· web viewboth non-aqueous and aqueous/organic conjugation conditions can be used. active esters:...

Reagents for Oligonucleotide Labeling

Fluorescent Dyes, Quenchers, Modified Nucleic Acid Bases and Minor Groove Binders

2016

21720 23rd Drive SE, Suite #150, Bothell, WA 98021Tel. 425-482-5177 or 425-482-5164 Fax 425-482-5550

Table of Contents

Dyes and labeling reagents ..…………………………………………………………….…………… 2Nucleic Acid Base analogues .……………………………………………………………..………… 8Epoch Blue 380 ………………………………………………………………………………….………… 136-FAM ……………………………………………..……………………………………………..…………… 19AquaPhluor 525 …..……………………………………………………………………..…..………….. 24Yakima Yellow …..…………………………………………………………………………….………….. 31AquaPhluor 559 …..…………………………………………………………………………..…………. 36AquaPhluor 570 …..………………………………………………………………………..……………. 41Redmond Red ………………………………………………………………………………….………….. 46AquaPhluor 593 …..……………………………………………………………………………………… 51AquaPhluor 639 …..…………………………………………………………………………...………… 56AquaPhluor 642 ……………………………………………………........................................ 59AquaPhluor 662 ……………………………………………….............................................. 63AquaPhluor 680 ……………………………………………….............................................. 67Quencher-470 ………………………………………………................................................. 71Eclipse Dark Quencher ………………………..……….................................................. 76Quencher-575 ………………………………………………................................................. 81Quencher-630 ………………………………………………................................................. 86Dye Calibrators .…………………………………………………………………………................... 91Super G ………………………………………………………................................................... 92Super A …………………………………………………........................................................ 95Super T ……………………………………………..…………………………….............................. 98Super I .............................................................................................................. 101CDPI3 Minor Groove Binder…………………………………………………………….….……….. 104Appendix A. Determination of the relative hydrophobicities of conjugated dyes using C18 column chromatography…………………………………… 108Appendix B. Determination of molar extinction coefficients of conjugated fluorescent dyes and quenchers………………................................... 109Appendix C. Determination of the pH dependence of fluorescence forvarious T8-conjugated dyes………………………………………………………………………….. 111Trademarks and Patents…………………………………………….…………….………………….. 112Quick reference guide………………………………………………………………………………….. 113

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Dyes and labeling reagents

ELITechGroup has been actively involved in the development of fluorescence-based nucleic acid probe technologies since the early 1990s. This experience has helped us better understand the challenges of nucleic acid probe manufacturing and the needs of end users. The proprietary reagents and techniques that we have been developing have two main goals in mind: first, to provide labeled probes with the desired physicochemical properties, and, second, to simplify the probes’ synthesis and purification. To achieve these goals we have put together a comprehensive toolbox for nucleic acid labeling which includes a wide range of fluorescent dyes and quenchers offered in a variety of reagent forms.

Our dyes are available in several fluorescent colors ranging from blue to far red. Their excitation and fluorescence emission spectra (Figures 1 and 2) have been optimized to match filter sets for most commercial fluorescence-based nucleic acid diagnostic instruments. With assay multiplexing in mind, the dyes have also been designed to maximize spectral separation and minimize channel cross-talk. The ELITechGroup’s non-fluorescent quenchers have absorption spectra covering fluorophores with emissions from 400 nm to 700 nm (Figure 3).

Figure 1 Absorption spectra of ELITechGroup’s fluorophores.

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Figure 2 Emission spectra of ELITechGroup’s fluorophores.

Figure 3 Absorption spectra of non-fluorescent quenchers: Quencher-470 (blue), Eclipse Dark Quencher (pink), Quencher-575 (Green), Quencher-630 (red).

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For most of our fluorophores and all quenchers we offer three types of labeling reagents: 2-cyanoethyl dye phosphoramidites, dye-modified oligonucleotide synthesis supports for on-line dye incorporation and amine-reactive active esters for post-synthesis conjugation. These three types of reagents are detailed below.

Phosphoramidites2-Cyanoethyl dye phosphoramidite chemistry is undeniably one of the most convenient methods for the preparation of labeled oligonucleotides. We offer dye phosphoramidites whenever they are synthetically accessible and the dyes are sufficiently stable during oligonucleotide synthesis and deprotection. Our internal phosphoramidites contain a multifunctional hydroxyprolinol linker with one primary hydroxyl, which is blocked by the DMT group, one secondary hydroxyl for phosphoramidite incorporation, and an amine for dye attachment via an amide bond. This design allows dye incorporation at any desired position within a probe’s sequence. In addition, if used in the last coupling cycle, the DMT group offers a convenient DMT-on purification handle, especially for dyes that do not significantly alter C18 HPLC retention.

Synthetically more accessible terminal phosphoramidites with a straight chain C6 linker are only suitable for single dye incorporation at the end of oligonucleotide synthesis but may be a viable alternative to the internal phosphoramidites depending on probe design and purification requirements.

Oligonucleotide Synthesis SupportsDye-labeled controlled pore glass (CPG) and polystyrene (PS) supports are very economical methods for dye labeling. Depending on whether 5’ or 3’-nucleoside phosphoramidites are used, this approach allows essentially quantitative 3’ or 5’ dye incorporation. All of our dye CPG or PS supports use the proprietary trifunctional hydroxyprolinol moiety, which provides a DMT-blocked primary hydroxyl (the starting point for oligonucleotide synthesis) and a secondary hydroxyl for connecting the dye to the synthesis support via a cleavable succinate linker.

Active estersMost of our dyes are available as amine-reactive pentafluorophenyl (PFP) esters or fused lactones with activities similar to well-known NHS esters. Both non-aqueous and aqueous/organic conjugation conditions can be used.

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Active esters: Post-synthesis conjugation using non-aqueous DMSO conditions

For conjugation applications one particularly useful property of oligonucleotides is the solubility of their trialkylammonium forms in organic solvents such as DMSO. These forms can be prepared in a variety of ways. For example, a triethylammonium form can be obtained by simple C18 reverse phase chromatography (which is usually done prior to a post-synthesis conjugation) using triethylammonium bicarbonate buffer. The volatile buffer can be readily removed by evaporation in vacuo. The resultant oligonucleotide (TEA form) is now soluble in DMSO and can be modified with a variety of agents under mostly anhydrous conditions. This approach allows use of conjugation reagents that are either insoluble or unstable in the presence of significant amounts of water, making the conjugation reaction more robust.

Another useful attribute of the non-aqueous protocol is that all oligonucleotide-related material can be easily precipitated by adding a solution of a sodium salt (such as iodide, perchlorate, etc.) in acetone, and separated from the bulk of unreacted dye, thus simplifying conjugate purification.

Overall, our experience indicates that when compared to aqueous/organic conditions, the non-aqueous conjugation procedure is more reproducible; it affords higher coupling yields and simplifies conjugate purification.

Active esters: Two-step conjugation proceduresPreparation of dye-labeled nucleic acid probes by post-synthesis conjugation may present significant challenges in terms of conjugation efficiency and separation of the resultant conjugate from unconjugated dye. The presence of unlabeled oligonucleotide will reduce the probe’s quality and ultimately its performance. On the other hand, even minor contamination with unconjugated dye may lead to an undesired increase in background signal.

Conjugates can often be separated from the starting oligonucleotide and unconjugated dye by reverse phase HPLC. It is therefore desirable, for purification purposes to use fairly hydrophobic dyes that provide significant changes in HPLC retention time. Excessive hydrophobicity, however, may lead to undesired dye aggregation and reduction in the probe’s performance. To address this dilemma we developed a two-step conjugation procedure. In the first step, the main conjugation reaction is done using a transiently protected, hydrophobic form of a

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dye followed by simple reverse phase HPLC purification. In the second step, the conjugate is treated with a deblocking agent releasing the dye in a more hydrophilic form, and purified again. Importantly, the dual HPLC purification procedure simplifies the removal of free, unincorporated dye. This approach is illustrated in the Figure 4 for a conjugation reaction between the AquaPhluor 525 lactone (M830683) and an amine-modified oligonucleotide. The AquaPhluor 525 dye, in its fully deprotected form, is highly hydrophilic and provides no useful change in the retention time for HPLC purification. On the other hand, its intermediate has the phosphonate group blocked by the TFAAB groups, which provide a very convenient purification handle. The blocking groups are removed at the next step by treatment with ammonium hydroxide generating the highly polar zwitterionic phosphonate.

PO

OO

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NH

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OO

OOHCl

ClO

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OM830683

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ClO

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NH4OH/ Deprotection

PO

O- O

NH3+

O OOHCl

Cl COO-

NH

OO P

O

O-

O ODN

Conjugation reaction

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Figure 4 Two-step post-synthesis incorporation of AquaPhluor 525 using its active lactone form (AP525 lactone, M830683).

AquaPhluor DyesOur proprietary AquaPhluor dyes have been developed for direct on-line incorporation of polar fluorescent dyes into oligonucleotide probes. In their form as labeling reagents, they possess latent phosphonate groups which are fully protected and neutral and, therefore, compatible with the phosphoramidite preparation and coupling chemistries. Upon completion of standard oligonucleotide synthesis, in their ultimate form the dyes are negatively charged and thus significantly more hydrophilic. The phosphonate chemistry of AquaPhluor dyes makes it possible to combine the versatility of automated oligonucleotide synthesis with the benefits of hydrophilicity. As an additional benefit, the phosphonate is a convenient attachment point for a variety of linking and functional groups. Based on how the phosphonate group is utilized the AquaPhluor dyes can be divided into two classes. The AquaPhluor 525, for example, has a phosphonate that is blocked with two trifluoroacetamidobutyl (TFAAB) groups, one of which is eventually removed producing a highly hydrophilic zwitterion (Figure 5).

Figure 5 Two types of AquaPhluor dyes.

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In the second class, which includes AP559, AP593, AP570, AP639, AP642, AP662 and AP680 dyes, a reactive linking group is integrated as one of the phosphonate blocking groups. After incorporation of the dye into an oligonucleotide and treatment with deblocking agents only the special blocking group is removed leaving the conjugation linkage intact. Both types of the dyes are compatible with phosphoramidite, active ester and oligonucleotide chemistries.

Nucleic Acid Base AnaloguesIn the field of molecular diagnostics, modified nucleobases are often utilized to modulate hybridization properties of natural nucleic acids (NAs). Short NA fragments are used as probes to identify specific NA targets and as primers to amplify these targets. Depending on the application it may be necessary to adjust duplex stability and specificity. For example, degeneracy of the genetic code, target polymorphism and a high rate of pathogen mutagenesis often imply strict requirements on assay design, necessitating very short probes and amplification primers. In this case, the hybridization strength of natural NA bases may not be sufficient to satisfy the assay requirements and, therefore, demand the use of some hybridization-enhancing techniques. For other applications, it may be imperative to ignore sequence variations (polymorphisms), which would require overcoming the fundamental specificity of the natural A:T and G:C base-pairs. ELITechGroup has developed a number of artificial NA base analogues to help fulfill those requirements.

Super G

Super G (8-aza-7-deazaguanosine) demonstrates approximately the same base pairing specificity as natural G. However, unlike natural G, due to the absence of the N-7 nitrogen atom, it cannot form non-canonical base pairs. As a result, Super G effectively eliminates undesired secondary structures inherent to natural G-rich sequences and improves hybridization efficiency. The Super G analogue is recommended for use in NA probes and primers to disrupt G stretches.

An additional benefit of Super G for fluorogenic hybridization probes is its low fluorescence quenching tendency, which is an intrinsic and often undesired property of natural G. The resultant Super G-modified probes demonstrate higher fluorescence efficiency and, therefore, assay sensitivity.

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Super G:C Super A:T Super T:A

NN N

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dRib

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Super A

Super A (7-hydroxybutynyl-2-amino-8-aza-7-deazaadenosine) is a duplex-stabilizing nucleoside analogue. The Super A:T base pair is much more stable than the natural A:T; it even surpasses the 2-aminoadenosine:T base pair and approaches the stability of the G:C pair. Depending on length and sequence, each Super A incorporation will increase a probe or primer’s melting temperature (Tm) by 2.5-5.5C. Generally, when Super A is located next to a G or C the stabilizing effect is larger (Ref. 3).

Super T

Super T (5-hydroxybutynyl-2’-deoxyuridine) is another duplex-stabilizing nucleoside analogue which forms a stabilized Super T:A base pair with an average Tm increase of about 1.5-3C per incorporation, presumably due to an increased stacking interaction between nucleotide bases (Ref. 1, 5). The stabilizing effect is highest when several Super Ts are incorporated consecutively.

Figure 6 Structures of Super G, Super A, and Super T and their corresponding complementary base pairs. Guidelines for incorporation of Super A and Super T bases into

PCR primersAn example of the typical benefits of primers containing modified bases is shown in Figure 7 where they are used to amplify the highly variable gag gene region of HIV. Only short sequences are conserved in that region and therefore available for primer design. The unmodified primer pair did not produce any visible PCR products on the agarose gel, whereas the primer pair stabilized by multiple incorporations of Super A and Super T (Forward ACCAAGGAAGC, Reverse

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1 2 3 4 5 6 7 8 9 10

CCTTCTGATAATGCTG) produced a good band of product with the expected 240 bp size. Other examples of successful applications of Super A and Super T can be found in research literature (Ref. 1-6)

Figure 7 Stabilizing moieties of Super A and Super T in short primers improve amplification of highly variable templates.Cloned HIV variants (gag gene region) were amplified with 0.5 µM each modified or unmodified primers using JumpStart™ Taq ReadyMix™ (Sigma, St. Louis, MO, USA) using the following cycling parameters: 2 min at 95°C, followed by 50 cycles of 15 s at 95°C and 1 min at 60°C. PCR products were run on a 4% agarose gel. Lanes 2-5 indicate the modified primer pair amplification and lanes 7-10 indicated the unmodified primer pair amplification. Lanes 2 &7 are no template controls. Lane 3 & 8 contain the pN43 (wild type) plasmid template. Lanes 4 & 9 contain the pN43 – K103N (mutant) plasmid template. Lanes 5 & 10 contain the pN43 – V108I (mutant) plasmid template. Lanes 1 & 6 contain a 100 bp DNA ladder.

It should be noted that PCR efficiency is also affected by factors beyond thermodynamic properties of primers. DNA polymerase must read through the modified bases without stuttering or pausing, and modified primers have to be efficiently extended. We have conducted experiments and summarized general guidelines for using Super A and Super T in PCR primers.

- Usual PCR Tm requirements should be used to ensure primer annealing.- Both Super A and Super T must not be used at the 3’ end.- Both Super A and Super T can be used in the second base from the 3’ end.- Super A should be used sparingly.- Multiple Super As should be separated by at least three natural bases or

Super T.- Super T can be used liberally.

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Super I:T Super I:C Super I:A

Super I:A Super I:G Super I:G

NN N

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dRib

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Super ISuper Inosine (Super I) is a nucleoside analogue that contains a quasi-universal nucleobase (7-aminobutynyl-8-aza-7-deazahypoxanthine) with improved hybridization and PCR enhancing properties (Ref. 7) as compared to commonly used 2’-deoxyinosine. It demonstrates improved ambiguity for pairing with A, T and C bases and its base pairing properties can be summarized as follows: X:C~X:A~X:T>X:G. Base pairing strength of Super I with A, T and C is approximately equal or slightly greater (depending on nearest neighbors) than the natural A:T but lower than the G:C, with the Super I:C base pair being slightly more stable than Super I:A and Super I:T. The improvement in PCR performance directly correlated with primers’ Tm. Primers with multiple Super I incorporations can be used without noticeable inhibition of Taq polymerase activity provided the modifications are not used in stretches and positioned more than 2 bases away from the 3’ end.

Figure 8 Possible hydrogen bonding patterns of Super I with natural nucleobases.

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Comparison of Super I and 2’-deoxyinosineWe have determined that stabilities of Super I:A, T and C base pairs are approximately equal or slightly greater (depending on nearest neighbors) than the natural A:T but lower than G:C, with Super I:C being slightly more stable than Super I:A and Super I:T. The beneficial stabilization effect of Super I pairs with A, T and C is especially pronounced for the multiply-substituted duplexes, which are generally more stable than the respective natural matched duplexes. Conversely, 2’-deoxyinosine forms base pairs with all four natural bases that are weaker than the A:T base pair. Moreover, multiple incorporations of 2’-deoxyinosine render modified duplexes much less stable than the respective natural matched duplexes. The much weaker Super I:G base pair is approximately equivalent to the dI:G, both of which significantly destabilize the DNA duplexes. For practical applications such as PCR primer design where maintaining primer melting temperature is imperative, these results imply that single and especially multiple substitutions of A, T and G bases with Super I would be thermodynamically favorable whereas substitutions of cytosines should be avoided.

REFERENCES1. Belousov, Y., Welch, R., Sanders, S., Mills, A., Kulchenko, A., Dempcy, R., Afonina, I., Walburger,

D., Glaser, C., Yadavalli, S., Vermeulen, N., and Mahoney, W. 2004. Single Nucleotide Polymorphism Genotyping by Two Color Melting Curve Analysis Using the MGB Eclipse™ Probe System in Challenging Sequence Environment. Human Genomics 1: 209-217.

2. Afonina, I., Belousov, Y., Metcalf, M., Mills, A., Sanders, S., Walburger, D., Mahoney, W., and Vermeulen, N. 2004. Single Nucleotide Polymorphism Detection with Fluorescent MGB Eclipse Probe Systems. In: A-Z of Quantitative PCR, Stephen A. Bustin Editor. IUL Biotechnology Series, International University Line, La Jolla, CA. (Chapter 23) pp 717-730.

3. Afonina, I., Mills, A., Sanders, S., Kulchenko, A., Dempcy, R., Lokhov, S., Vermeulen, N., and Mahoney, W. 2007. Improved Biplex Quantitative Real-Time Polymerase Chain Reaction with Modified Primers for Gene Expression Analysis. Oligonucleotides 16(4):401-409.

4. Afonina, I., Ankoudinova, I., Mills, A., Lokhov, S., Huynh, P., and Mahoney, W. 2007. Primers with 5’-flaps Improve Real-Time PCR. BioTechniques. Dec; 43(6):770-773.

5. Hymas, W., Stevenson, J., Taggart, E. and Hillyard, D. 2005. Use of lyophilized standards for the calibration of a newly developed real time PCR assay for human herpes type six (HHV6) variants A and B. Journal of Virological Methods. 128(1–2):143–50.

6. Hymas, W., Atkinson, A., Stevenson, J., and Hillyard, D. 2007. Use of modified oligonucleotides to compensate for sequence polymorphisms in the real-time detection of norovirus. Journal of Virological Methods. 142(1–2):10–4.

7. Vorobiev, A V., Scarr, N. K., Belousov, Y. and Lukhtanov, E. A. 2013. 7-Aminobutynyl-8-aza-7-deazahypoxanthine as a quasi-universal nucleobase. Nucleosides, Nucleotides and Nucleic Acids. 32(8), 421-438.

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Epoch Blue 380Acronym: EB380

ONO

O

Epoch Blue 380 is a UV light-excitable blue fluorescent aminocoumarin dye with excitation and emission properties similar to those of Marina Blue and Alexa Fluor 350. It is fully compatible with oligonucleotide synthesis and deprotection.

Absorbance maximum* 381 nmEmission maximum* 460 nmExtinction coefficient (260 nm) 7,000 M-1cm-1

Extinction coefficient (260 nm) 17,200 M-1cm-1 (Epoch Blue 380-HEG)Extinction coefficient (381 nm) 20,000 M-1cm-1

Fluorescence quantum yield**() 0.24Hydrophobicity by C18 chromatography*** 22.0 (moderately hydrophobic)pH dependence of dye fluorescence Independent between pH of 5 and 8 (Appendix C)

Available Epoch Blue 380 products Product numberEpoch Blue 380 PFP ester M830726Epoch Blue 380 Internal Amidite M830192Epoch Blue 380-HEG Amidite M830727Epoch Blue 380 CPG M830669Epoch Blue 380 Polystyrene **** M830670 (bulk), M100441 (columns)

*Measured for T8-Epoch Blue 380 conjugate (50 mM Tris-HCl pH 8.5)**Measured for T8-Epoch Blue 380 conjugate (0.1 M Sodium tetraborate)***Percent acetonitrile required for elution of T8-EB380-HEG Dye Calibrator (M300585) from C18 column (see page 108 for details)****Suitable for ABI 3900 DNA synthesizer

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Epoch Blue 380 PFP esterProduct number: M830726

ONO

OO

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O-

OConjugation reaction

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ONH

OP

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O

Formula C21H16F5NO4

Molecular weight (reagent) 441.35 DaMolecular weight (incorporated) 257.28 DaPurity* (reverse phase HPLC) >90%Structure identity test 1H NMRRecommended coupling conditions See conjugation protocolDeprotection conditions N/ARecommended storage conditions/Stability Desiccated at < -10C/ 1 yearAvailable documents Certificate of analysis, conjugation protocols

*Purity of this active derivative is verified by HPLC, NMR and a test reaction with an aliphatic amine and usually exceeds 90%. Some loss of activity, however, may occur during packaging and storage, especially for small (1-10 mg) unit sizes.

Available unit size 1 mg 10 mg bulk

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Epoch Blue 380 Internal AmiditeProduct number: M830192

ONO

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Formula C50H61N4O8PMolecular weight (reagent) 877.03 DaMolecular weight (incorporated) 436.39 DaPurity (reverse phase HPLC) >90%Purity of test oligo (reverse phase HPLC) >85%Identity before/after coupling 1H, 31P NMR /Mass spectroscopyRecommended coupling conditions Double amidite addition and extended coupling timeRecommended deprotection conditions 30% NH4OH 55C 17 h or 70C 2 hRecommended storage conditions/Stability Desiccated at < -10C/ 2 yearsAvailable documents Certificate of analysis

Available unit size 50 µmol 100 µmol 250 mg 1 g Bulk

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Epoch Blue 380-HEG AmiditeProduct number: M830727

Oligo Synthesis

and deprotection

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Formula C69H90N5O15PMolecular weight (reagent) 1260.45 DaMolecular weight (incorporated) 819.83 DaPurity (reverse phase HPLC) >90%Purity of test oligo (reverse phase HPLC) >85%Identity before/after coupling 1H, 31P NMR /Mass-spectroscopyRecommended coupling conditions Double amidite addition and extended coupling time Recommended deprotection conditions 30% NH4OH 55C 17 h or 70C 2 hRecommended storage conditions/Stability Desiccated at < -10C/ TBDAvailable documents Certificate of analysis


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Epoch Blue 380 CPGProduct number: M830669

Oligo Synthesis

and deprotection

ONO

ON

O

DMTO

O

NH-CPGO

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ON

OH

OPOO

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O

Formula N/AMolecular weight (reagent) N/AMolecular weight (incorporated) 436.39 DaCapacity by DMT loading >15 µmol/gPore size ~1000 ÅPurity of test oligo (reverse phase HPLC) >85%Recommended coupling conditions N/ARecommended deprotection conditions 30% NH4OH 55C 17 h or 70C 2 hRecommended storage conditions/Stability Desiccated at 4C/ TBDAvailable documents Certificate of analysis

Available unit size 0.1 g 250 mg 1 g Bulk

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Epoch Blue 380 PolystyreneProduct numbers: M830670 (bulk), M100441 (columns)

Oligo Synthesis

and deprotection

ONO

ON

O

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OH

OPOO

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O

Formula N/AMolecular weight (reagent) N/AMolecular weight (incorporated) 436.39 DaCapacity by DMT loading >10 µmol/gPurity of test oligo (reverse phase HPLC) >85%Recommended coupling conditions N/ARecommended deprotection conditions 30% NH4OH 55C 17 h or 70C 2 hRecommended storage conditions/Stability Desiccated at 4C/ TBDAvailable documents Certificate of analysis

Available unit size Pack of 3 columns* Pack of 10 columns* Bulk

*ABI 3900 type columns, ~200 nmol/column

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6-FAM

O OOH

COOH

O

FAM (6-Carboxyfluorescein) is a popular green fluorescent dye compatible with standard oligonucleotide synthesis and deprotection.

Absorbance maximum* 496 nmEmission maximum* 517 nmExtinction coefficient (260 nm) 33,300 M-1cm-1 (FAM-HEG)Extinction coefficient (496 nm) 80,400 M-1cm-1

Fluorescence quantum yield**() 0.88Hydrophobicity by C18 chromatography*** 19.2 (hydrophilic)pH dependence of dye fluorescence pKa 6.8-7.0 (see Appendix C)

Available FAM products Product number6-FAM Amidite M100011FAM-HEG Amidite M830100FAM-HEG CPG M830719FAM-HEG Polystyrene **** M830718 (bulk), M100442 (columns)

*Measured for T8-FAM-HEG conjugate (50 mM Tris-HCl pH 8.5)**Measured for T8-FAM-HEG conjugate (0.1 M Sodium tetraborate)***Percent acetonitrile required for elution of T8-FAM-HEG Dye Calibrator (M300586) from C18 column (see page 108 for details)****Suitable for ABI 3900 DNA synthesizer

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6-FAM AmiditeProduct number: M100011

O OO

O

NHO

PN

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and deprotection

O OOH

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O-

O

Formula C46H58N3O10PMolecular weight (reagent) 843.94 DaMolecular weight (incorporated) 537.45 DaPurity (reverse phase HPLC) >90%Purity of test oligo (reverse phase HPLC) >85%Identity before/after coupling 1H, 31P NMR /Mass-spectroscopyRecommended coupling conditions Double amidite addition and extended coupling time Recommended deprotection conditions 30% NH4OH 55C 17 h or 70C 2 hRecommended storage conditions/Stability Desiccated at < -10C / 7 yearsAvailable documents Certificate of analysis


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FAM-HEG AmiditeProduct number: M830100

O OO

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Formula C85H101N4O20PMolecular weight (reagent) 1529.7 DaMolecular weight (incorporated) 920.84 DaPurity (reverse phase HPLC) >90%Purity of test oligo (reverse phase HPLC) >85%Identity before/after coupling 1H, 31P NMR /Mass-spectroscopyRecommended coupling conditions Double amidite addition and extended coupling time Recommended deprotection conditions 30% NH4OH 55C 17 h or 70C 2 hRecommended storage conditions/Stability Desiccated at < -10C/ 5 yearsAvailable documents Certificate of analysis


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FAM-HEG CPGProduct number: M830719

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Available unit size 100 mg 250 mg 1 g Bulk

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FAM-HEG PolystyreneProduct numbers: M830718 (bulk), M100442 (columns)

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AquaPhluor 525Acronym: AP525

O OCl

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Cl OP

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NH3+

AquaPhluor 525 is a hydrophilic fluorescent dye with excitation and emission properties similar to VIC, JOE, HEX and Alexa Fluor 532. It possesses a zwitterionic 4-aminobutyl- phosphonate group for increased hydrophilicity and reduced aggregation. The latent phosphonate is conveniently protected in the reactive forms of the dye.

Absorbance maximum* 527 nmEmission maximum* 549 nmExtinction coefficient (260 nm) 41,000 M-1cm-1 Extinction coefficient (527 nm) 99,300 M-1cm-1

Fluorescence quantum yield**() 0.73Hydrophobicity by C18 chromatography*** 18.6 (hydrophilic)pH dependence of dye fluorescence pKa 6.8-7.0 (see Appendix C)

Available AquaPhluor 525 Products Product numberAquaPhluor 525 Lactone M830683AquaPhluor 525 Internal Amidite M830199AquaPhluor 525 Terminal Amidite M830714AquaPhluor 525-HEG Amidite M100104AquaPhluor 525 CPG M830671AquaPhluor 525 Polystyrene**** M830672 (bulk), M100276 (columns)*Measured for T8-AP525 conjugate (50 mM Tris-HCl pH 8.5)**Measured for T8-AP525 conjugate (0.1 M Sodium tetraborate)***Percent acetonitrile required for elution of T8-AP525 Dye Calibrator (M300587) from C18 column (see page 108 for details)****Suitable for ABI 3900 DNA synthesizer

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AquaPhluor 525 LactoneProduct number: M830683

PO

OO

NHF3C

NH

O

OO

OOHCl

ClO

F3C O

O

NH2O P

O

O-

O

PO

OO

NHF3C

NH

O OOHCl

ClO

F3C O

COO-

NH

OO P

O

O-

O

NH4OH/ Deprotection

PO

O- O

NH3+

O OOHCl

Cl COO-

NH

OO P

O

O-

O


Formula C41H33Cl2F6N2O11PMolecular weight (reagent) 945.58 DaMolecular weight (incorporated) 682.44 DaPurity* (reverse phase HPLC) >90%Coupling efficiency test >90%Structure identity test 1H NMRRecommended coupling conditions See conjugation protocolRecommended deprotection conditions See conjugation protocolRecommended storage conditions/Stability Desiccated at < -10C / 1 yearAvailable documents Certificate of analysis, conjugation protocols



26

AquaPhluor 525 Internal AmiditeProduct number: M830199

Oligo Synthesis

and deprotection

N

O

DMTO

PN

O

N

O OO

OO

OCl

O

PO

ONH

O

NH

CF3

O

O CF3

O

Cl

PO

O-

O

N

O

O

O OOH

COO-

Cl

PO

-

OO

NH3+

O

Cl

Formula C86H95Cl2F6N5O18P2

Molecular weight (reagent) 1733.54 DaMolecular weight (incorporated) 861.55 DaPurity (reverse phase HPLC) >90%Purity of test oligo (reverse phase HPLC) >85%Identity before/after coupling 1H, 31P NMR /Mass-spectroscopyRecommended coupling conditions Double amidite addition and extended coupling time Recommended deprotection conditions 30% NH4OH 55C 17 h or 70C 2 hRecommended storage conditions/Stability Desiccated at < -10C / 2.5 yearsAvailable documents Certificate of analysis


27

AquaPhluor 525 Terminal AmiditeProduct number: M830714

Oligo Synthesis

and deprotection

O OCl

O

ClP

O

OO

OO

OO

NH

NHF3C

O

O CF3

NH

OO

PN

O

N

O OCl

OH

COO-

ClP

O

O- O

NH3+

NH

OO P

O

O-O


Molecular weight (reagent) 1431.23 DaMolecular weight (incorporated) 861.59 DaPurity (reverse phase HPLC) >90%Purity of test oligo (reverse phase HPLC) >85%Identity before/after coupling 1H, 31P NMR /Mass-spectroscopyRecommended coupling conditions Double amidite addition and extended coupling time Recommended deprotection conditions 30% NH4OH 55C 17 h or 70C 2 hRecommended storage conditions/Stability Desiccated at < -10C / 2 yearsAvailable documents Certificate of analysis


28

AquaPhluor 525-HEG AmiditeProduct number: M100104

Oligo Synthesis

and deprotection

O

NHO

OO

OO

O

O

N

O

DMTO

O OO

OO

OCl

O

PO

ONH

O

NH

CF3

O

O CF3

PN

O

N

Cl

PO

O-

O

O

NHO

OO

OO

O

O

N

O

O

O OOH

COO-

Cl

PO

-

OO

NH3+Cl


Molecular weight (reagent) 2116.98 DaMolecular weight (incorporated) 1244.98 DaPurity (reverse phase HPLC) >90%Purity of test oligo (reverse phase HPLC) >85%Identity before/after coupling 1H, 31P NMR /Mass-spectroscopyRecommended coupling conditions Double amidite addition and extended coupling time Recommended deprotection conditions 30% NH4OH 55C 17 h or 70C 2 hRecommended storage conditions/Stability Desiccated at < -10C / 5 yearsAvailable documents Certificate of analysis


29

AquaPhluor 525 CPGProduct number: M830671

Oligo Synthesis

and deprotection

N

O

DMTO

O

O OO

OO

OCl

O

PO

ONH

O

NH

CF3

O

O CF3

O

ONH-CPG

Cl

N

OH

O

O OOH

COO-

Cl

PO

-

OO

NH3+

O

Cl

POO

-

O



30

AquaPhluor 525 PolystyreneProduct numbers: M830672 (bulk), M100276 (columns)

Oligo Synthesis

and deprotection

N

O

DMTO

O

O OO

OO

OCl

O

PO

ONH

O

NH

CF3

O

O CF3

O

ONH-PS

Cl

N

OH

O

O OOH

COO-

Cl

PO

-

OO

NH3+

O

Cl

POO

-

O

Formula N/AMolecular weight (reagent) N/AMolecular weight (incorporated) 861.55 DaCapacity by DMT loading >10 µmol/gPurity of test oligo (reverse phase HPLC) >85%Recommended coupling conditions N/ARecommended deprotection conditions 30% NH4OH 55C 17 h or 70C 2 hRecommended storage conditions/Stability Desiccated at 4C/ 5 years (bulk)Available documents Certificate of analysis



31

Yakima Yellow®Acronym: YY

OCH3

OH OCl

ClOCOOH

ClCl

Yakima Yellow is a fluorescent dye with excitation and emission properties similar to VIC, JOE, HEX and Alexa Fluor 532. It is fully compatible with standard oligonucleotide synthesis and deprotection.


Extinction coefficient (525 nm) 86,500 M-1cm-1

Fluorescence quantum yield**() 0.57Hydrophobicity by C18 chromatography*** 25.6 (Moderately hydrophobic)pH dependence of dye fluorescence Independent above pH of 7 (see Appendix C)

Available Yakima Yellow Products Product numberYakima Yellow Lactone M830684Yakima Yellow Phosphoramidite M830552Yakima Yellow CPG M830395Yakima Yellow Polystyrene**** M400063 (bulk), M100443 (columns)

*Measured for T8-YY conjugate (50 mM Tris-HCl pH 8.5)**Measured for T8-YY conjugate (0.1 M Sodium tetraborate)***Percent acetonitrile required for elution of T8-YY Dye Calibrator (M830730) from C18 column (see page 108 for details)****Suitable for ABI 3900 DNA synthesizer

32

Yakima Yellow LactoneProduct number: M830684

OCH3

OH OCl

Cl

ClCl

O

OO

NH2 O P

O

O-

O


OCH3

OH OCl

Cl

ClCl

COO-

O

NHO P

O

O-

O

Formula C24H12Cl4O6

Molecular weight (reagent) 538.16 DaMolecular weight (incorporated) 538.16 Da Purity* (reverse phase HPLC) >90%Coupling efficiency test >90%Structure identity test 1H NMRRecommended coupling conditions See conjugation protocolDeprotection conditions N/ARecommended storage conditions/Stability Desiccated at < -10C / 1 yearAvailable documents Certificate of analysis, conjugation protocols



33

Yakima Yellow PhosphoramiditeProduct number: M830552

OCH3

OH OCl

Cl

ClCl

COO-

O

NHO P

O

O-

O

OCH3

O OCl

Cl

ClCl

O

NHO

PO

N

N

OO

OO

Oligo Synthesis

and deprotection

Formula C49H60Cl4N3O10PMolecular weight (reagent) 1023.81 DaMolecular weight (incorporated) 717.31 DaPurity (reverse phase HPLC) >90%Purity of test oligo (reverse phase HPLC) >85%Identity before/after coupling 1H, 31P NMR /Mass-spectroscopyRecommended coupling conditions Double amidite addition and extended coupling time Recommended deprotection conditions 30% NH4OH 55C 17 h or 70C 2 hRecommended storage conditions/Stability Desiccated at < -10C / 5 yearsAvailable documents Certificate of analysis

Available unit size 50 µmol 100 µmol 250 mg 1 g

34

Yakima Yellow CPGProduct number: M830395

Oligo Synthesis

and deprotection

OCH3

OH OCl

Cl

ClCl

COO-

O

N

OH

O P

O

O-

O

OCH3

O OCl

Cl

ClCl

O

OO

OO

N

O

ODMT

O

NH-CPGO

Formula N/AMolecular weight (reagent) N/AMolecular weight (incorporated) 717.27 DaCapacity by DMT loading >15 µmol/gPore size ~1000 ÅPurity of test oligo (reverse phase HPLC) >85%Recommended coupling conditions N/ARecommended deprotection conditions 30% NH4OH 55C 17 h or 70C 2 hRecommended storage conditions/Stability Desiccated at 4C/ 5 yearsAvailable documents Certificate of analysis


35

Yakima Yellow PolystyreneProduct numbers: M400063 (bulk), M100443 (columns)

Oligo Synthesis

and deprotection

OCH3

OH OCl

Cl

ClCl

COO-

O

N

OH

O P

O

O-

O

OCH3

O OCl

Cl

ClCl

O

OO

OO

N

O

ODMT

O

NH-PSO

Formula N/AMolecular weight (reagent) N/AMolecular weight (incorporated) 717.27 DaCapacity by DMT loading >10 µmol/gPurity of test oligo (reverse phase HPLC) >85%Recommended coupling conditions N/ARecommended deprotection conditions 30% NH4OH 55C 17 h or 70C 2 hRecommended storage conditions/Stability Desiccated at 4C/ 5 yearsAvailable documents Certificate of analysis



36


OP

O- O

ON

N

O

OCl

AquaPhluor 559 is a fluorescent dye with excitation and emission properties similar to TAMRA and NED. The dye is compatible with oligonucleotide synthesis and deprotection.



Fluorescence quantum yield**() 0.64Hydrophobicity by C18 chromatography*** 25.6 (Moderately hydrophobic)pH dependence of dye fluorescence Weakly dependent between pH of 5 and 8 (see

Appendix C)

Available AquaPhluor 559 Products Product numberAquaPhluor 559 Internal Amidite M830195AquaPhluor 559 Terminal Amidite M830698AquaPhluor 559 CPG M830673AquaPhluor 559 Polystyrene **** M830674 (bulk), M100278 (columns)

*Measured for T8-AP559 conjugate (50 mM Tris-HCl pH 8.5)**Measured for T8-AP559 conjugate (0.1 M Sodium tetraborate)***Percent acetonitrile required for elution of T8-AP559 Dye Calibrator (M301430) from C18 column (see page 108 for details)****Suitable for ABI 3900 DNA synthesizer

37


OO

PO

NHO

O CF3

ON

N

O

ClO

PN

ON

Oligo Synthesis

and deprotectionO

OPO

- O

ON

N

O

ClO

PO

-

OO

Formula C51H67ClF3N5O9P2

Molecular weight (reagent) 1048.5 DaMolecular weight (incorporated) 743.11 DaPurity (reverse phase HPLC) >75%Purity of test oligo (reverse phase HPLC) >80%Identity before/after coupling 1H, 31P NMR /Mass-spectroscopyRecommended coupling conditions Multiple amidite additions and extended coupling time Recommended deprotection conditions 30% NH4OH 55C 17 h or 70C 2 hRecommended storage conditions/Stability Desiccated at < -10C / 2 yearsAvailable documents Certificate of analysis


38


Oligo Synthesis

and deprotection

OPO

O

ON

N

O

ClO

O

N

O

DMTO

NH

O CF3

PN

O

NO

PO

- O

ON

N

O

ClO

O

N

O

O

P OOO

-


Molecular weight (reagent) 1463.98 DaMolecular weight (incorporated) 856.23 DaPurity (reverse phase HPLC) >80%Purity of test oligo (reverse phase HPLC) >50%Identity before/after coupling 1H, 31P NMR /Mass-spectroscopyRecommended coupling conditions Multiple amidite additions and extended coupling time Recommended deprotection conditions 30% NH4OH 55C 17 h or 70C 2 hRecommended storage conditions/Stability Desiccated at < -10C / TBDAvailable documents Certificate of analysis


39


Oligo Synthesis

and deprotection

OPO

O

ON

N

O

ClO

O

N

O

DMTO

O

NH-CPGO

NH

O CF3

OPO

- O

ON

N

O

ClO

O

N

OH

OPO

-

OO

Formula N/AMolecular weight (reagent) N/AMolecular weight (incorporated) 856.23 DaCapacity by DMT loading >15 µmol/gPore size ~1000 ÅPurity of test oligo (reverse phase HPLC) >85%Coupling efficiency test N/ARecommended coupling conditions N/ARecommended deprotection conditions 30% NH4OH 55C 17 h or 70C 2 hRecommended storage conditions/Stability Desiccated at 4C/ TBDAvailable documents Certificate of analysis


40


Oligo Synthesis

and deprotection

OPO

O

ON

N

O

ClO

O

N

O

DMTO

O

NH-PSO

NH

O CF3

OPO

- O

ON

N

O

ClO

O

N

OH

OPO

-

OO

Formula N/AMolecular weight (reagent) N/AMolecular weight (incorporated) 856.23 DaCapacity by DMT loading >10 µmol/gPurity of test oligo (reverse phase HPLC) >85%Recommended coupling conditions N/ARecommended deprotection conditions 30% NH4OH 55C 17 h or 70C 2 hRecommended storage conditions/Stability

Desiccated at 4C/ TBD

Available documents Certificate of analysis



41


OP

O- O

ON

N

O

OCl

AquaPhluor 570 is a fluorescent dye with excitation and emission properties similar to Redmond Red. Unlike Redmond Red it is pH insensitive and fully compatible with standard oligonucleotide synthesis and deprotection.



Fluorescence quantum yield**() 0.68Hydrophobicity by C18 chromatography*** 29.4 (Hydrophobic)pH dependence of dye fluorescence Weakly dependent between pH of 5 and 8

(Appendix C)

Available AquaPhluor 570 Products Product numberAquaPhluor 570 Internal Amidite M830675AquaPhluor 570 Terminal Amidite M830699AquaPhluor 570 CPG M830676AquaPhluor 570 Polystyrene **** M830677 (bulk), M100444 (columns)


42


OO

PO

NHO

O CF3

ON

N

O

ClO

PN

ON OO

PO

- O

ON

N

O

ClO

PO

-

OO

Oligo Synthesis

and deprotection


Molecular weight (reagent) 1124.59 DaMolecular weight (incorporated) 819.2 DaPurity (reverse phase HPLC) >85%Purity of test oligo (reverse phase HPLC) >85%Identity before/after coupling 1H, 31P NMR /Mass-spectroscopyRecommended coupling conditions Double amidite addition and extended coupling time Recommended deprotection conditions 30% NH4OH 55C 17 h or 70C 2 hRecommended storage conditions/Stability Desiccated at < -10C / TBDAvailable documents Certificate of analysis


43


Oligo Synthesis

and deprotection

OPO

O

ON

N

O

ClO

O

N

O

DMTO

NH

O CF3

PN

O

NO

PO

- O

ON

N

O

ClO

O

N

O

O

P OOO

-


Molecular weight (reagent) 1540.08 DaMolecular weight (incorporated) 932.33 DaPurity (reverse phase HPLC) >85%Purity of test oligo (reverse phase HPLC) >85%Identity before/after coupling 1H, 31P NMR /Mass-spectroscopyRecommended coupling conditions Double amidite addition and extended coupling time Recommended deprotection conditions 30% NH4OH 55C 17 h or 70C 2 hRecommended storage conditions/Stability Desiccated at < -10C / TBDAvailable documents Certificate of analysis


44


Oligo Synthesis

and deprotection

OPO

O

ON

N

O

ClO

O

N

O

DMTO

O

NH-CPGO

NH

O CF3

OPO

- O

ON

N

O

ClO

O

N

OH

OPO

-

OO



45


Oligo Synthesis

and deprotection

OPO

O

ON

N

O

ClO

O

N

O

DMTO

O

NH-PSO

NH

O CF3

OPO

- O

ON

N

O

ClO

O

N

OH

OPO

-

OO




46

Redmond Red®Acronym: RR

O

N

OH O

O

Redmond Red is a fluorescent dye with excitation and emission properties similar to those of AquaPhluor 570. It is compatible with oligonucleotide synthesis. Mild deprotection conditions are recommended.



Fluorescence quantum yield**() 0.46Hydrophobicity by C18 chromatography*** 17.9 (Hydrophilic)pH dependence of dye fluorescence Highly pH-dependent, pKa ~7 (see Appendix C)

Available Redmond Red Products Product numberRedmond Red Lactone M830728Redmond Red Phosphoramidite M830035Redmond Red CPG M830390Redmond Red Polystyrene**** M830729 (bulk), M100445 (columns)

*Measured for T8-RR conjugate (50 mM Tris-HCl pH 8.5)**Measured for T8-RR conjugate (0.1 M Sodium tetraborate)***Percent acetonitrile required for elution of T8-RR Dye Calibrator (M830731) from C18 column (see page 108 for details)****Suitable for ABI 3900 DNA synthesizer

47

Redmond Red LactoneProduct number: M830728

O

N

O O O

NH2 O P

O

O-

O


O

N

OH O

O

NHO P

O

O-

O

Formula C15H9NO4

Molecular weight (reagent) 267.24 DaMolecular weight (incorporated) 267.24 Da Purity* (reverse phase HPLC) >90%Coupling efficiency test >90%Structure identity test 1H NMRRecommended coupling conditions See conjugation protocolDeprotection conditions N/ARecommended storage conditions/Stability Desiccated at < -10C / 1 yearAvailable documents Certificate of analysis, conjugation protocols



48

Redmond Red PhosphoramiditeProduct number: M830035

Oligo Synthesis

and deprotectionO

N

O O

O

O

N

O

ODMT

PN

O

N

O

N

OH O

O

N

O

O

P

O

O-

O

Formula C55H63N4O10PMolecular weight (reagent) 971.08 DaMolecular weight (incorporated) 446.34 DaPurity (reverse phase HPLC) >90%Purity of test oligo (reverse phase HPLC) >85%Identity before/after coupling 1H, 31P NMR /Mass-spectroscopyRecommended coupling conditions Double amidite addition and extended coupling time Recommended deprotection conditions tert-BuNH2:MeOH:H2O (1:2:1, v:v:v), 17 h at 50-55CRecommended storage conditions/Stability Desiccated at < -10C / 5 yearsAvailable documents Certificate of analysis


49

Redmond Red CPGProduct number: M830390

Oligo Synthesis

and deprotection

O

N

OH O

O

N

OH

O P

O

O-

O

O

N

O O

O

O

N

O

ODMT

O

NH-CPGO

Formula N/AMolecular weight (reagent) N/AMolecular weight (incorporated) 446.34 DaCapacity by DMT loading >15 µmol/gPore size ~1000 ÅPurity of test oligo (reverse phase HPLC) >85%Recommended coupling conditions N/ARecommended deprotection conditions tert-BuNH2:MeOH:H2O (1:2:1, v:v:v), 17 h at 50-55CRecommended storage conditions/Stability Desiccated at 4C/ 3 yearsAvailable documents Certificate of analysis


50

Redmond Red PolystyreneProduct numbers: M830729 (bulk), M100445 (columns)

Oligo Synthesis

and deprotection

O

N

OH O

O

N

OH

O P

O

O-

O

O

N

O O

O

O

N

O

ODMT

O

NH-PSO

Formula N/AMolecular weight (reagent) N/AMolecular weight (incorporated) 446.34 DaCapacity by DMT loading >10 µmol/gPurity of test oligo (reverse phase HPLC) >85%Coupling efficiency test N/ARecommended coupling conditions N/ARecommended deprotection conditions tert-BuNH2:MeOH:H2O (1:2:1, v:v:v), 17 h at 50-55CRecommended storage conditions/Stability Desiccated at 4C/ 3 yearsAvailable documents Certificate of analysis



51


OPO

- O

ON

N

O

N+ AquaPhluor 593 is a fluorescent dye with

excitation and emission properties similar to ROX and Texas Red. The dye is stable under the standard oligonucleotide synthesis and deprotection conditions.



Fluorescence quantum yield**() 0.61Hydrophobicity by C18 chromatography*** 29.9 (Hydrophobic)pH dependence of dye fluorescence Weakly dependent between pH 5 and 8

(Appendix C)

Available AquaPhluor 593 Products Product numberAquaPhluor 593 PFP ester M830196AquaPhluor 593 Terminal Amidite M830736AquaPhluor 593 CPG M830189AquaPhluor 593 Polystyrene **** M830732 (bulk), M100257 (columns)


52

AquaPhluor 593 PFP esterProduct number: M830196

OPO

- O

ON

N

O

N+

O

PFPO

NHO

PO

- O

ON

N

O

N+

OOP

O-

OO

NH2OP

O-

OO


Formula C48H49F5N3O7PMolecular weight (reagent) 905.88 DaMolecular weight (incorporated) 721.82 DaPurity* (reverse phase HPLC) @590 nm >80%Structure identity test 1H NMRRecommended coupling conditions: See conjugation protocolDeprotection conditions N/ARecommended storage conditions/Stability Desiccated at -20C/ 2 yearsAvailable documents Certificate of analysis, conjugation protocols



53


OO

PO

NHO

O CF3

ON

N

O

N+

PN

ON OO

PO

- O

ON

N

O

N+

PO

-

OO

Oligo Synthesis

and deprotection

PF6-

Formula C57H78F9N6O8P3

Molecular weight (reagent) 1239.17 DaMolecular weight (incorporated) 787.8 DaPurity (reverse phase HPLC) ≥85%Purity of test oligo (reverse phase HPLC) ≥80%Identity before/after coupling 1H, 31P NMR /Mass-spectroscopyRecommended coupling conditions Double amidite addition and extended coupling time Recommended deprotection conditions 30% NH4OH 55C 17 h or 70C 2 hRecommended storage conditions/Stability Desiccated at < -10C / 1.5 yearsAvailable documents Certificate of analysis


54


Oligo Synthesis

and deprotection

OPO

- O

ON

N

O

N+

O

N

O

DMTO

O

NH-CPGO

OPO

- O

ON

N

O

N+

O

N

OH

OPO

-

OO



55


Oligo Synthesis

and deprotection

OPO

- O

ON

N

O

N+

O

N

O

DMTO

O

NH-PSO

OPO

- O

ON

N

O

N+

O

N

OH

OPO

-

OO




56


OP

O- O

ON

N

OSO3-

N+

Cl

AquaPhluor 639 is a fluorescent dye with excitation and emission properties similar to Cy5, Quasar 670 and AP642. It features sequence-independent absorption and fluorescence spectra and improved chemical and photo-chemical stability as compared to the spectrally-similar AquaPhluor 642 dye. AP639 is compatible with both standard and mild oligonucleotide deprotection conditions.



Fluorescence quantum yield**() 0.28Hydrophobicity by C18 chromatography*** TBDpH dependence of dye fluorescence Independent between pH of 5 and 8 (Appendix C)

Available AquaPhluor 639 Products Product numberAquaPhluor 639 Terminal Amidite M830741AquaPhluor 639 Polystyrene **** M830743 (bulk), M100440 (columns)

*Measured for T8-AP639 conjugate (50 mM Tris-HCl pH 8.5)**Measured for T8-AP639 conjugate (0.1 M Sodium tetraborate)***Percent acetonitrile required for elution of T8-AP639 Dye Calibrator (M301715) from C18 column (see p107 for details)****Suitable for ABI 3900 DNA synthesizer

57


OO

PO

NHO

O CF3

ON

N

OSO3-

N+

PN

ON Cl OO

PO

- O

ON

N

OSO3-

N+

PO

-

O ClO

Oligo Synthesis

and deprotection

Formula C62H82ClF3N6O11P2SMolecular weight (reagent) 1273.81 DaMolecular weight (incorporated) 968.42 DaPurity (reverse phase HPLC) ≥85%Purity of test oligo (reverse phase HPLC) ≥80%Identity before/after coupling 1H, 31P NMR /Mass-spectroscopyRecommended coupling conditions Double amidite addition and extended coupling time Recommended deprotection conditions 30% NH4OH 55C 17 h or 70C 2 hRecommended storage conditions/Stability Desiccated at < -10C / TBDAvailable documents Certificate of analysis


58


Oligo Synthesis

and deprotection

OPO

NHO

O CF3

ON

N

OSO3-

N+

Cl

O

N

O

DMTO

O

NH-PSO

OPO

- O

ON

N

OSO3-

N+

Cl

O

N

OH

OPO

-

OO




59


N+

O-

O

N

PO

O-O

AquaPhluor 642 is a fluorescent dye with excitation and emission properties similar to Cy5. Mild deprotection conditions are recommended for products carrying this dye.



Fluorescence quantum yield**() 0.13Hydrophobicity by C18 chromatography*** 33.9 (hydrophobic)pH dependence of dye fluorescence Independent between pH of 5 and 8 (Appendix C)

Available AquaPhluor 642 Products Product numberAquaPhluor 642 PFP ester M830197AquaPhluor 642 CPG M830190AquaPhluor 642 Polystyrene **** M830678 (bulk), M100279 (columns)


60


N+

O-

O

N

PO

O-O

O

OF

FF

F

F

NH2OP

O

O-

O Conjugation reaction

N+

O-

O

N

PO

O-O

O

NHOP

O

O-

O

Formula C46H42F5N2O7P (zwitterionic form)Molecular weight (reagent) 860.80 DaMolecular weight (incorporated) 676.73 DaPurity* (reverse phase HPLC) >80%Structure identity test 1H NMRRecommended coupling conditions See conjugation protocolDeprotection conditions N/ARecommended storage conditions/Stability Desiccated at < -10C / 1 yearAvailable documents Certificate of analysis, conjugation protocols



61


N+

O-

O

N

PO

O

O

NO

NH

O CF3

O

DMTO

O

NH-CPGO

Oligo Synthesis

and deprotection

N+

O-

O

N

PO

O-

O

NO

OH

OPO

O-

O

Formula N/AMolecular weight (reagent) N/AMolecular weight (incorporated) 855.84 DaCapacity by DMT loading >15 µmol/gPore size ~1000 ÅPurity of test oligo (reverse phase HPLC) >70%Coupling efficiency test N/ARecommended coupling conditions N/ARecommended deprotection conditions tert-BuNH2:MeOH:H2O (1:2:1, v:v:v), 17 h at 50-55CRecommended storage conditions/Stability Desiccated at 4C/ TBDAvailable documents Certificate of analysis


62


N+

O-

O

N

PO

O

O

NO

NH

O CF3

O

DMTO

O

NH-PSO

Oligo Synthesis

and deprotection

N+

O-

O

N

PO

O-

O

NO

OH

OPO

O-

O

Formula N/AMolecular weight (reagent) N/AMolecular weight (incorporated) 855.84 DaCapacity by DMT loading >10 µmol/gPurity of test oligo (reverse phase HPLC) >70%Recommended coupling conditions N/ARecommended deprotection conditions tert-BuNH2:MeOH:H2O (1:2:1, v:v:v), 17 h at 50-55CRecommended storage conditions/Stability Desiccated at 4C/ TBDAvailable documents Certificate of analysis



63


N+

O-

O

N

PO

O-O

AquaPhluor 662 is a fluorescent dye with excitation and emission properties similar to those of Cy5. Mild deprotection conditions are recommended for products carrying this dye.



Fluorescence quantum yield**() 0.14Hydrophobicity by C18 chromatography*** 35.0 (Hydrophobic)pH dependence of dye fluorescence Independent between pH of 5 and 8 (Appendix C)



64


PO

O-O

O

OF

FF

F

F

N+

O-

O

N

NH2OP

O

O-


PO

O-O

O

NHOP

O

O-

O

N+

O-

O

N

Formula C48H42F5N2O7P (zwitterionic form)Molecular weight (reagent) 884.82 DaMolecular weight (incorporated) 700.75 DaPurity* (reverse phase HPLC) >85%Structure identity test 1H NMRRecommended coupling conditions See conjugation protocolDeprotection conditions N/ARecommended storage conditions/Stability Desiccated at < -10C / 1.5 yearsAvailable documents Certificate of analysis, conjugation protocols



65


PO

O

O

NO

NH

O CF3

O

DMTO

O

NH-CPGO

N+

O-

O

N

Oligo Synthesis

and deprotection

PO

O-

O

NO

OH

OPO

O-

ON

+

O-

O

N

Formula N/AMolecular weight (reagent) N/AMolecular weight (incorporated) 879.86 DaCapacity by DMT loading > 15µmol/gPore size ~1000 ÅPurity of test oligo (reverse phase HPLC) >65%Recommended coupling conditions N/ARecommended deprotection conditions tert-BuNH2:MeOH:H2O (1:2:1, v:v:v), 17 h at 50-55CRecommended storage conditions/Stability Desiccated at 4C/ TBDAvailable documents Certificate of analysis


66


PO

O

O

NO

NH

O CF3

O

DMTO

O

NH-PSO

N+

O-

O

N

Oligo Synthesis

and deprotection

PO

O-

O

NO

OH

OPO

O-

ON

+

O-

O

N




67


N+

PO

O-O

NAquaPhluor 680 is a fluorescent dye with excitation and emission properties similar to those of Cy5.5. Mild deprotection conditions are recommended for products carrying this dye.

Absorbance maximum* 682 nmEmission maximum* 704 nmExtinction coefficient (260 nm) 16,100M-1cm-1


Fluorescence quantum yield**() 0.07Hydrophobicity by C18 chromatography 35.8** (Hydrophobic)pH dependence of dye fluorescence Independent between pH of 5 & 8 (Appendix C)



68


PO

O-O

O

OF

FF

F

F

N+

N

NH2OP

O

O-


PO

O-O

O

NHOP

O

O-

O

N+

N

Formula C47H44F5N2O5P (zwitterionic form)Molecular weight (reagent) 842.83 DaMolecular weight (incorporated) 658.76 DaPurity* (reverse phase HPLC) >90%Structure identity test 1H NMRRecommended coupling conditions See conjugation protocolDeprotection conditions N/ARecommended storage conditions/Stability Desiccated at < -10C / 1 yearAvailable documents Certificate of analysis, conjugation protocols



69


PO

O

O

NO

NH

O CF3

O

DMTO

O

NH-CPGO

N+

N

Oligo Synthesis

and deprotection

PO

O-

O

NO

OH

OPO

O-

ON

+

N

Formula N/AMolecular weight (reagent) N/AMolecular weight (incorporated) 837.88 DaCapacity by DMT loading >15 µmol/gPore size ~1000 ÅPurity of test oligo (reverse phase HPLC) >85%Recommended coupling conditions N/ARecommended deprotection conditions tert-BuNH2:MeOH:H2O (1:2:1, v:v:v), 17 h at 50-55CRecommended storage conditions/Stability Desiccated at 4C/ TBDAvailable documents Certificate of analysis


70


PO

O

O

NO

NH

O CF3

O

DMTO

O

NH-PSO

N+

N

Oligo Synthesis

and deprotection

PO

O-

O

NO

OH

OPO

O-

ON

+

N




71

Quencher-470Acronym: Q-470

NN

O

NQuencher-470 is a Dabcyl-based non-fluorescent quencher. Its principle application is a quencher for blue and green fluorescent donor dyes for FRET probes. The quencher is fully compatible with standard oligonucleotide synthesis and deprotection.

Absorbance maximum* 470 nmExtinction coefficient (260 nm) 9,150 M-1cm-1


*Measured for T8-Q470 conjugate (50 mM Tris-HCl pH 8.5)

Available Q470 products Product numberQuencher-470 PFP ester M830572Quencher-470 Internal Amidite M830565Quencher-470 CPG M830562Quencher-470 Polystyrene ** M830568 (bulk), M100446 (columns)

** Suitable for ABI 3900 DNA synthesizer

72

Quencher-470 PFP esterProduct number: M830572

NH2OP

O

O-

O


NN

O

N

OF

F

FF

F

NN

O

N

NHOP

O

O-

O

Formula C21H14F5N3O2

Molecular weight (reagent) 435.35 DaMolecular weight (incorporated) 251.28 DaPurity* (reverse phase HPLC) >90%Structure identity test 1H NMRRecommended coupling conditions See conjugation protocolDeprotection conditions N/ARecommended storage conditions/Stability Desiccated at < -10C / 1 yearAvailable documents Certificate of analysis, conjugation protocols

*Purity of this active derivative is verified by HPLC, NMR and a test reaction with an aliphatic amine and usually exceeds 90%. Some loss of activity, however, may occur during packaging and storage, especially for small (10 mg) unit sizes.

Available unit size 10 mg 100 mg 1 g bulk

73

Quencher-470 Internal AmiditeProduct number: M830565

NN

O

N

N

O

DMTO

PO

N

N

Oligo Synthesis

and deprotectionN

N

O

N

N

O

O

P

O

O-

O

Formula C50H59N6O6PMolecular weight (reagent) 871.01 DaMolecular weight (incorporated) 430.39 DaPurity (reverse phase HPLC) >90%Purity of test oligo (reverse phase HPLC) >85%Identity before/after coupling 1H, 31P NMR /Mass-spectroscopyRecommended coupling condition Double amidite addition and extended coupling time Recommended deprotection conditions 30% NH4OH 55C 17 h or 65C 2 hRecommended storage conditions/Stability Desiccated at -20C/ TBDAvailable documents Certificate of analysis


74

Quencher-470 CPGProduct number: M830562

Oligo Synthesis

and deprotection

P

O

O-

O

NN

O

N

N

OH

O

NN

O

N

N

O

DMTO

O

ONH-CPG



75

Quencher-470 PolystyreneProduct numbers: M830568 (bulk), M100446 (columns)

Oligo Synthesis

and deprotection

P

O

O-

O

NN

O

N

N

OH

O

NN

O

N

N

O

DMTO

O

ONH-PS

Formula N/AMolecular weight (reagent) N/AMolecular weight (incorporated) 430.39 DaCapacity by DMT loading > 10 µmol/gPurity of test oligo (reverse phase HPLC) >85%Coupling efficiency test N/ARecommended coupling conditions N/ARecommended deprotection conditions 30% NH4OH 55C 17 h or 70C 2 hRecommended storage conditions/Stability Desiccated at 4C/ TBDAvailable documents Certificate of analysis



76

Eclipse Dark QuencherSynonyms: Eclipse Quencher, EDQ

NN

N

NO2

Cl

O

Eclipse dark quencher is a universal non-fluorescent quencher for FRET probes. The quencher is fully compatible with standard oligonucleotide synthesis and deprotection.



*Measured for T8-Eclipse Quencher conjugate (50 mM Tris-HCl pH 8.5)

Available Eclipse Quencher products Product numberEclipse Dark Quencher PFP ester M830573Eclipse Dark Quencher Phosphoramidite M830028Eclipse Dark Quencher CPG M830386Eclipse Dark Quencher Polystyrene ** M830486 (bulk), M100447 (columns)


77

Eclipse Dark Quencher PFP ester Product number: M830573

NH2OP

O

O-


NN

N

NO2

ClO

O

F

FF

F

F

NN

N

NO2

Cl

O

NHOP

O

O-

O

Formula C23H16ClF5N4O4

Molecular weight (reagent) 542.84 DaMolecular weight (incorporated) 358.77 DaPurity* (reverse phase HPLC) >90%Structure identity test 1H NMRRecommended coupling conditions See conjugation protocolDeprotection conditions N/ARecommended storage conditions/Stability Desiccated at < -10C / 1 yearAvailable documents Certificate of analysis, conjugation protocols



78

Eclipse Dark Quencher PhosphoramiditeProduct number: M830028

N

O

DMTO

PO

N

N

NN

N

NO2

Cl

O

Oligo Synthesis

and deprotection

N

O

O

P

O

O-

ON

N

N

NO2

Cl

O

Formula C52H61ClN7O8PMolecular weight (reagent) 978.51 DaMolecular weight (incorporated) 537.89 DaPurity (reverse phase HPLC) >90%Purity of test oligo (reverse phase HPLC) >85%Identity before/after coupling 1H, 31P NMR /Mass-spectroscopyRecommended coupling conditions Double amidite addition and extended coupling time Recommended deprotection conditions 30% NH4OH 55C 17 h or 70C 2 hRecommended storage conditions/Stability Desiccated at < -10C / 5 yearsAvailable documents Certificate of analysis


79

Eclipse Dark Quencher CPGProduct number: M830386

Oligo Synthesis

and deprotection

N

O

DMTO

O NN

N

NO2

Cl

O

NH-CPGO

P

O

O-

O

N

OH

O

NN

N

NO2

Cl

O

Formula N/AMolecular weight (reagent N/AMolecular weight (incorporated) 537.89 DaCapacity by DMT loading >15 µmol/gPore size ~1000 ÅPurity of test oligo (reverse phase HPLC) >85%Recommended coupling conditions N/ARecommended deprotection conditions 30% NH4OH 55C 17 h or 70C 2 hRecommended storage conditions/Stability Desiccated at 4C/ 5 yearsAvailable documents Certificate of analysis


80

Eclipse Dark Quencher PolystyreneProduct numbers: M830486 (bulk), M100447 (columns)

Oligo Synthesis

and deprotection

N

O

DMTO

O NN

N

NO2

Cl

O

NH-PSO

P

O

O-

O

N

OH

O

NN

N

NO2

Cl

O

Formula N/AMolecular weight (reagent) N/AMolecular weight (incorporated) 537.89 DaCapacity by DMT loading > 10 µmol/gPurity of test oligo (reverse phase HPLC) >85%Recommended coupling conditions N/ARecommended deprotection conditions 30% NH4OH 55C 17 h or 70C 2 hRecommended storage conditions/Stability Desiccated at 4C/5 yearsAvailable documents Certificate of analysis



81


NN

N

NO2

Cl

OOCH3

H3COQuencher-575 quencher is a universal non-fluorescent quencher for FRET probes. The quencher is compatible with standard oligonucleotide synthesis. Mild deprotection is recommended.






82

Quencher-575 PFP ester Product number: M830574

NH2OP

O

O-


NN

N

NO2

ClO

O

F

FF

F

FH3CO

OCH3

NN

N

NO2

Cl

O

NHOP

O

O-

O

H3CO

OCH3

Formula C26H22ClF5N4O6

Molecular weight (reagent) 616.92 DaMolecular weight (incorporated) 432.85 DaPurity* (reverse phase HPLC) >90%Structure identity test 1H NMRRecommended coupling conditions See conjugation protocolDeprotection conditions N/ARecommended storage conditions/Stability Desiccated at < -10C/ 1 yearAvailable documents Certificate of analysis, conjugation protocols



83


N

O

DMTO

PO

N

N

NN

N

NO2

Cl

OOCH3

H3CO

Oligo Synthesis

and deprotection

N

O

O

P

O

O-

ON

N

N

NO2

Cl

OOCH3

H3CO

Formula C55H67ClN7O10PMolecular weight (reagent) 1052.59 DaMolecular weight (incorporated) 611.96 DaPurity (reverse phase HPLC) >90%Purity of test oligo (reverse phase HPLC) >85%Identity before/after coupling 1H, 31P NMR /Mass-spectroscopyRecommended coupling conditions Double amidite addition and extended coupling time Recommended deprotection conditions tert-BuNH2:MeOH:H2O (1:2:1, v:v:v), 17 h at 50-55CRecommended storage conditions/Stability Desiccated at < -10C/ 3 yearsAvailable documents Certificate of analysis


84


Oligo Synthesis

and deprotection

N

O

DMTO

O NN

N

NO2

Cl

O

NH-CPGO

OCH3

H3CO

P

O

O-

O

N

OH

O

NN

N

NO2

Cl

OOCH3

H3CO



85


Oligo Synthesis

and deprotection

N

O

DMTO

O NN

N

NO2

Cl

O

NH-PSO

OCH3

H3CO

P

O

O-

O

N

OH

O

NN

N

NO2

Cl

OOCH3

H3CO




86


NN

NO

S N

NO2

Quencher-630 is the most red-shifted quencher currently available from ELITechGroup, and it is especially suitable for quenching red and far red fluorophores including the AP680 dye. The quencher is compatible with standard oligonucleotide synthesis. Mild deprotection is recommended.

Absorbance maximum* 630 nmEmission maximum* N/AExtinction coefficient (260 nm) 11,160 M-1cm-1





87

Quencher-630 PFP ester Product number: M830575

NH2OP

O

O-


NN

NO

O

F

FF

F

F

S N

NO2

NN

NO

NHOP

O

O-

O

S N

NO2

Formula C24H16F5N5O4SMolecular weight (reagent) 565.47 DaMolecular weight (incorporated) 381.40 DaPurity* (reverse phase HPLC) >90%Structure identity test 1H NMRRecommended coupling conditions See conjugation protocolDeprotection conditions N/ARecommended storage conditions/Stability Desiccated at < -10C/ 1 yearAvailable documents Certificate of analysis, conjugation protocols

* Purity of this active derivative is verified by HPLC, NMR and a test reaction with an aliphatic amine and usually exceeds 90%. Some loss of activity, however, may occur during packaging and storage, especially for small (10 mg) unit sizes.


88


Oligo Synthesis

and deprotection

N

O

DMTO

PO

N

N

NN

NO

SN

O2N

N

O

O

P

O

O-

ON

N

NO

SN

O2N

Formula C53H61N8O8PSMolecular weight (reagent) 1001.14 DaMolecular weight (incorporated) 560.51 DaPurity (reverse phase HPLC) >90%Purity of test oligo (reverse phase HPLC) >85%Identity before/after coupling: 1H, 31P NMR /Mass-spectroscopyRecommended coupling conditions Double amidite addition and extended coupling time Recommended deprotection conditions tert-BuNH2:MeOH:H2O (1:2:1, v:v:v), 17 h at 50-55CRecommended storage conditions/Stability Desiccated at < -10C/ 2 yearsAvailable documents Certificate of analysis

Available unit size 50 µmol 100 µmol 250 mg 1 g bulk

89


Oligo Synthesis

and deprotection

N

O

DMTO

O NN

NO

NH-CPGO

SN

O2N

P

O

O-

O

N

OH

O

NN

NO

SN

O2N



90


Oligo Synthesis

and deprotection

N

O

DMTO

O NN

NO

NH-PSO

SN

O2N

P

O

O-

O

N

OH

O

NN

NO

SN

O2N




91

Dye Calibrators

OOH

O

N

NH

O

O

POH

O O O

O

N

NH

O

O

POOOH

NOH

Dye

7

Dye calibrators are available for evaluation of our fluorescent dyes’ properties and instrument calibrations. The calibrators are T8 conjugates with the dyes attached to the 3’-terminus via the hydroxyprolinol linker. All conjugates are carefully purified and formulated as 0.1 mM solution in 1x TE buffer.

Purity by mass-spectroscopy 90%Identity by mass-spectroscopy ± 0.5% of calculated MWRecommended storage conditions: -20C, protect from lightStability 3 years at -20CAvailable documents Certificate of analysis

Product Number

Dye Calibrator

Abs max (nm)

Em max (nm)

ε260 (M-1

*cm-1)ελ max

(M-1*cm-1)

MW Hydro-phobicity*

M300585 T8-EB380-HEG 381 460 82,600 20,000 3191.50 22.0M300586 T8-FAM-HEG 496 517 98,700 80,400 3292.44 19.2M300587 T8-AP525 527 549 117,900 99,300 3233.15 18.6M830730 T8-YY 525 547 94,000 86,500 3088.87 25.6M301430 T8-AP559 559 581 93,700 86,500 3227.83 25.6M830700 T8-AP570 570 592 109,500 101,100 3303.93 29.4M830731 T8-RR 580 594 75,200 64,500 2817.94 17.9M300392 T8-AP593 593 613 85,080 115,240 3272.53 29.9M301715 T8-AP639 639 655 24,650 129,300 3453.14 TBDM300589 T8-AP642 641 653 85,100 283,200 3227.44 33.9M830724 T8-AP662 662 676 81,000 241,100 3251.46 35.0M300753 T8-AP680 682 704 81,500 221,300 3209.48 35.8

All absorption and emission data were obtained in 50 mM Tris-HCl pH 8.5.* Acetonitrile concentration (%) required to elute a dye calibrator from C18 column (see Appendix A, Figure 9 for details).

92

Super G®

OOH

OH

N

NH

NN

O

NH2

Super G (8-aza-7-deazaguanosine) eliminates undesired secondary structures inherent to natural G-rich sequences and provides improved hybridization efficiency. It is recommended for use in NA probes and primers to disrupt G stretches. An additional benefit of Super G for use in fluorogenic hybridization probes is its low fluorescence quenching tendency, which is an intrinsic and often undesired property of natural G. The resultant Super G-modified probes demonstrate higher fluorescence efficiency and, therefore, assay sensitivity.


Available Super G reagents Product numberSuper G 3’-phosphoramidite M830006Super G 5’-phosphoramidite M830031

93

Super G 3’-phosphoramiditeProduct number: M830006

ODMTO

O

N

NH

NN

O

N N

PN

ON

Oligo Synthesis

and deprotectionOO

O

N

NH

NN

O

NH2

P OO

-O

Formula C43H53N8O7PMolecular weight (reagent) 824.93 DaMolecular weight (incorporated) 329.21 DaPurity (reverse phase HPLC) >90%Purity of test oligo (reverse phase HPLC) >85%Identity 1H, 31P NMRRecommended coupling conditions Double amidite addition and extended coupling time Recommended deprotection conditions 30% NH4OH 55C 17 h or 70C 2 hRecommended storage conditions/Stability Desiccated at < -10C/ 5 yearsAvailable documents Certificate of analysis, SDS


94

Super G 5’-phosphoramiditeProduct number: M830031

Oligo Synthesis

and deprotectionOO

ODMT

N

NH

NN

O

N NPO

N

N OO

O

N

NH

NN

O

NH2PO

O-

O

Formula C43H53N8O7PMolecular weight (reagent) 824.93 DaMolecular weight (incorporated) 329.21 DaPurity (reverse phase HPLC) >90%Purity of test oligo (reverse phase HPLC) >85%Identity 1H, 31P NMRRecommended coupling conditions Double amidite addition and extended coupling time Recommended deprotection conditions 30% NH4OH 55C 17 h or 70C 2 hRecommended storage conditions/Stability Desiccated at < -10C/ 5 yearsAvailable documents Certificate of analysis, SDS


95

Super A®

OOH

OH

N

N

NN

NH2

OH

NH2

Super A (7-hydroxybutynyl-2-amino-8-aza-7-deazaadenosine) is a duplex-stabilizing nucleoside analogue. The Super A:T base pair is more stable than natural A:T and even surpasses the 2-aminoadenine:T base pair and approaches the stability of G:C pair. Depending on length and sequence each Super A incorporation will increase probe’s or primer’s melting temperature (Tm) by 2.5-5.5C.


Available Super A reagents Product numberSuper A 3’-phosphoramidite M830249Super A 5’-phosphoramidite M830250

96

Super A 3’-phosphoramiditeProduct number: M830249

ODMTO

O

N

N

NN

NH

NH

PN

ON

O

O

O

O

F

F

FF

Oligo Synthesis

and deprotectionOO

OP OO

-O

N

N

NN

NH2

NH2

OH

Formula C60H59F4N8O10PMolecular weight (reagent) 1159.14 DaMolecular weight (incorporated) 396.3 DaPurity (reverse phase HPLC) >90%Purity of test oligo (reverse phase HPLC) >85%Identity 1H, 31P NMRRecommended coupling conditions Double amidite addition and extended coupling time Recommended deprotection conditions 30% NH4OH 55C 17 h or 70C 2 h

Extra deprotection time may be needed for multiple incorporations

Recommended storage conditions/Stability Desiccated at < -10C/ 5 yearsAvailable documents Certificate of analysis, SDS


97

Super A 5’-phosphoramiditeProduct number: M830250

Oligo Synthesis

and deprotectionOO

ODMT

PON

NN

N

NN

NH

NH

O

O

O

O

F

F

FF

OO

O

PO

O-

O N

N

NN

NH2

NH2

OH

Formula C60H59F4N8O10PMolecular weight (reagent) 1159.14 DaMolecular weight (incorporated) 396.3 DaPurity (reverse phase HPLC) >90%Purity of test oligo (reverse phase HPLC) >85%Identity 1H, 31P NMRRecommended coupling conditions Double amidite addition and extended coupling time Recommended deprotection conditions 30% NH4OH 55C 17 h or 70C 2 h

Extra deprotection time may be needed for multiple incorporations

Recommended storage conditions/Stability Desiccated at < -10C/ 5 yearsAvailable documents Certificate of analysis, SDS


98

Super T®

OOH

OH

N

NH

O

O

OH Super T (5-hydroxybutynyl-2’-deoxyuridine) is another duplex-stabilizing nucleoside analogue which forms a stabilized Super T:A base pair with an average Tm increase of about 2C per each incorporation. The Super T nucleobase can be used liberally in both probe and primer designs with little or no adverse effect on amplification efficiency.


Available Super T reagents Product numberSuper T 3’-phosphoramidite M830040Super T 5’-phosphoramidite M830041

99

Super T 3’-phosphoramiditeProduct number: M830040

ODMTO

OPN

ON

N

NH

O

O

O

O

Cl

Oligo Synthesis

and deprotectionOO

OP OO

-O

N

NH

O

O

OH

Formula C50H54ClN4O10PMolecular weight (reagent) 937.41 DaMolecular weight (incorporated) 358.24 DaPurity (reverse phase HPLC) >90%Purity of test oligo (reverse phase HPLC) >85%Identity 1H, 31P NMRRecommended coupling conditions Double amidite addition and extended coupling time Recommended deprotection conditions 30% NH4OH 55C 17 h or 70C 2 hRecommended storage conditions/Stability Desiccated at < -10C/ 5 yearsAvailable documents Certificate of analysis, SDS


100

Super T 5’-phosphoramiditeProduct number: M830041

Oligo Synthesis

and deprotectionOO

O

PO

O-

O N

NH

O

O

OH

OO

ODMT

PNO

N

N

NH

O

O

O

O

Cl

Formula C50H54ClN4O10PMolecular weight (reagent) 937.41 DaMolecular weight (incorporated) 358.24 DaPurity (reverse phase HPLC) >90%Purity of test oligo (reverse phase HPLC) >85%Identity 1H, 31P NMRRecommended coupling conditions Double amidite addition and extended coupling time Recommended deprotection conditions 30% NH4OH 55C 17 h or 70C 2 hRecommended storage conditions/Stability Desiccated at < -10C/ 5 yearsAvailable documents Certificate of analysis, SDS


101

Super I

OOH

OH

N

NH

NN

O

NH2

Super inosine (Super I) is a nucleoside analogue that contains a quasi-universal nucleobase (7-aminobutynyl-8-aza-7-deazahypoxanthine) with improved hybridization and polymerase chain reaction (PCR) enhancing properties as compared to commonly used 2’-deoxyinosine. It demonstrates improved ambiguity for pairing with A, T and C bases and its base pairing properties can be summarized as follows: X:C~X:A~X:T>X:G. Base pairing strength of Super I with A, T and C is approximately equal or slightly greater (depending on nearest neighbors) than the natural A:T but lower than the G:C, with the Super I:C base pair being slightly more stable than Super I:A and Super I:T. The improvement in PCR performance directly correlated with primers’ Tm. Primers with multiple Super I incorporations can be used without noticeable inhibition of Taq polymerase activity provided the modifications are not used in stretches or positioned more than 2 bases away from the 3’ end.


Available Super I reagents Product numberSuper I 3’-phosphoramidite M830193Super I 5’-phosphoramidite M830712

102

Super I 3’-phosphoramiditeProduct number: M830193

Oligo Synthesis

and deprotectionOO

OP OO

-O

N

NH

NN

O

NH2

ODMTO

O

N

NH

NN

O

PN

ON

NH

O

CF3

Formula C46H51F3N7O8PMolecular weight (reagent) 917.91 DaMolecular weight (incorporated) 381.28 DaPurity (reverse phase HPLC) >90%Purity of test oligo (reverse phase HPLC) >85%Identity 1H, 31P NMRRecommended coupling conditions Double amidite addition and extended coupling time Recommended deprotection conditions Pre-treatment with 5% N,N,N’,N’-tetramethylguanidine

in CH3CN followed by 30% NH4OH 55C 17 h or 70C 2 hRecommended storage conditions/Stability Desiccated at < -10C/ 5 yearsAvailable documents Certificate of analysis, SDS


103

Super I 5’-phosphoramiditeProduct number: M830712

Oligo Synthesis

and deprotectionOO

O

PO

O-

O N

NH

NN

O

NH2

OO

ODMT

PON

NN

NH

NN

O

NH

O

CF3

Formula C46H51F3N7O8PMolecular weight (reagent) 917.93 DaMolecular weight (incorporated) 381.28 DaPurity (reverse phase HPLC) >90%Purity of test oligo (reverse phase HPLC) >85%Identity NMRRecommended coupling conditions Double amidite addition and extended coupling time Recommended deprotection conditions Pre-treatment with 5% N,N,N’,N’-tetramethylguanidine

in CH3CN followed by 30% NH4OH 55C 17 h or 70C 2 hRecommended storage conditions/Stability Desiccated at < -10C/ 5 yearsAvailable documents Certificate of analysis, SDS


104

CDPI3 Minor Groove BinderAcronym: CDPI3 MGB

NH

N

O NH

N

O

NH

N

O

OCDPI3 MGB is an efficient duplex-stabilizing agent. This ligand improves background fluorescence and mismatch discrimination abilities of DNA probes. When attached to the 5’-end of a probe it inhibits the 5’ – 3’ exonuclease activity of DNA polymerases.

Absorbance maximum* 340 nmEmission maximum* N/AExtinction coefficient (260 nm) 37,900 M-1cm-1**Extinction coefficient (340 nm) 59,300 M-1cm-1

*Measured for T10-CDPI3 conjugate (50 mM Tris-HCl pH 8.5)** CDPI3 contribution only

Available CDPI3 MGB products Product numberCDPI3 MGB Terminal Amidite M830252CDPI3 MGB CPG M830739CDPI3 MGB Polystyrene *** M830203 (bulk), M100103 (columns)

*** Suitable for ABI 3900 DNA synthesizer

105

CDPI3 MGB Terminal AmiditeProduct number: M830252

O

N

N

O N

N

O

N

N

O

O

ON

O

N OP ON

N

O

O

O

O

F

FF

NH

N

O NH

N

O

NH

N

ON

O

NH OP OOH

OOligo Synthesis

and deprotection

Formula C67H82F3N10O13PMolecular weight (reagent) 1323.4 DaMolecular weight (incorporated) 872.94 DaPurity (reverse phase HPLC) >85%Purity of test oligo (reverse phase HPLC) >80%Identity before/after coupling 1H, 31P NMR /Mass-spectroscopyRecommended coupling conditions Double amidite addition and extended coupling time Recommended deprotection conditions 30% NH4OH 55C 17 h or 70C 2 hRecommended storage conditions/Stability Desiccated at < -10C / TBDAvailable documents Certificate of analysis


106

CDPI3 MGB CPGProduct number: M830739

NH

N

O NH

N

O

NH

N

ONH

O

ODMT

OO

O

NH-CPGO

NH

N

O NH

N

O

NH

N

ONH

O

O

OH

P

O

O-

OOligo Synthesis

and deprotection



107

CDPI3 MGB PolystyreneProduct numbers: M830203 (bulk), M100103 (columns)

NH

N

O NH

N

O

NH

N

ONH

O

ODMT

OO

O

NH-PSO

NH

N

O NH

N

O

NH

N

ONH

O

O

OH

P

O

O-

OOligo Synthesis

and deprotection

Formula N/AMolecular weight (reagent) N/AMolecular weight (incorporated) 831.87 DaCapacity by DMT loading 10-17 µmol/gPurity of test oligo (reverse phase HPLC) >85%Recommended coupling conditions N/ARecommended deprotection conditions 30% NH4OH 55C 17 h or 70C 2 hRecommended storage conditions/Stability Desiccated at 4C/ 5 years (bulk)Available documents Certificate of analysis



108

1

2 3

4

5, 6

7

8

910

11

Appendix A. Determination of the relative hydrophobicities of conjugated dyes using C18 column chromatography

Figure 9 HPLC analysis of a mixture of fluorescent dye calibrators. 1 - T8-RR (M830731); 2 - T8-AP525 (M830722); 3 - T8-FAM (M830721); 4 - T8-EB380 (M830720); 5 - T8-YY (M830730); 6 - T8-AP559 (M830723); 7 - T8-AP570 (M830700) ; 8 - T8-AP593 (M300392) ; 9 - T8-AP642 (M830589) ; 10 - T8-AP662 (M830724) ; 11 - T8-AP680 (M300753).Column conditions: C18 Luna (4.6x100 mm), Phenomenex (00D-4041-E0). Sample loop – 20 µL. Flow rate – 1ml/min, Solvent A – 0.1 M triethylammonium acetate pH 7.5. Solvent B – CH 3CN. Gradient – 0-40% B in 19 min. Detection – 260 nm.

Product number

Product name Retention(min)

Hydrophobicity(% CH3CN)

M830720 T8-EB380 Dye Calibrator 11.77 22.0M300586 T8-FAM Dye Calibrator 10.46 19.2M300587 T8-AP525 Dye Calibrator 10.14 18.6M830730 T8-YY Dye Calibrator 13.49 25.6M301430 T8-AP559 Dye Calibrator 13.49 25.6M830700 T8-AP570 Dye Calibrator 15.30 29.4M830731 T8-RR Dye Calibrator 9.85 17.9M300392 T8-AP593 Dye Calibrator 15.56 29.9M300589 T8-AP642 Dye Calibrator 17.43 33.9M830724 T8-AP662 Dye Calibrator 17.99 35.0M300753 T8-AP680 Dye Calibrator 18.35 35.8

109

Appendix B. Determination of molar extinction coefficients ofconjugated fluorescent dyes and quenchers

Determination of extinction coefficients for a conjugated dye may be challenging due to the sensitivity of absorption and fluorescence spectra to the dye’s environment and potentially complex dye-conjugate interactions. In the case of oligonucleotide conjugates, for instance, dyes may interact with nucleobases as well as other conjugated moieties. To take into account these effects, an optimal measurement procedure should employ a dye-oligonucleotide conjugate. However, determination of dye-oligonucleotide concentration by pure conjugate mass, which is required for the direct extinction coefficient measurement, is often impractical. We have overcome this problem by using a concentration-independent approach which utilizes the known extinction coefficients of oligothymidylates and the dye absorption as an internal reference. In this approach, a series of dye-oligothymidylate conjugates is prepared with a variable number of thymidine units. The molar extinction coefficient for oligothymidylate can be described by a simple formula E260 = 8100n + 600, where n is the number of thymidine residues. Assuming that the dye conjugation does not affect this dependence, the combined extinction coefficient for dye-oligoT conjugate can be represented as E260 (conjugate) = 8100n + 600 + E260(dye). To obtain E260(dye), one must measure A260 (260 nm absorption), Aλmax(dye) (absorption at the dye maximum) and plot A260(conjugate)/Aλmax(dye) values over the number of thymidine units. These data are then analyzed by linear regression to generate a line of best fit and its corresponding equation y = ax + b. Since T oligomers’ slope parameter a equals 8100, the equation can be rewritten as y = 8100n + b/a8100.

110

C E260(dye) = 0.1746 * 8100 / 0.0698 – 600 = 19661.6 (M-1cm-1)

Figure 10 Extinction coefficient (E260) determination for the AP593 dye conjugated to oligothymidylates. (A) Normalized (at 593 nm) absorption spectra of T12-, T10-, T8- and T6-AP593 conjugates. (B) Line of best fit (with equation and R2 value) for the normalized A260 values versus number of thymidine units. (C) Calculation of the E260(dye) coefficient using the parameters a and b from the best fit linear equationThe y-intercept (b/a8100) of this line represents E260(dye) + 600. Therefore, E260(dye) = b8100/a – 600. The described procedure is illustrated in Figure 10. In this example, the described approach was used to determine an extinction coefficient for a Tn-conjugated AP593 dye.

It should be noted that the success of this method relies on several assumptions and requirements listed below:

1) Dye-oligonucleotide interactions are independent on the length of the oligomers;2) All conjugates must be carefully purified to ensure accurate A260 measurements;3) The linear fit of all data points should have a high (>0.99) R2 value to ensure accurate y-

intercept determination.

111

0

0.2

0.4

0.6

0.8

1

1.2

220 270 320 370 420 470 520 570 620 670

nm

Nor

mal

ized

abs

orpti

on

y = 0.0698x + 0.1746R2 = 0.9988

0

0.2

0.4

0.6

0.8

1

1.2

0 2 4 6 8 10 12 14

Number of T-Residues

Nor

mal

ized

Abso

rptio

n at

260

nm

A B

T12-AP593

T10-AP593

T8-AP593

T6-AP593

Nor

mal

ized

A 260

Appendix C. Fluorescence dependence on pH for various T8-conjugated dyes.

Figure 11 Dependence of the fluorescence signal on pH for various T8-Dye calibrators. Experimental conditions: Instrument - Varian Carry Eclipse; 5 nm slit width; Buffer - 40 mM phosphate buffer (pH 5.6 -8.15); Temperature – 20C.

112

Figure 11 Continued

NOTICE TO PURCHASER: LIMITED LICENSE

Materials in this publication may be subject to proprietary rights of ELITechGroup (formerly Epoch Biosciences). There is no implied license for commercial use with respect to the products. A license must be obtained directly from ELITechGroup with respect to any lease, license or other transfer of the products or any material derived or produced from them, the sale, lease, license or other grant of rights to use products or any materials derived or produced from them, or the use of products to perform services for a fee for third parties (including contract research).

Trademark InformationSuper Inosine is also known as Super D™. AquaPhluor, Yakima Yellow, Redmond Red, Eclipse

, Super A, Super G and Super T are registered Trademarks of ELITechGroup S.A.S.

PatentsThe dye- and modified base- reagents are covered by one or more of U.S. Patents Nos. US6,653,473, US6,699,975, US6,727,356, US6,790,945, US6,972,339, US7,112,684, US7,601,851, US7,662,942, US7,671,218, US7,767,834, US7,897,736, US8,008,522, US8,163,910, US8,389,745, RE38,416, US6,127,121, US6,485,906, US6,949,367, US7,045,610, US7,368,549, US6,683,173, US7,751,982, US7,715,989, US6,660,845, US6,972,328, US6,962,991 and Foreign Patents Nos., EP1,235,938, AUS782,204, JP5,019,688, JP5,214,967 EP1068358 , EP1975256 , EP1261616, EP1232157, AU759944, CA2327547, CA 2392033 and JP4558932 as well as applications that are currently pending. The minor Groove Binder reagents and their applications are covered by one or more U.S. Patents Nos. US5801155, US6084102, US6,312,894, US6653473, US6699975, US6,492,346, US6,426,408, US6,486,308, US6727356, US6790945, US6884584, US7205105, US7381818, US7485442, US 7556923, US7662942, US7759126, US 7794945, US 8465921, US9,056,887

113

QUICK REFERENCE GUIDE: FLUORESCENT DYES

Name

AbsMax(nm)

Emax

(M-1cm-1)

Emmax(nm)

pHSensi-tivity

Hydro-phobicity* Available Reagents

Epoch Blue 380 381 20,000 460 0.24not

sensitive22

Active ester, amidite, solid support

6-FAM 496 80,400 517 0.88 pKa~7 19.2 Amidite, solid support

AquaPhluor 525 527 99,300 549 0.73 pKa~7 18.6Lactone, amidite, solid support

Yakima Yellow 525 86,500 547 0.57 pKa~6 25.6Lactone, amidite, solid support

AquaPhluor 559 559 86,500 581 0.64not

sensitive25.6 Amidite, solid support

AquaPhluor 570 570 101,100 592 0.68not

sensitive29.4 Amidite, solid support

Redmond Red 580 64,500 594 0.46 pKa~7.5 17.9Lactone, amidite, solid support

AquaPhluor 593 593 115,240 613 0.61not

sensitive29.9

Active ester, amidite, solid support

AquaPhluor 639 639 129,300 655 0.28not

sensitiveTBD Amidite, solid support

AquaPhluor 642 642 283,200 653 0.13not

sensitive33.9

Active ester, solid support

AquaPhluor 662 662 241,100 672 0.14not

sensitive35


AquaPhluor 680 682 221,300 704 0.07not

sensitive35.8


All physicochemical parameters are for T8-Dye conjugates at 20oC * Percent (%) CH3CN required to elute T8-Dye conjugate from C18 HPLC column

114

· web viewboth non-aqueous and aqueous/organic conjugation conditions can be used. active esters:...

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