 pg o templateor less (2)

RT-PCR (600 bp frag-ment, human GAPDHgene, template dilu-tion series)





NA

Contamination withbacterial or humanDNA

not detectable not detectable not detectable not detectable not detectable

DNases, RNases,Nicking Activity

not detectable not detectable not detectable not detectable not detectable

Proteases not detectable not detectable not detectable not detectable not detectable

(1) Extinction coefcients: Cavaluzzi & Borer (2004) Nucleic Acids Res. 32(1):e13 (2) For dUTP: Low Copy PCR (1 kb, lambda DNA, template dilution series)

Specifications


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Jena Bioscience is one o only a ew primary manuacturers o premium quality dNTPs or PCR. Our dNTPs are

synthesized by experienced personnel in Germany using enzymatic & chemical technologies ollowed by chroma-

tographic cascades. While dTTP is synthesized by chemical phosphorylation o thymidine, dATP, dCTP, dGTP, and

dUTP are manuactured rom their corresponding Ribo-NTPs in a single step employing the highly specifc enzyme

Ribonucleotide Reductase (Fig. 1). Jena Biosciences annual manuacturing capacity is multiple kilograms per year

corresponding to more than 100 L o 100 mM dNTP solutions.

Figure 1

The bacterial enzyme ribonucleotide reductase selectively reduces the 2-OH-group of NTP raw materials resulting

in their corresponding dNTPs. This enzymatic synthesis is performed on the kg-scale and reaches turnover of nearly

100% within a few days.

With increasing level o sophistication o PCR processes and regulatory requirements, highest dNTP quality with

minimal batch-to-batch variations is simply inevitable. According to Jena Biosciences DIN ISO 9001:2000 certifedquality management system each dNTP lot is assayed by most stringent criteria regarding purity and unctionality:

PurityPurity o dNTPs is one important parameter or assessing their suitability or PCR reactions run by clinical/diagnostic

laboratories or molecular biologists. Even minute amounts o impurities present in dNTP preparations may interere

with PCR, and appropriate technology or detecting and eliminating such impurities is pivotal. In principle, there are

three types o impurities that need to be addressed:

I. Nucleosidic Contaminants: Include other dNTPs (e.g. deaminated/methylated dNTPs or dNTPs with a

dierent base moiety), NTPs, and other deoxynucleoside phosphates such as dNMP, dNDP, or their tetra- and

polyphosphates.II. Inorganic Species:Include chemicals used during dNTP production/purifcation such as chloride, acetate, or

pyrophosphate as well as contaminants potentially present in raw materials (e.g. heavy metals) or resulting rom

dNTP hydrolysis (phosphate).

B = ADENINE, CYTOSINE, GUANINE, URACIL

RIBONUCLEOTIDEREDUCTASE

Production Technology

Quality


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III. Macromolecular Contaminants:These include nucleic acids (DNA or RNA) as well as traces o enzymatic

activities (DNases, RNases, Proteases, and DNA nicking activities).

(I) Nucleosidic Contaminants alongside other UV absorbing species are detected by Reversed Phase HPLC(RP-HPLC). dNTP preparations showing RP-HPLC purity o 99% (UV-absorbing contaminants 1%, respectively) are

considered state o the art. Consistent and reproducible detection o impurities in the sub-percent range however, requires

modern instrumentation as well as optimized methodology (Fig. 2). Only then reliable RP-HPLC purities o 99% can be

reported and guaranteed.

Figure 2

At Jena Bioscience a separate HPLC system (2008 Shimadzu Prominence with diode array detector SPD 20, Prontosil

column 120-5-C18 250 4.6 mm) is dedicated solely for detection of impurities in dNTP preparations. This system was

used for analysis of dATP purchased from a competitor in Q3/2009 using 0.1 M TEAA buffer/acetonitrile for elution.

Under standard conditions (pH 6.5) the apparent purity of the material is 99.1%. When adjusting buffer to pH 7.9

the resolution improves and the assay reveals that purity of dATP (96.8%) is clearly below specification. Major impurities

were separated and analyzed by mass spectrometry and identified as dADP (1.0%) and Deoxyadenosine-tetraphosphate

(1.3%).



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(II) Inorganic Species: Presence o critical concentrations o inorganic species may result rom contaminated rawmaterials and inadequate manuacturing processes. Since they interere with PCR they are commonly termed PCR

inhibitors. They do not absorb in the UV and would escape detection by RP-HPLC. Hence, Jena Bioscience uses

Analytical Anion Chromatography, GC/FID, and ICP-MS or analyses (Fig. 3).

Figure 3

Jena Biosciences maximum tolerated concentrations of common PCR inhibitors ensure their presence in a PCR reac-

tion at levels several orders of magnitude below critical values. The species are determined with a 2006 ShimadzuIon Chromatograph with a CDD 6A detector (chloride and phosphate), with a 2005 Shimadzu Gas Chromatograph

17A (acetate), a 2003 Thermo Fisher ICP-MS (all metals), and pyrophosphate is measured photometrically.

(III) Macromolecular Contaminants: The most critical macro-molecules potentially present in dNTPs are human DNA and bacterial

DNA. Enzymes o bacterial origin are commonly used during enzymatic

dNTP synthesis, and human DNA is ubiquitously present during dNTP

handling. Since the presence o only a ew copies o such genomic

DNA may result in alse positive PCR, Jena Bioscience analyses each

dNTP lot at the very end o the production process using qPCR (Fig. 4).

Testing or residual enzymatic activities is perormed with FRET-probes

(DNases, RNases, Nicking activities) and a colorimetric UV-assay is

used or detection o proteases.

SpeciesConcentration reported tobe critical in PCR

Maximum tolerated concentration inJena Biosciences dNTPs

in PCR mixture(0.2 mM = dilution 1:500) in 100 mM dNTPsolutions

Chloride Cl- 25 mM 0.004 mM 2 mM

Acetate CH3COO- 5 mM 0.004 mM 2 mM

Phosphate PO4

3- no systematic data available 0.0004 mM 0.2 mM

Pyrophosphate P2O

6

4- 0.1...0.3 mM 0.0001 mM 0.05 mM

Magnesium Mg2+

1.5 mM (1) 0.0005 mM 0.25 mM

Calcium Ca2+ 1 mM 0.0005 mM 0.25 mM

Total Heavy Metals (2) no systematic data available 0.01 g ml-1 5 g ml-1

(1) Standard concentration in PCR(2) Ba, Cd, Co, Cr, Cu, Fe, Mn, Mo, Ni, Pb, Sn, U


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Figure 4

A multiplex qPCR assay verifies absence of contaminating bacterial or human DNA. Bacterial DNA is amplified

using primers and probes for the 16S rRNA gene. Human DNA is detected by amplification of a beta-actin gene

fragment. Traces of contaminating DNA are typically detected at ct-values in between 35 and 45.

DETECTION OF HUMAN DNADETECTION OF BACTERIAL DNA

HUMAN DNABACTERIAL DNA

10 pg human DNA

100 pg human DNA

1 ng human DNA

10 ng human DNA

dATP Lot 100.846

1 pg E. coli DNA

10 pg E. coli DNA

100 pg E. coli DNA

1 ng E. coli DNA

dATP Lot 100.846

Cycle

Fluorescence

Fluorescence

Cycle



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Figure 5

Each dNTP lot is assayed under defined conditions in

an 18 kb PCR reaction with dilution of template. Amount

of template starts at 1,000 pg and is reduced to zero

(no template = negative control). Batches yielding a

visible PCR product with 100 pg of template or less are

accepted.

A: Conforming dNTP mix yielding a visible PCR product with

20 pg of template.

B: Non-conforming dNTP mix yielding a visible PCR product

with 200 pg of template.

Figure 6

Since Reverse Transcription PCR (RT-PCR) is considered

one of the most stringent assays for dNTP quality, Jena

Bioscience assays each lot in a RT-PCR template dilution

series. From decreasing amounts of human genomic

RNA (1,000 pg to zero) a 600 bp fragment of the

human GAPDH gene is amplified. dNTP lots yielding a

visible RT-PCR product with 100 pg of template or less

are accepted.

A: Conforming dNTP mix yielding a visible PCR product with

20 pg of template.

B: Non-conforming dNTP mix yielding a visible PCR product

with 200 pg of template.

Decreasing amount of template (pg) Decreasing amount of template (pg)

M 1,000 500 200 100 50 20 0 M M 1,000 500 200 100 50 20 0 M

AA

BB

Ater ensuring premium purity o dNTPs at the end o their production process, samples o each lot are subsequently

tested by a Low Copy Long Range PCR and a Reverse Transcription PCR. Both assays are perormed with limiting amount

o template which allows detection o even minute variations in dNTP perormance. (Fig. 5 & 6)

Functionality


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Jena Bioscience GmbH

Loebstedter Str. 80

07749 Jena

Germany

Phone +49 (0)3641- 628 - 5000

Fax +49 (0)3641- 628 - 5100

[email protected]

www.jenabioscience.com


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For inquiries or further information,

please contact: [email protected]

Jena Bioscience GmbH

Loebstedter Str. 8007749 Jena, Germany

Phone +49 (0)3641- 628 - 5000

Fax +49 (0)3641- 628 - 5100

How to find us in Germany

For the complete Jena Bioscience product portfolio please view

www.jenabioscience.com

Hamburg

Frankurt

Munich

Jena

Berlin

Jena

Erurt Weimar

Thuringia

Eisenach

Suhl

A71

A4

A9

A38

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dNTP Guide Web