section 8.2 - labeling oligonucleotides and nucleic acids

287

Cat # Product Name Unit SizeS-7563 SYBR® Green I nucleic acid gel stain *10,000X concentrate in DMSO* ............................................................................................................... 500 µLS-7567 SYBR® Green I nucleic acid gel stain *10,000X concentrate in DMSO* ............................................................................................................... 1 mLS-7585 SYBR® Green I nucleic acid gel stain *10,000X concentrate in DMSO* *special packaging* .............................................................................. 20 x 50 µLS-7564 SYBR® Green II RNA gel stain *10,000X concentrate in DMSO* ......................................................................................................................... 500 µLS-7568 SYBR® Green II RNA gel stain *10,000X concentrate in DMSO* ......................................................................................................................... 1 mLS-7586 SYBR® Green II RNA gel stain *10,000X concentrate in DMSO* *special packaging* ........................................................................................ 20 x 50 µLS-7580 SYBR® Green Nucleic Acid Gel Stain Starter Kit ................................................................................................................................................... 1 kitS-21500 SYBR® 101, succinimidyl ester ............................................................................................................................................................................ 1 mgS-21501 SYBR® 102, succinimidyl ester ............................................................................................................................................................................ 1 mgS-21502 SYBR® 103, succinimidyl ester ............................................................................................................................................................................ 1 mgS-11340 SYTO® Red Fluorescent Nucleic Acid Stain Sampler Kit *SYTO® dyes 17 and 59–64* *50 µL each* ................................................................. 1 kitS-11348 SYTOX® Blue nucleic acid stain *5 mM solution in DMSO* ................................................................................................................................. 250 µLS-7020 SYTOX® Green nucleic acid stain *5 mM solution in DMSO* .............................................................................................................................. 250 µLS-11368 SYTOX® Orange nucleic acid stain *5 mM solution in DMSO* ............................................................................................................................ 250 µLT-3602 TO-PRO®-1 iodide (515/531) *1 mM solution in DMSO* .................................................................................................................................... 1 mLT-3605 TO-PRO®-3 iodide (642/661) *1 mM solution in DMSO* .................................................................................................................................... 1 mLT-7596 TO-PRO®-5 iodide (745/770) *1 mM solution in DMSO* .................................................................................................................................... 1 mLT-3600 TOTO®-1 iodide (514/533) *1 mM solution in DMSO* ........................................................................................................................................ 200 µLT-3604 TOTO®-3 iodide (642/660) *1 mM solution in DMSO* ........................................................................................................................................ 200 µLY-3603 YO-PRO®-1 iodide (491/509) *1 mM solution in DMSO* .................................................................................................................................... 1 mLY-3607 YO-PRO®-3 iodide (612/631) *1 mM solution in DMSO* .................................................................................................................................... 1 mLY-3601 YOYO®-1 iodide (491/509) *1 mM solution in DMSO* ........................................................................................................................................ 200 µLY-3606 YOYO®-3 iodide (612/631) *1 mM solution in DMSO* ........................................................................................................................................ 200 µL

8.2 Labeling Oligonucleotides andNucleic Acids

To facilitate the preparation of optimally labeled nucleic acids, Molecular Probes andits distributors exclusively supply many unique and important reagents and kits. Thesuperior properties of our proprietary dyes ensure that the labeled nucleic acids are thebest that can be prepared by each method. Our available technologies include:

• ChromaTide dUTP, ChromaTide OBEA-dCTP 1 and ChromaTide UTP nucleotides,which provide researchers with a large selection of fluorophore- and hapten-labelednucleotides that can be enzymatically incorporated into DNA or RNA probes forFISH (fluorescence in situ hybridization), DNA arrays and microarrays and otherhybridization techniques (see Legal Notice for ChromaTide UTP and dUTP Nucleo-tides).

• ULYSIS Nucleic Acid Labeling Kits, which employ a fast, simple and reliable chemi-cal method for labeling nucleic acids without enzymatic incorporation.

• ARES DNA Labeling Kits, which employ a versatile, two-step method for labelingDNA with fluorescent dyes to achieve a uniformity and consistency of labeling that isdifficult to obtain with conventional enzymatic incorporation of labeled nucleotides.

• Alexa Fluor Oligonucleotide Amine Labeling Kits, which use familiar chemical label-ing of amine-terminated oligonucleotides to prepare the best singly labeled fluorescentconjugates.

Custom conjugations of some of our proprietary dyes to oligonucleotides for personalresearch use are available from several authorized sources. A variety of additional meth-ods for preparing labeled oligonucleotides and nucleic acids and using them in nucleicacid sequencing are described in this section. Section 8.5 describes use of labeled nucleicacids as hybridization reagents for microarrays, FISH and real-time PCR assays. Section8.5 also includes a discussion of our important ELF and TSA technology for amplifyingFISH signals.

Legal Notice forChromaTide UTP and dUTPNucleotidesFor Research Use Only. These NEN-brandproducts are distributed and sold under anagreement between Enzo Diagnostics, Inc., andPerkinElmer Life Sciences, Inc. (formerly NENLife Science Products, Inc.), for researchpurposes only by the end-user in the researchmarket and are not intended for diagnostic ortherapeutic use. Purchase does not include orcarry any right or license to use, develop orotherwise exploit this product commercially.Any commercial use, development or exploita-tion of this product without the express priorwritten authorization of Enzo Diagnostics, Inc.,and PerkinElmer Life Sciences, Inc., is strictlyprohibited. This product or the use of thisproduct may be covered by one or more Enzopatents, including the following:

US 4,707,440; US 4,952,685; US 5,002,885;US 5,013,831; US 5,328,824; US 5,449,767;and DK 164 407 8; and by one or more Per-kinElmer patents, including US 5,047,519; US5,151,507; and US 5,608,063.

Section 8.2

288 Chapter 8 — Nucleic Acid Detection and Genomics Technology www.probes.com

Alexa Fluor Dyes for Labeling Nucleic Acids

TECHNICAL NOTE

Molecular Probes offers a variety of products for labeling nucleic acids with ourfamily of superior Alexa Fluor dyes (Section 8.2). The Alexa Fluor dyes span the visiblelight spectrum and beyond (Figure 1.14, Figure 1.21, Figure 1.30) making them idealtools for multicolor applications such as fluorescence in situ hybridization (FISH) andmicroarray experiments (Section 8.5). The spectral diversity provides an enormousamount of flexibility in choosing a label that is compatible with a particular opticaldetection system or multicolor experiment. The Alexa Fluor dyes have several propertiesthat make them superior to other fluorescent dyes:

• High water solubility. The Alexa Fluor dyes are highly water soluble, making themideal for hybridization experiments. Nucleic acids labeled with the Alexa Fluor dyesdo not aggregate or precipitate, even in high-salt conditions.

• pH independence. Fluorescence of the Alexa Fluor conjugates is not pH sensitive inthe ranges used for hybridization solutions and microscopy mounting media (Figure1.11).

• Resistance to photobleaching. The enhanced photostability of Alexa Fluor dyesmakes them ideal for applications requiring imaging, such as FISH and microarrays(Figure 1.10).

Bright Fluorescence When Bound to Nucleic AcidsNucleic acids labeled with Alexa Fluor dyes show brighter fluorescence than nucleic

acids labeled with similar dyes. We have observed that nucleic acids labeled with theCy3 dye exhibit abnormal quenching and absorption shifts to shorter wavelengths(Figure 1) that are correlated with a decrease in fluorescence emission (Figure 2). Nucle-ic acids labeled with the spectrally similar Alexa Fluor 546 dye do not show this absorp-tion shift (Figure 1); therefore, they are far brighter than nucleic acids labeled with theCy3 dye. Nucleic acids labeled with the Cy5 dye show a similar absorption shift andaccompanying loss of fluorescence, whereas nucleic acids labeled with the spectrallysimilar Alexa Fluor 647 dye do not (Figure 1). Therefore, nucleic acids labeled with theAlexa Fluor 647 dye exhibit much brighter fluorescence (Figure 2). The absorption shiftsand fluorescence quenching phenomena appear to increase with the level of Cy dyelabeling and are reversible upon treatment with nucleases. Thus, higher levels of label-ing with Cy dyes do not result in appreciable increase in fluorescence, markedly limitingthe brightness achievable with these dyes (Figure 3). Because Alexa Fluor dyes do notshow this absorption shift, nucleic acids can be very highly labeled to produce excep-tionally bright and sensitive nucleic acid probes and labeled samples.

Figure 1 Change in absorption of Alexa Fluor dyesversus Cy dyes. DNA was modified using nicktranslation in the presence of aminoallyl dUTP(A-21664) and then labeled with Alexa Fluor 546or 647 succinimidyl ester (A-21677, A-21676) orwith Cy3 or Cy5 reactive dye. The absorptionspectra of the Cy3 and Cy5 dye–labeled DNAshow additional shorter-wavelength absorptionpeaks when compared to the spectrum of the freedye. However, light absorbed by the Cy dye–la-beled DNA at this wavelength does not result influorescence. DNA labeled with the spectrallysimilar Alexa Fluor dyes does not exhibit thisspectral anomaly and therefore exhibit muchgreater fluorescence at similar degrees of labeling.

continued on next page

Figure 2 Fluorescence emission of DNA la-beled with Alexa Fluor dyes versus Cydyes. DNA was amine modified by reversetranscription in the presence of aminoallyldUTP (A-21664). The modified DNA wasthen labeled with Alexa Fluor 555 or 647succinimidyl ester (A-21677, A-21676) orwith Cy3 or Cy5 reactive dye to a level opti-mal for hybridization. The fluorescenceemission spectra for DNA samples labeledto the same degree show that the AlexaFluor dye–labeled DNA was consistentlybrighter than the spectrally similar Cy dye–labeled DNA.

289

Figure 3 Change in signal brightness with level of labeling forAlexa Fluor versus Cy dye–labeled DNA. Chromosome 17 α-sat-ellite DNA was amine-modified by nick translation in the presenceof varying ratios of aminoallyl dUTP (A-21664) to dTTP. Thepools of modified DNA were split into equal parts and labeledwith Alexa Fluor 555 or Alexa Fluor 647 succinimidyl ester(A-21677, A-21676) or with Cy3 or Cy5 reactive dye. The degreeof labeling was calculated for each reaction; each probe was thenhybridized to human metaphase chromosomes and the bright-ness of each signal measured. The brightness of the signal wasplotted against the degree of labeling for each dye. At higher lev-els of labeling, the Alexa Fluor dyes become brighter, whereas thecorresponding Cy dyes become quenched.

continued from previous page

ChromaTide Nucleotides

Molecular Probes offers a series of uridine triphosphates (UTP,Table 8.5) and deoxyuridine or deoxycytidine triphosphates (dUTP,OBEA-dCTP; Table 8.6) conjugated to an extensive selection ofhaptens and fluorophores, including several that incorporate oursuperior Alexa Fluor dyes (see Alexa Fluor Dyes for LabelingNucleic Acids). These ChromaTide nucleotides are useful forgenerating labeled nucleic acids for molecular biology and molecu-lar cytogenetics applications, including chromosome and mRNAFISH experiments 2–5 (Figure 8.76), gene expression studies andmutation detection on arrays and microarrays 6–16 (Figure 8.91),in situ PCR and RT-PCR. The biotin and DNP nucleotides areuseful for signal amplification in FISH and microarrays and fordetection of probes hybridized to blots (Section 8.5). Our extensiveselection of fluorescent labels provides the ideal tools for multicol-or techniques such as spectral karyotyping, 17–23 multilocus FISHanalysis,24 “chromosome painting” 25 and comparative genomehybridization 26,27 (Section 8.5).

Structures of the ChromaTide NucleotidesThe ChromaTide UTP and dUTP nucleotides (see Legal

Notice for ChromaTide UTP and dUTP Nucleotides) are modifiedat the C-5 position of uridine via a unique aminoalkynyl linker(Figure 8.38). The C-5 position of UTP and dUTP is not involvedin Watson–Crick base-pairing and so interferes little with probehybridization. The aminoalkynyl linker 28 between the fluoro-phore and the nucleotide in the ChromaTide UTP and dUTPnucleotides is designed to reduce the fluorophore’s interactionwith enzymes or target binding sites. In addition to this four-atombridge, several of these nucleotides contain a seven- to ten-atomspacer, which further separates the dye from the base. The num-ber in the product’s name (e.g., the “12” in ChromaTide fluores-cein-12-dUTP) indicates the net length of the spacer in atoms.Longer spacers typically result in brighter conjugates and in-creased hapten accessibility for secondary detection reagents.

The ChromaTide OBEA-deoxycytidine triphosphates 1 (OBEA-dCTP, Table 8.6) are modified at the N-4 position of cytosine using

Figure 8.38 Structure of ChromaTide BODIPY FL-14-dUTP (C-7614).This structure is representative of our other ChromaTide labeled dUTPnucleotides. Fluorophore labels are attached via a four-atom aminoalky-nyl spacer (between arrows A and B) to either deoxyuridine triphosphate(dUTP) or uridine triphosphate (UTP). Fluorophore labels for otherChromaTide nucleotides, which also include OBEA-dCTP nucleotides,are indicated in Table 8.5 and Table 8.6.

Table 8.5 Characteristics of ChromaTide UTP nucleotides.

Section 8.2


a patented 1 2-aminoethoxyethyl (OBEA) linker (Figure 8.39). OurOBEA-dCTP also has a built-in seven-atom spacer that reducespossible interference in enzyme activity caused by the presence ofthe dye on the nucleotide.

Biotin and Dinitrophenyl (DNP) ChromaTide NucleotidesNucleic acid probes labeled with biotin have generally been the

most common nonisotopic probes used in hybridization techniques.Biotinylated probes are detected with fluorophore or enzyme con-jugates of avidins or streptavidins (Table 7.17; Section 7.6, Section8.5), providing amplification of the signal (Figure 8.78). Biotin canalso be detected with anti-biotin antibodies, which we provideunconjugated (A-11242, Section 7.4), or conjugated to the brightgreen-fluorescent Alexa Fluor 488 dye (A-11243, Section 7.4). Thesignal from biotin-labeled hybridization probes can be considerablyamplified, while retaining excellent spatial resolution, by combina-tion with Enzyme-Labeled Fluorescence (ELF) technology (Sec-tion 8.5, Figure 8.85) or tyramide signal amplification (TSA)technology (Section 8.5, Figure 8.83). In addition, biotinylatednucleic acids can be adsorbed onto streptavidin or CaptAvidinagarose (S-951, C-21386; Section 7.6; Figure 7.85), bound to thestreptavidin conjugate of Captivate ferrofluid superparamagneticparticles (C-21476, Section 7.6) or detected with NANOGOLD orAlexa Fluor FluoroNanogold streptavidin 29–31 (N-24918, A-24926,A-24927; Section 7.6). Our ChromaTide dinitrophenyl-11-dUTP(DNP-11-dUTP, C-7610) provides a hapten that can be combinedwith fluorophores, biotin or other haptens in double-labeling exper-iments. The DNP hapten can be detected with our rabbit anti–DNP-KLH antibody, which is available unlabeled (A-6430, Section7.4) or labeled with the Alexa Fluor 488 dye (A-11097, Section7.4) or fluorescein (A-6423, Section 7.4). Both ChromaTide biotin-11-dUTP (C-11411) and ChromaTide DNP-11-dUTP (C-7610) canbe incorporated into DNA probes using a variety of enzymatictechniques (Table 8.6).

Fluorescent ChromaTide NucleotidesThe spectral diversity of our ChromaTide dUTP and Chroma-

Tide OBEA-dCTP nucleotides (Table 8.6) and of the ChromaTideUTP nucleotides (Table 8.5) gives researchers significant flexibil-ity in choosing a label that is compatible with a particular opticaldetection system or multicolor experiment. Probes made from thefluorescent ChromaTide nucleotides can be imaged directly;alternatively, some fluorophores can be used as a hapten forsignal amplification, as described in Section 8.5 (see Legal Noticefor ChromaTide UTP and dUTP Nucleotides). In many cases, theTSA (Section 6.2) or ELF technologies (Section 6.3) can be usedto significantly amplify the signal of dye-labeled hybridizationprobes in cells and tissues (Section 8.5). Combination of the TSAand ELF technologies promises to yield the most sensitive detec-tion of in situ hybridization that is currently possible.32 The AlexaFluor conjugates of UTP, OBEA-dCTP and dUTP provide fluoro-phore labels with demonstrably superior fluorescence properties,compared to conventional dyes (see Alexa Fluor Dyes for Label-ing Nucleic Acids). Likewise, Alexa Fluor 488-5-dUTP, AlexaFluor 568-5-dUTP and Alexa Fluor 594-5-dUTP are spectrallysimilar to the fluorescein, Lissamine rhodamine B and Texas Redconjugates, respectively, but exhibit superior spectral and chemi-cal properties. ChromaTide OBEA-dCTP nucleotides have beenprepared from four of our best dyes — the Alexa Fluor 488,Alexa Fluor 546, Alexa Fluor 594 and Alexa Fluor 647 dyes —with spectra virtually identical to those of fluorescein, Cy3, TexasRed and Cy5 dyes, respectively (see Alexa Fluor Dyes for Label-ing Nucleic Acids).

The ChromaTide Alexa Fluor dUTP and ChromaTide AlexaFluor OBEA-dCTP nucleotides are highly water soluble, as areDNA probes that contain them. Thus, Alexa Fluor dye–labeledDNA probes do not aggregate or precipitate, even in high-salthybridization solutions. Fluorescence of the Alexa Fluor conju-gates is not pH sensitive in the ranges used for hybridizationsolutions or found in microscopy mounting media. Additionally,the enhanced photostability of these conjugates makes them idealfor applications requiring imaging.

We also have available the Oregon Green 488, RhodamineGreen and Texas Red conjugates of dUTP (C-7630, C-7629,C-7631, C-7608). When compared with the correspondingfluorescein conjugates (C-7603, C-7604), the Oregon Green488 and Rhodamine Green conjugates have similar fluorescencespectra but superior photostability (Section 1.5). The TexasRed-12-dUTP (C-7631) has an emission spectrum in solutionthat is narrower and about 25% more intense than that of TexasRed-5-dUTP (C-7608). For certain multicolor applications, werecommend conjugates of the BODIPY dyes because they havenarrow emission bandwidths with minimal spectral overlap. TheBODIPY 630/650-14-dUTP (C-11395) and BODIPY 650/665-14-dUTP (C-11396) are well suited to excitation by the 633 nmspectral line of the He–Ne laser and the 647 nm spectral line ofthe Ar–Kr laser, respectively. Oregon Green 488-5-dUTP hasbeen microinjected into unfertilized oocytes to follow DNAsynthesis in oocytes following fertilization.33 Other microinject-ed fluorescent nucleotides have been utilized to follow thedynamics of chromosome formation and cell proliferation inlive cells.34,35

Figure 8.39 Structure of ChromaTide Alexa Fluor 488-7-OBEA-dCTP(C-21555). This structure is representative of our ChromaTide OBEA la-beled OBEA-dCTP nucleotides. Fluorophore labels are attached via theOBEA spacer (between arrows A and B) to deoxycytidine triphosphate(dCTP). Fluorophore labels for other ChromaTide nucleotides are indi-cated in Table 8.5 and Table 8.6.

291

Table 8.6 Characteristics of ChromaTide dUTP and ChromaTide OBEA-dCTP nucleotides.

Using ChromaTide Nucleotides in Enzymatic Labeling MethodsThe ChromaTide nucleotides can be incorporated into DNA and RNA using conven-

tional enzymatic labeling techniques (Table 8.5, Table 8.6). Protocols for many of thesetechniques are provided with the ChromaTide nucleotides. Enzymes that we have usedsuccessfully include:

• Taq polymerase in polymerase chain reaction (PCR) assays 36 (Note: we have ob-served that the long-wavelength BODIPY dye conjugates (C-11395, C-11396)do not serve as Taq polymerase substrates and appear to inhibit the Taq polymerasereaction.)

• DNA polymerase I in nick-translation and primer-extension assays• Klenow polymerase in random-primer labeling

Section 8.2

Technical Assistanceat Our Web Site(www.probes.com)

Molecular Probes’ Web sitecontains the complete text of thisHandbook as well as a number of otherfeatures to assist the researcher,including:

• Product searches by product nameor catalog number

• Color photomicrographs andmovies that show our products inaction

• Bibliographies for all products forwhich we have references

• Keyword searches of our entirebibliography of over 44,000references

• Product information sheets formany kits and reagents

• Technical bulletins, includingBioProbes newsletters and otherproduct literature

• Chemical structures, technicaldata and material safety datasheets

Check our Web site frequently forinformation on our newest productsand the most recent additions to ourbibliography, as well special offers onfeatured products.

Additional information on thescientific and technical background ofour products can be obtained bycontacting our Technical AssistanceDepartment:

In the U.S. and CanadaPhone: (541) 465-8353Fax: (541) 465-4593E-mail: [email protected]

In EuropePhone: +31-71-5233431Fax: +31-71-5241883E-mail: [email protected]


• Terminal deoxynucleotidyl transferase (TdT) for 3′-end labeling• Reverse transcriptase for synthesizing DNA from RNA templates• SP6 RNA polymerase, T3 RNA polymerase and T7 RNA polymerase for in vitro

transcription

Please note that not all ChromaTide nucleotides have been tested in all applications.Refer to Table 8.5 and Table 8.6 for information on applications of individual Chroma-Tide nucleotides.

ChromaTide nucleotides have also been used in the TUNEL assay for detecting DNAfragmentation in apoptotic cells 37–40 (Section 15.5, Figure 15.70). Microinjected fluores-cent nucleotides are incorporated into cellular nucleic acids where they assemble intochromosomes and persist through cell replication.34

Aminoallyl UTP and Aminoallyl dUTP

Aminoallyl UTP 41–43 (5-(3-aminoallyl)uridine 5′-triphosphate, A-21663) and ami-noallyl dUTP 44 (5-aminoallyl-2′-deoxyuridine 5′-triphosphate, A-21664) can be incor-porated into RNA and DNA, respectively, using conventional enzymatic incorporationtechniques, as described above for the ChromaTide UTP, OBEA-dCTP and dUTP nucle-otides.45 The resulting amine-modified nucleic acid can then be labeled using any of theamine-reactive dyes and other reagents that are described in Chapter 1. Lacking bulkydye groups, the aminoallyl-modified nucleotides can be incorporated to extremely highand consistent levels. Subsequent reaction of the amine-modified nucleic acid with anexcess of amine-reactive reagent achieves correspondingly high and consistent labelingefficiencies, regardless of the labeling reagent chosen. We typically obtain labeling effi-ciencies of 1 dye for every 12–20 bases. This two-step labeling method also eliminatesthe need to optimize an enzymatic reaction to accommodate different dye-modifiednucleotides, which may incorporate at very different rates. This labeling method is idealfor both FISH probes (Figure 8.40, Figure 8.77) and microarray-based experiments (Fig-ure 8.87). Aminoallyl dUTP labeling can be achieved easily using our convenient ARESDNA Labeling Kits (see below), which provide aminoallyl dUTP, premeasured aliquotsof our best reactive dyes and carefully tested protocols.

5-Bromo-2′-Deoxyuridine, 5-Bromo-dUTP (BrdUTP) and5-Bromo-UTP (BrUTP)

Cells can naturally incorporate the thymidine analog, 5-bromo-2′-deoxyuridine (BrdU,B-23151) into their DNA during cell division, making this nucleoside analog an excellentmarker of both cell cycle and cell proliferation.46 Analysis of incorporated BrdU can beeither by direct detection with an antibody to BrdU-modified DNA or by modification ofthe fluorescence of a nucleic acid stain. For instance, the fluorescence of TO-PRO-3 andLDS 751 is considerably enhanced by the presence of BrdU in DNA,47 whereas that ofthe Hoechst dyes is specifically quenched.48 5-Bromo-2′-deoxyuridine 5′-triphosphate(BrdUTP, B-21550) is commonly used in TUNEL-based methods to detect proliferating orapoptotic cells,38,39 as in the ABSOLUTE-S SBIP Cell Proliferation Assay Kit (A-23150,Section 15.4, Figure 15.51) and the Apo-BrdU TUNEL Assay Kit (A-23210, Section15.5). In addition, this nucleotide is a substrate for reverse transcriptase 49,50 and has beenused in a sensitive nonisotopic assay for detection of HIV-1–associated reverse transcript-ase activity.49,51 Similarly, the corresponding brominated ribonucleotide, 5-bromouridine5′-triphosphate (BrUTP, B-21551) is an excellent substrate for RNA polymerase 52 and hasbeen used to monitor nucleolar transcription in situ.53,54

BrdUTP is readily incorporated into apoptotic cells by terminal deoxynucleotidyltransferase (TdT), as in the Apo-BrdU TUNEL Assay Kit (A-23210, Section 15.5), and isapparently metabolized in cells like thymidine 5′-triphosphate. Furthermore, UV light–induced photolysis of nucleic acids that have incorporated BrdU from either 5-bromo-2′-deoxyuridine or BrdUTP are susceptible to photolytic cleavage, which is the basis fornucleic acid labeling and detection in the ABSOLUTE-S SBIP Cell Proliferation AssayKit 38,39,55 (A-23150, Section 15.4). BrdUTP can also be used to detect excision and repairof strand breaks in UV light–damaged DNA in cells.56 BrdUTP is a substrate for reverse

Figure 8.40 Expression of snail RNA in an early-stage fruit fly embryo visualized by FISH. A 1.7 kbRNA probe corresponding to the snail gene was la-beled by in vitro transcription using aminoallyl UTP(A-21663) followed by reaction with Alexa Fluor488 carboxylic acid, succinimidyl ester (A-20000,A-20100). The probe was hybridized to Drosophilamelanogaster embryos and imaged using confocallaser-scanning microscopy. Image contributed byDavid Kosman and Ethan Bier, University of Califor-nia, San Diego.

Figure 8.41 Schematic diagram of the labelingmethod provided in our ULYSIS Nucleic Acid La-beling Kits (Table 8.7). The ULS reagent in theULYSIS Nucleic Acid Labeling Kits reacts with theN-7 position of guanine residues to provide a sta-ble coordination complex between the nucleic acidand the fluorophore label.

293

transcriptase 49,50 and Klenow polymerase.57,58 BrdUTP has alsobeen used in a sensitive nonisotopic assay for detecting HIV-1–associated reverse transcriptase activity.49,51 Nucleic acids contain-ing halogenated bases can be photocrosslinked to proteins withwhich they interact.59,60 BrUTP and BrdUTP can serve as low-costbuilding blocks for nucleic acid probes, in the manner of thefluorescent ChromaTide nucleotides (see below), with detectionby labeled anti-BrdU antibodies (Section 15.4). BrUTP is reportedto be a better substrate for RNA polymerase than is UTP itself.52

BrUTP that has been microinjected into cells is incorporated intoRNA of a nucleolar compartment.53,54 For an especially efficientand low-cost method of producing large quantities of DNA probe,BrdUTP can be incorporated into DNA while cells or plasmid-containing bacteria are growing in the presence of BrdU.61

Molecular Probes offers anti-BrdU mouse monoclonal anti-bodies conjugated with several of our superior Alexa Fluor dyes(Section 15.4). Because incorporation of BrdU and the relatedBrdUTP into DNA is specific, use of the labeled anti-BrdU anti-body permits unequivocal detection of DNA in cells. Also, ourfluorescently labeled anti-BrdU antibody crossreacts with ribonu-cleic acids that have incorporated bromouridine or BrUTP, thuspermitting the only method of specifically detecting transcribedRNA in cells with a fluorescent dye.

ULYSIS Nucleic Acid Labeling Kits

ULYSIS Nucleic Acid Labeling Kits (Table 8.7) combinesome of Molecular Probes’ best fluorescent dyes with the versa-tile, patented Universal Linkage System (ULS) platinum-basedchemistry developed at KREATECH Diagnostics,113 resulting ina simple, fail-safe method for producing bright, fluorophore-labeled hybridization probes.22,62,63 The ULS labeling techniquedirectly labels nucleic acids without the need for enzymatic incor-poration of modified nucleotides. The ULS method is based onthe use of a platinum–dye complex, patented by KREATECHBiotechnology BV, that forms a stable adduct with the N-7 posi-tion of guanine and, to a lesser extent, adenine bases in DNA,RNA, PNA and oligonucleotides (Figure 8.41). The labelingreaction requires only 15 minutes, and separation of the labeled

Figure 8.42 Nucleic acid labeling method provided in our ULYSIS Nucleic Acid Labeling Kits (Table 8.7).

nucleic acids from the unreacted ULS complex can be accom-plished through the use of a simple spin-column procedure(Figure 8.42). DNA longer than ~1000 base pairs requires a10-minute DNase digestion before labeling, which both optimizeslabeling and fragments the probe for efficient hybridization.

The ULYSIS Kits allow researchers to label DNA with a widevariety of our exceptionally bright and photostable Alexa Fluordyes (see Alexa Fluor Dyes for Labeling Nucleic Acids) and theOregon Green 488 dye (Table 8.7). Probes labeled using theULYSIS Kits are stable indefinitely and hybridize effectively totarget DNA. The ULS method has been used to prepare labeledprobes for dot, Southern and Northern blot analysis, RNA andDNA in situ hybridization, multicolor fluorescence in situ hybrid-ization (FISH, Section 8.5, Figure 8.43, Figure 8.74, Figure 8.75),comparative genome hybridization (CGH) and microarray analy-sis (Figure 8.90). We maintain a bibliography of references forthe ULS technology that use our ULS reagents and those previ-ously described (U-24832). Combination of the Oregon Green488 dye or Alexa Fluor 488 dye with antibodies to these dyes(Section 7.4) permits detection by chemiluminescent, chromoge-nic or fluorogenic enzyme-linked methods — including signalamplification schemes that use the TSA and ELF technologies(Section 8.5) or the BOLD APB chemiluminescent substrate foralkaline phosphatase (B-21901, Section 8.4).

Each ULYSIS Nucleic Acid Labeling Kit provides:

• The ULS labeling reagent and an appropriate solvent• Labeling buffer• Deoxyribonuclease I (DNase I), for digesting DNA longer

than 1000 base pairs prior to labeling• DNase I storage buffer• Concentrated DNase I reaction buffer• Control DNA from calf thymus• Nuclease-free H2O• A detailed procedure for preparing fluorescent DNA hybrid-

ization probes optimized for chromosome in situ hybridizationand dot-blot hybridization

Sufficient materials are supplied in each kit for 20 labelings of1 µg DNA each.

Section 8.2


Table 8.7 Spectral characteristics of the fluorescent dyes available in Molecular Probes’ ULYSIS NucleicAcid Labeling Kits.

Cat # Fluorophore Ex/Em * Similar Dyes

U-21658 Pacific Blue 410/455 SpectrumAqua

U-21650 Alexa Fluor 488 490/520 Fluorescein (FITC or FAM), SpectrumGreen

U-21659 Oregon Green 488 495/520 Fluorescein (FITC or FAM), SpectrumGreen

U-21651 Alexa Fluor 532 525/550 Rhodamine 6G

U-21652 Alexa Fluor 546 555/570 Cy3 dye, tetramethylrhodamine (TRITC), SpectrumOrange

U-21653 Alexa Fluor 568 575/600 Lissamine rhodamine B dye

U-21654 Alexa Fluor 594 590/615 Texas Red dye, SpectrumRed

U-21660 Alexa Fluor 647 650/670 Cy5 dye

U-21656 Alexa Fluor 660 660/690 Cy5 dye, Cy5.5 dye

U-21657 Alexa Fluor 680 680/700 Cy5.5 dye

* Excitation (Ex) and Emission (Em) maxima, in nm.

Figure 8.43 Centromere probes to chromosome 1,chromosome 15 and chromosome 17 were labeledwith the ULYSIS Alexa Fluor 546 (U-21652), AlexaFluor 594 (U-21654) and Oregon Green 488 Nucle-ic Acid (U-21659) Labeling Kits, respectively, andhybridized to human metaphase chromosomes.The chromosomes were then counterstained withHoechst 33342 (H-1399, H-3570, H-21492).

ARES DNA Labeling Kits

ARES DNA Labeling Kits (Table 8.8) provide a versatile, two-step method for label-ing DNA with fluorescent dyes (Figure 8.44). This method achieves a uniformity andconsistency of labeling that is difficult to obtain with conventional enzymatic incorpora-tion of labeled nucleotides. In the first step, an amine-modified nucleotide, 5-(3-aminoal-lyl)-dUTP, is incorporated into DNA using conventional enzymatic labeling methods.This step ensures relatively uniform labeling of the probe with primary amine groups.The aminoallyl dUTP substrate used in this reaction is taken up efficiently by reversetranscription or nick translation, for which we provide the protocols; other enzymaticmethods are also very likely to be compatible. In the second step, the amine-modifiedDNA is chemically labeled using an amine-reactive fluorescent dye. This chemical reac-tion varies little in its efficiency from dye to dye, so that it is possible to use any combi-nation of the ARES Kits, with their broad selection of the brightest and most photostabledyes, and obtain consistent labeling for every DNA sample. The labeling protocols pro-

Figure 8.45 Fluorescent probes generated withARES DNA Labeling Kits hybridized to humanmetaphase chromosome spreads. Centromereprobes specific for chromosomes 17, 1 and 15were prepared by nick translation and labeled withkits containing green-fluorescent Alexa Fluor 488(A-21665), red-orange–fluorescent Alexa Fluor 546(A-21667) and red-fluorescent Alexa Fluor 594(A-21669) dyes. DNA was counterstained with theblue-fluorescent Hoechst 33342 dye (H-1399,H-3570, H-21491), and the slides were mountedusing the ProLong Antifade Kit (P-7481). This mul-tiple-exposure image was obtained with bandpassfilter sets appropriate for fluorescein, rhodamine,Texas Red dye and DAPI.

Figure 8.44 Schematic diagram of the labeling method provided in our ARES DNA Labeling Kits (Ta-ble 8.8). The ARES DNA Labeling Kits use a two-step method to label DNA. Step 1) The aminoallyldUTP is enzymatically incorporated. Step 2) A reactive fluorophore is used to label the incorporatedaminoallyl group.

Figure 8.46 A-21664 5-(3-aminoallyl)-2′-deoxyuri-dine 5′-triphosphate, trisodium salt.

295

Table 8.8 Spectral characteristics of the fluorescent dyes available in Molecular Probes’ ARESDNA Labeling Kits.

Cat # Fluorophore Ex/Em * Spectrally Similar Dyes

A-21675 Alexa Fluor 350 346/442 AMCA

A-21673 Pacific Blue 410/455 DEAC, SpectrumAqua

A-21665 Alexa Fluor 488 490/520 Fluorescein (FITC), SpectrumGreen

A-21674 Oregon Green 488 495/520 Fluorescein (FITC), SpectrumGreen

A-21666 Alexa Fluor 532 525/550 Rhodamine 6G

A-21667 Alexa Fluor 546 555/570 Cy3 dye, tetramethylrhodamine (TRITC), SpectrumOrange

A-21677 Alexa Fluor 555 555/565 Cy3 dye, tetramethylrhodamine (TRITC), SpectrumOrange

A-21668 Alexa Fluor 568 575/600 Lissamine rhodamine B dye

A-21669 Alexa Fluor 594 590/615 Texas Red dye, SpectrumRed

A-21676 Alexa Fluor 647 650/670 Cy5 dye

A-21671 Alexa Fluor 660 660/690 Cy5 dye, Cy5.5 dye

A-21672 Alexa Fluor 680 680/700 Cy5.5 dye

* Excitation (Ex) and Emission (Em) maxima, in nm.

vided generally result in about one dye per 12–20 bases, whichwe have determined to be optimal for FISH and dot-blot hybrid-ization. Nucleic acids labeled using this method are ideal forFISH (Figure 8.45) or microarray experiments (Figure 8.87).

The ARES Kits are supplied with some of our best fluorescentdyes (Table 8.8). The Alexa Fluor dyes (Section 1.3) have proper-ties superior to conventional dyes for labeling nucleic acids (seeAlexa Fluor Dyes for Labeling Nucleic Acids). The OregonGreen 488 dye is a modification of fluorescein with reduced pHsensitivity and higher photostability (Figure 1.11, Figure 1.57).The signal of nucleic acids labeled with this dye can be amplifiedusing our anti-fluorescein/Oregon Green antibodies, as describedin Section 8.5. Our ARES Pacific Blue DNA Labeling Kit(A-21673), provides a blue-green–fluorescent label whose fluo-rescence can be distinguished from that of DAPI — a commonlyused nucleic acid counterstain.

Each ARES DNA Labeling Kit provides:

• 5-(3-Aminoallyl)-dUTP• The amine-reactive fluorescent dye and an appropriate solvent• Sodium bicarbonate• Nuclease-free H2O• A detailed protocol for labeling DNA using reverse tran-

scriptase or nick translation

Sufficient materials are supplied for 5–10 labelings, eachcontaining 1–5 µg DNA. The 5-(3-aminoallyl)-dUTP (A-21664,Figure 8.46) and succinimidyl ester dyes are also available asstandalone reagents. Enzymatic incorporation of 5-(3-aminoal-lyl)-dUTP permits incorporation of almost any amine-reactivedye in Chapter 1 into nucleic acids.44

Labeled OligonucleotidesDNA can also be labeled from RNA templates by reverse

transcription using fluorophore-labeled random oligonucleotideprimers and unlabeled deoxynucleotide triphosphates. Molecular

Figure 8.47 Absorption spectrum of 5′-QSY 7–labeled M13 primer in pH 7.5 TE buffer.

Probes provides two types of labeled oligodeoxynucleotides thatcan be used for this purpose. Our dT18 oligodeoxynucleotides(O-21560, O-21561, O-21562, O-21563) are labeled at the 5′-terminus with one of four of our popular Alexa Fluor dyes(Table 8.13). The labeled dT18 oligodeoxynucleotides hybridizeto poly(A) tails in RNA samples, providing primers for reversetranscription or hybridization probes for poly(A)-terminatedmRNA in cell- and solution-based assays. Our Panomer 9random-sequence oligodeoxynucleotides (Section 8.5) arecovalently labeled on the 5′-terminus with one of our propri-etary fluorescent dyes, with a nonfluorescent QSY 7 quencherdye (Figure 8.47) or with biotin (Table 8.13). The Panomer 9oligonucleotides are also useful as primers for synthesizinglabeled DNA via Klenow DNA polymerase or reverse tran-scriptase. In these reactions, the primer provides the fluorescentlabel, whereas unlabeled nucleotides are incorporated by theenzyme. This labeling strategy ensures efficient and unbiasedincorporation of nucleotides, because the bulky dye moleculedoes not interfere with nucleotide incorporation. However,because the synthesized probe contains only a single fluoro-phore, the labeling efficiency will typically be lower than thatachieved by incorporating fluorophore-labeled nucleotides.

Chemical Labeling of Oligonucleotides

Amine or thiol groups may be incorporated into a chemicallysynthesized oligonucleotide. These groups can then be directlyconjugated to an amine-reactive (Chapter 1) or thiol-reactive (Chap-ter 2) fluorophore or hapten (Figure 8.48). Fluorophore-labeledoligonucleotides are extensively used as primers for sequencing orPCR reactions (see below). Double-labeled oligonucleotides areused to produce fluorescence resonance energy transfer (FRET, seeSection 1.3) or quenched reporters for real-time PCR assays (Sec-tion 8.5). Labeled oligonucleotides can also be used as probes forfluorescence in situ hybridization (Section 8.5).

Section 8.2


Figure 8.48 Schematic diagram of labeling amine-modified oligodeoxynucleotides with succinimidylester dyes.

Figure 8.49 Fluorescence quenching of 5′-tetra-methylrhodamine–labeled M13 primers by nonfluo-rescent dyes attached at the 3′-end. The comparisonrepresents equal concentrations of oligonucleotideswith (1) no 3′-quencher (control), (2) 3′-dabcylquencher, (3) 3′-QSY 7 quencher.

Table 8.9 Oligonucleotide Amine Labeling Kits.

Cat # Fluorophore Ex/Em *

A-20190 Alexa Fluor 350 345/440

A-20191 Alexa Fluor 488 490/520

A-20192 Alexa Fluor 532 525/550

A-20193 Alexa Fluor 546 555/570

A-20197 Alexa Fluor 555 555/565

A-20194 Alexa Fluor 568 575/600

A-20195 Alexa Fluor 594 590/615

A-20196 Alexa Fluor 647 650/670

* Approximate excitation (Ex) and emission (Em)maxima, in nm.

Alexa Fluor Oligonucleotide Amine Labeling KitsThe Alexa Fluor Oligonucleotide Amine Labeling Kits (Table 8.9) provide the re-

agents required for labeling synthetic oligonucleotides that have amine groups incorpo-rated at their 5′- or 3′-terminus. Our outstanding Alexa Fluor dyes (Section 1.3) haveproperties superior to conventional dyes, including exceptionally high quantum yieldsand large extinction coefficients, excellent photostability, reduced pH sensitivity andimproved water solubility (see Alexa Fluor Dyes for Labeling Nucleic Acids). Thesedyes have excitation and emission wavelengths that span much of the visible-light spec-trum (Figure 1.14, Figure 1.21, Figure 1.30). Following purification by standard chro-matographic or electrophoretic procedures, these singly labeled oligonucleotides canserve as primers for a variety of applications. The dye-labeled oligonucleotides may alsoserve as fluorescence resonance energy transfer (FRET, see Section 1.3) acceptors ordonors in hybridization reactions. Each Alexa Fluor Oligonucleotide Amine Labeling Kitprovides the reagents required for three labelings of 50 µg each, a detailed protocol andrecommendations for methods to purify and characterize the conjugates.

Other Reactive Fluorescent Dyes and QuenchersSeveral other dyes in Chapter 1 and Chapter 2, including the dyes for sequencing

applications (see below), carboxydichlorofluorescein, carboxyrhodamine 6G, RhodamineGreen and Texas Red derivatives, can be used to label primers, deoxynucleotides ordideoxynucleotides for nucleic acid sequencing and other applications. Oligonucleotideslabeled with multiple dyes that form excited-state energy transfer pairs enhance thedetection in sequencing applications that depend on the argon-ion laser for excitation andare particularly useful for the molecular beacon, TaqMan and related technologies 64

(Section 8.5). The Rhodamine Green and BODIPY 505/515 dyes (see below) are usefulfor DNA sequencing using fluorescence lifetime decay measurements, a technique thatuses the different lifetimes of the dyes to separate the fluorescence signals.65 For optimallabeling of oligonucleotides that will be used with secondary detection reagents, werecommend amine-reactive haptens and fluorophores that contain aminohexanoyl spacers(“X”) to reduce the label’s interaction with the oligonucleotide and enhance its accessi-bility to secondary detection reagents. Oregon Green 488-X, Rhodamine Green-X,Rhodamine Red-X, Texas Red-X, fluorescein-X, tetramethylrhodamine-X, biotin-XX,DSB-X biotin and DNP-X succinimidyl esters (Table 4.2) are reactive forms of popularfluorescent dyes or haptens with aminohexanoyl spacer arms.

Our nonfluorescent QSY 7, QSY 9 and QSY 21 dyes (Section 1.6) have absorption inthe visible and near-infrared spectrum (Figure 1.66, Figure 8.47), making them excellentenergy transfer acceptors from a wide variety of dyes that emit in the visible range, in-cluding fluoresceins, Oregon Green dyes, rhodamines, Texas Red, Cy3 and several ofthe Alexa Fluor dyes for various assays that use fluorescence resonance energy transfer(FRET, see Section 1.3) (Figure 8.49, Table 1.8). Conjugates of the QSY 35 dye havesomewhat shorter-wavelength absorption (Figure 1.66) and are useful as quenchers ofUV light–excited fluorescent dyes. Oligonucleotide conjugates of the nonfluorescentdabcyl succinimidyl ester (D-2245, Figure 1.107) have also been extensively used forFRET-based and quencher-based assays.66–69 Several applications of QSY and dabcylconjugates of oligonucleotides are discussed in Section 8.5. A unique method that usesQSY dye–conjugated oligonucleotides to reduce the background from “primer dimers”in real-time PCR assays is described in Section 8.3 (Figure 8.59).

The amine-reactive succinimidyl esters of the SYBR 101, SYBR 102 and SYBR 103dyes (S-21500, S-21501, S-21502) can be coupled with amine-derivatized oligonucle-otides. The conjugates may fluoresce green as the result of intramolecular or intermolec-ular association of the dye with the nucleic acid backbone; however, changes in intensityand fluorescence polarization may occur during hybridization reactions. Similar amine-reactive versions of the cyanine dyes have been utilized to label peptide–nucleic acidconjugates (PNA) and to detect their hybridization to target nucleic acids in solution.70–73

Dyes for Sequencing ApplicationsMolecular Probes is the primary manufacturer of most of the dyes that are used direct-

ly or indirectly in nucleic acid sequencing and provides these dyes in reactive forms for

Several companies are licensed byMolecular Probes to prepare oligo-nucleotides using our patented dyes,including the BODIPY dyes, foruse in noncommercial applications.Our current licensees are listed atwww.probes.com/about/license.

297

preparing conjugates (Table 8.10). Because the electrophoreticseparation step during sequencing is highly sensitive to the chem-ical structure of the fragments, the use of single-isomer labels isessential. In addition to providing high-purity reactive succini-midyl esters of the common FAM, JOE, TET, HEX, TAMRA andROX dyes, Molecular Probes also prepares amine-reactive singleisomers of carboxyrhodamine 6G (CR 6G) (Table 8.10). The 6-isomer of the CR 6G dye has spectroscopic and electrophoreticproperties that are superior to the JOE dye often used for auto-mated DNA sequencing. Contact our Custom and Bulk SalesDepartment for information about availability of any of our reac-tive dyes in bulk.

Certain BODIPY dyes (Section 1.4) have been shown to bevery useful for DNA sequencing,74–76 in part because the dyeshave a minimal effect on the mobility of the fragment duringelectrophoresis and also exhibit well-resolved spectra with nar-row bandwidths (Figure 1.37). Using BODIPY dye energy trans-fer pairs further improves sensitivity. The patented BODIPY dyesare all high-purity, pH-insensitive single isomers.

BODIPY FL-X, BODIPY TMR-X and BODIPY TR-Xsuccinimidyl esters (Section 1.4) are reactive versions of ourpatented BODIPY fluorophores with emission properties similarto those of fluorescein, tetramethylrhodamine and Texas Reddyes, respectively (Figure 1.39). The BODIPY fluorophoresexhibit high extinction coefficients, excellent quantum yields anda fluorescence emission that is quite photostable and insensitiveto pH (Section 1.4). The narrow absorption and emission band-widths of these BODIPY fluorophores (Figure 1.37) make themparticularly well suited to multicolor applications. BODIPY630/650-X succinimidyl ester and the BODIPY 650/665 analogprovide long-wavelength fluorophores that match filter setsoptimized for the Alexa Fluor 647 and Cy5 dyes (Table 24.6).Oligonucleotide conjugates of the BODIPY FL, BODIPY R6G,BODIPY 564/570 and BODIPY FL, BODIPY R6G, BODIPY564/570 and BODIPY 581/591 have been found to be particularlyuseful for automated DNA sequencing.74–76

Labeling Phosphate-Modified Oligonucleotides

A fluorophore or hapten containing an aliphatic amine may beconjugated to the 3′- or 5′-phosphate group of an oligonucleotideby using the zero-length crosslinker EDAC (E-2247, Section 3.3)in an N-methylimidazole buffer at pH 9. This reaction results in aphosphoramidate bond that is stable in most molecular biologyassays. The method can be used in combination with T4 polynu-cleotide kinase to fluorescently label oligonucleotides lacking a5′-phosphate, or to double-label radioactively labeled oligonucle-otides. We have found that this reaction is very efficient — label-ing over 90% of the oligonucleotides that contain a phosphategroup — and much easier than conventional methods for modify-ing terminal phosphate groups, which typically require multistepsynthesis.77–79 For this reaction we recommend cadaverine-conju-gated fluorophores (Section 3.3) and biotins (Section 4.2).

It has also been reported that DNA can be reacted quantitative-ly with carbonyl diimidazole and a diamine (such as ethylenedi-amine) or a carbohydrazide to yield a phosphoramidate that has afree primary amine and that this amine can then be modified withamine-reactive reagents of the type described in Chapter 1.77–81

Fluorescent or biotinylated amines have been coupled to the 5′-phosphate of tRNA using dithiodipyridine and triphenylphos-phine.82 Wang and Giese 83 have reported a general method thatemploys an imidazole derivative prepared from our BODIPY FLhydrazide (D-2371, Section 3.2) to label phosphates, includingnucleotides, for capillary electrophoresis applications.

Other Chemical Labeling Methods forNucleic Acids

Labeling Cytidine ResiduesDNA and RNA can be modified by reacting their cytidine

residues with sodium bisulfite to form sulfonate intermediatesthat can then be directly coupled to hydrazides or aliphaticamines.84–86 For example, biotin hydrazides (Section 4.2) havebeen used in a bisulfite-mediated reaction to couple biotin to

Table 8.10 Amine-reactive dyes for nucleic acid sequencing.

Cat # Reactive Dye * Ex/Em § HandbookLocation

C-2210 5-FAM, SE † 494/518 Section 1.5

C-6164 6-FAM, SE 494/518 Section 1.5

C-20092 6-TET, SE 521/536 Section 1.5

C-6127 5-CR 6G, SE 525/555 Section 1.6

C-6128 6-CR 6G, SE 525/555 Section 1.6

C-6171 6-JOE, SE † 522/550 Section 1.5

C-20091 6-HEX, SE 535/556 Section 1.5

C-2211 5-TAMRA, SE 555/580 Section 1.6

C-6123 6-TAMRA, SE † 555/580 Section 1.6

C-6125 5-ROX, SE 580/605 Section 1.6

C-6126 6-ROX, SE † 580/605 Section 1.6

D-2184 BODIPY FL, SE ‡ 505/513 Section 1.4

D-6140 BODIPY FL, SSE 505/513 Section 1.4

D-6102 BODIPY FL-X, SE 505/513 Section 1.4

D-6180 BODIPY R6G, SE ‡ 528/550 Section 1.4

D-6117 BODIPY TMR-X, SE 542/574 Section 1.4

D-2222 BODIPY 564/570, SE ‡ 565/571 Section 1.4

D-2228 BODIPY 581/591, SE ‡ 584/592 Section 1.4

D-6116 BODIPY TR-X, SE 589/617 Section 1.4

* FAM = carboxyfluorescein; TET = 6-carboxy-2′,4,7,7′-tetrachlorofluorescein;CR 6G = carboxyrhodamine 6G; JOE = 6-carboxy-4′,5′-dichloro-2′,7′-dimethoxy-fluorescein; HEX = 6-carboxy-2′,4,4′,5′,7,7′-hexachlorofluorescein; TAMRA =carboxytetramethylrhodamine; ROX = carboxy-X-rhodamine; BODIPY = a sub-stituted 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene derivative (Figure 1.38);SE = succinimidyl ester; SSE = water-soluble sulfosuccinimidyl ester. † These arethe most widely used isomers for DNA sequencing (Anal Biochem 223, 39 (1994);Nucleic Acids Res 20, 2471 (1992); Proc Natl Acad Sci U S A 86, 9178 (1989);Genome Res 6, 995 (1996)). ‡ These BODIPY derivatives were reported to beuseful for automated DNA sequencing, in part because the dyes have a minimaleffect on the mobility of the fragment during electrophoresis and also exhibit well-resolved spectra with narrow bandwidths (Science 271, 1420 (1996)). § Excitation(Ex) and emission (Em) maxima, in nm.

Section 8.2


cytidine residues in oligonucleotides.87 The fluorescent, biotinyl-ated and other hydrazides and aliphatic amines in Chapter 3 andChapter 4, except possibly the BODIPY derivatives, might beuseful in this reaction. The bisulfite-activated cytidylic acid canalso be coupled to aliphatic diamines such as ethylenediamine.88

The amine-modified DNA or RNA can then be modified withany of the amine-reactive dyes described in this section or inChapter 1.

Labeling the 3′-Terminus of RNASelective oxidation of the 3′-terminus of RNA by sodium

metaperiodate yields a dialdehyde. This dialdehyde can then becoupled with a fluorescent or biotin hydrazide reagent 81,89–94

(Section 3.2, Section 4.2).

Labeling Abasic Sites with ARPThe biotinylated hydroxylamine ARP (Aldehyde-Reactive

Probe, A-10550) has been used to modify abasic sites in DNA —apurinic sites and apyrimidinic lesions thought to be importantintermediates in carcinogenesis 95–97 (Figure 8.132). Once thealdehyde group in an abasic site is modified with ARP, the result-ing biotinylated DNA can be detected with the avidin and strept-avidin conjugates described in Section 7.6 (Table 7.17). ARP ispermeant to cell membranes, permitting detection of abasic sitesin living cells.98

References

1. Manufactured for Molecular Probes by Sero-logicals Corporation and provided for researchuse only. OBEA dCTP is covered by US patent5,684,142. 2. Trends Genet 13, 475 (1997);3. Bioessays 19, 75 (1997); 4. Mol Pathol 51, 62(1998); 5. Cell Vis 5, 49 (1998); 6. Nat Genet 21,48 (1999); 7. Nat Genet 21, 42 (1999); 8. NatGenet 21, 33 (1999); 9. Nat Genet 21, 25 (1999);10. Nat Genet 21, 20 (1999); 11. Nat Genet 21, 15(1999); 12. Nat Genet 21, 10 (1999); 13. NatGenet 21, 5 (1999); 14. Mol Psychiatry 3, 483(1998); 15. Nat Biotechnol 16, 45 (1998);16. Biotechniques 19, 442 (1995); 17. Nat Genet14, 312 (1996); 18. Histochem Cell Biol 108, 299(1997); 19. Genes Chromosomes Cancer 28, 318(2000); 20. Cytometry 35, 214 (1999); 21. GenesChromosomes Cancer 27, 418 (2000); 22. Eur JHum Genet 7, 2 (1999); 23. Science 273, 494(1996); 24. Am J Hum Genet 61, 16 (1997);25. Cytobios 90, 7 (1997); 26. J Mol Med 75, 801(1997); 27. J Cell Biochem Suppl 17G, 139(1993); 28. US 5,047,519; 29. Eur J Histochem42, 111 (1998); 30. Cell Vis 5, 83 (1998); 31. AmJ Pathol 150, 1553 (1997); 32. J HistochemCytochem 48, 1593 (2000); 33. Dev Biol 206, 232(1999); 34. J Cell Biol 144, 813 (1999); 35. Bio-phys J 77, 2871 (1999); 36. The PCR process iscovered by patents owned by Hoffmann-LaRoche,Inc. Purchase of these products does not convey alicense under these patents. Information aboutlicenses for PCR can be obtained from PE Biosys-tems or Roche Molecular Systems, Inc. 37. Cy-tometry 27, 1 (1997); 38. Exp Cell Res 222, 28(1996); 39. Cell Prolif 28, 571 (1995); 40. Cytom-etry 20, 172 (1995); 41. J Clin Microbiol 29, 583

(1991); 42. Histochemistry 93, 191 (1989);43. Biotechniques 5, 660 (1987); 44. Biotech-niques 28, 518 (2000); 45. Proc Natl Acad Sci US A 90, 4206 (1993); 46. Methods Cell Biol 41,297 (1994); 47. Cytometry 17, 310 (1994);48. Exp Cell Res 173, 256 (1987); 49. BiotechnolAppl Biochem 29, 241 (1999); 50. BiotechnolAppl Biochem 23, 95 (1996); 51. J Virol Methods31, 181 (1991); 52. J Biochem (Tokyo) 96, 1501(1984); 53. Histochem Cell Biol 113, 181 (2000);54. Mol Biol Cell 10, 211 (1999); 55. Cell Prolif29, 539 (1996); 56. Mutat Res 193, 167 (1988);57. Exp Cell Res 234, 498 (1997); 58. Biochem J253, 637 (1988); 59. J Cell Biol 150, 797 (2000);60. Eur J Biochem 236, 389 (1996); 61. Biotech-niques 21, 82 (1996); 62. Genes ChromosomesCancer 25, 301 (1999); 63. Cytogenet Cell Genet87, 47 (1999); 64. Molecular Probes’ proprietarymaterials and methods have been found useful inthe practice of a variety of patents. Although weindicate the usefulness of our materials andmethods for the practice of third party technology,we emphasize that purchase of our products doesnot include a license to practice any third partypatent, unless a license under such a patent isclearly indicated in our product literature.65. Anal Chem 69, 2392 (1997); 66. Anal Bio-chem 276, 177 (1999); 67. Biotechniques 27,1116 (1999); 68. Biotechniques 26, 552 (1999);69. J Am Chem Soc 121, 2921 (1999); 70. Bio-chemistry 39, 4327 (2000); 71. Anal Biochem281, 26 (2000); 72. Anal Biochem 287, 179(2000); 73. J Am Chem Soc 123, 803 (2001);74. Biotechniques 25, 446 (1998); 75. Science

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Specialized Methods for Nucleic Acid ModificationA few other specialized methods have been developed for

nucleic acid modification. These include:

• Synthesis of DNA using fluorescent 2′- or 3′-acyl derivativesof uridine 5′-triphosphate and terminal deoxyribonucleotidetransferase 99

• Use of a fluorescent iodoacetamide or maleimide, along withT4 polynucleotide kinase and ATP-γ-S (ATP with a sulfur inthe terminal phosphate) to introduce a thiophosphate at the 5′-terminus of 5′-dephosphorylated RNA 90 or DNA

• Introduction of 4-thiouridine at the 3′-terminus of DNA usingcalf thymus terminal deoxynucleotidyl transferase followed bytreatment with ribonuclease and reaction with thiol-reactiveprobes 100,101

• Direct reaction of thiol-reactive reagents with 4-thiouridineresidues in nucleic acids 82,102–105

• Direct reaction of amine- or thiol-reactive reagents with ami-noacyl tRNA or thioacetylated aminoacyl tRNA 82,106,107

• Reaction of the X-base of tRNA with isothiocyanates 92 orreplacement of other uncommon bases in tRNA by fluoro-phores 108–110

• Photolabeling of plasmid DNA with fluorescent 4-azido-2,3,5,6-tetrafluorobenzyl derivatives 111

• Coupling of labeled diazonium salts to nucleic acids 112

299

Product information for ChromaTide nucleotide products is given in Table 8.5 and Table 8.6.

Cat # Product Name Unit SizeA-20190 Alexa Fluor® 350 Oligonucleotide Amine Labeling Kit *3 labelings* ..................................................................................................................... 1 kitA-20191 Alexa Fluor® 488 Oligonucleotide Amine Labeling Kit *3 labelings* ..................................................................................................................... 1 kitA-20192 Alexa Fluor® 532 Oligonucleotide Amine Labeling Kit *3 labelings* ..................................................................................................................... 1 kitA-20193 Alexa Fluor® 546 Oligonucleotide Amine Labeling Kit *3 labelings* ..................................................................................................................... 1 kitA-20197 Alexa Fluor® 555 Oligonucleotide Amine Labeling Kit *3 labelings* ..................................................................................................................... 1 kitA-20194 Alexa Fluor® 568 Oligonucleotide Amine Labeling Kit *3 labelings* ..................................................................................................................... 1 kitA-20195 Alexa Fluor® 594 Oligonucleotide Amine Labeling Kit *3 labelings* ..................................................................................................................... 1 kitA-20196 Alexa Fluor® 647 Oligonucleotide Amine Labeling Kit *3 labelings* ..................................................................................................................... 1 kitA-21664 5-(3-aminoallyl)-2′-deoxyuridine 5′-triphosphate, trisodium salt (aminoallyl dUTP) *2 mM in TE* ..................................................................... 500 µLA-21663 5-(3-aminoallyl)uridine 5′-triphosphate, trisodium salt (aminoallyl UTP) *2 mM in TE* ...................................................................................... 500 µLA-10550 N-(aminooxyacetyl)-N′-(D-biotinoyl) hydrazine, trifluoroacetic acid salt (ARP) .................................................................................................... 10 mgA-21675 ARES™ Alexa Fluor® 350 DNA Labeling Kit *5–10 labelings* .............................................................................................................................. 1 kitA-21665 ARES™ Alexa Fluor® 488 DNA Labeling Kit *5–10 labelings* .............................................................................................................................. 1 kitA-21666 ARES™ Alexa Fluor® 532 DNA Labeling Kit *5–10 labelings* .............................................................................................................................. 1 kitA-21667 ARES™ Alexa Fluor® 546 DNA Labeling Kit *5–10 labelings* .............................................................................................................................. 1 kitA-21677 ARES™ Alexa Fluor® 555 DNA Labeling Kit *5–10 labelings* .............................................................................................................................. 1 kitA-21668 ARES™ Alexa Fluor® 568 DNA Labeling Kit *5–10 labelings* .............................................................................................................................. 1 kitA-21669 ARES™ Alexa Fluor® 594 DNA Labeling Kit *5–10 labelings* .............................................................................................................................. 1 kitA-21676 ARES™ Alexa Fluor® 647 DNA Labeling Kit *5–10 labelings* .............................................................................................................................. 1 kitA-21671 ARES™ Alexa Fluor® 660 DNA Labeling Kit *5–10 labelings* .............................................................................................................................. 1 kitA-21672 ARES™ Alexa Fluor® 680 DNA Labeling Kit *5–10 labelings* .............................................................................................................................. 1 kitA-21674 ARES™ Oregon Green® 488 DNA Labeling Kit *5–10 labelings* .......................................................................................................................... 1 kitA-21673 ARES™ Pacific Blue™ DNA Labeling Kit *5–10 labelings* ................................................................................................................................... 1 kitB-23151 5-bromo-2′-deoxyuridine (BrdU) .......................................................................................................................................................................... 100 mgB-21550 5-bromo-2′-deoxyuridine 5′-triphosphate (BrdUTP) *10 mM in TE buffer* ......................................................................................................... 25 µLB-21551 5-bromouridine 5′-triphosphate (BrUTP) *10 mM in TE buffer* .......................................................................................................................... 25 µLO-21560 oligodeoxythymidine-18, Alexa Fluor® 488 conjugate (Alexa Fluor® 488 dT18) .................................................................................................... 10 nmolO-21561 oligodeoxythymidine-18, Alexa Fluor® 555 conjugate (Alexa Fluor® 555 dT18) .................................................................................................... 10 nmolO-21562 oligodeoxythymidine-18, Alexa Fluor® 594 conjugate (Alexa Fluor® 594 dT18) .................................................................................................... 10 nmolO-21563 oligodeoxythymidine-18, Alexa Fluor® 647 conjugate (Alexa Fluor® 647 dT18) .................................................................................................... 10 nmolS-21500 SYBR® 101, succinimidyl ester ............................................................................................................................................................................ 1 mgS-21501 SYBR® 102, succinimidyl ester ............................................................................................................................................................................ 1 mgS-21502 SYBR® 103, succinimidyl ester ............................................................................................................................................................................ 1 mgU-21650 ULYSIS® Alexa Fluor® 488 Nucleic Acid Labeling Kit *20 labelings* ................................................................................................................... 1 kitU-21651 ULYSIS® Alexa Fluor® 532 Nucleic Acid Labeling Kit *20 labelings* ................................................................................................................... 1 kitU-21652 ULYSIS® Alexa Fluor® 546 Nucleic Acid Labeling Kit *20 labelings* ................................................................................................................... 1 kitU-21653 ULYSIS® Alexa Fluor® 568 Nucleic Acid Labeling Kit *20 labelings* ................................................................................................................... 1 kitU-21654 ULYSIS® Alexa Fluor® 594 Nucleic Acid Labeling Kit *20 labelings* ................................................................................................................... 1 kitU-21660 ULYSIS® Alexa Fluor® 647 Nucleic Acid Labeling Kit *20 labelings* ................................................................................................................... 1 kitU-21656 ULYSIS® Alexa Fluor® 660 Nucleic Acid Labeling Kit *20 labelings* ................................................................................................................... 1 kitU-21657 ULYSIS® Alexa Fluor® 680 Nucleic Acid Labeling Kit *20 labelings* ................................................................................................................... 1 kitU-21659 ULYSIS® Oregon Green® 488 Nucleic Acid Labeling Kit *20 labelings* ............................................................................................................... 1 kitU-21658 ULYSIS® Pacific Blue™ Nucleic Acid Labeling Kit *20 labelings* ........................................................................................................................ 1 kit

Product List — 8.2 Labeling Oligonucleotide and Nucleic Acids

Section 8.2

The full citations and, in most cases, links to PubMed for all references in thisHandbook are available at our Web site (www.probes.com/search).

section 8.2 - labeling oligonucleotides and nucleic acids

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