Molecular Probes

Section 8.2 — Labeling Oligonucleotides and Nucleic Acids

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

• ChromaTide dUTP, ChromaTide OBEA-dCTP and ChromaTide UTP nucleotides, which provide researchers with a large selection of fluorophore- and hapten-labeled nucleotides that can be enzymatically incorporated into DNA or RNA probes for FISH (fluorescence in situ hybridization), DNA arrays/microarrays and other hybridization techniques.
• Unlabeled aminoallyl derivatives of dUTP and UTP, as well as unlabeled and labeled aminohexylacrylamide (aha) derivatives of dUTP and dCTP, that are easy to incorporate into nucleic acids for subsequent conjugation with any of our amine-reactive probes (Chapter 1).
• ULYSIS Nucleic Acid Labeling Kits, which employ a fast, simple and reliable chemical method for labeling nucleic acids without enzymatic incorporation of labeled nucleotides.
• ARES DNA Labeling Kits, which employ a versatile, two-step method for labeling DNA with fluorescent dyes to achieve a uniformity and consistency of labeling that is difficult to obtain with conventional enzymatic incorporation of labeled nucleotides.
• Alexa Fluor Oligonucleotide Amine Labeling Kits, which use familiar chemical labeling of amine-terminated oligonucleotides to prepare the best singly labeled fluorescent oligonucleotide conjugates.

Custom conjugations of most of our proprietary dyes to oligonucleotides for personal research use are available from several authorized sources (Licensing). A variety of additional methods for preparing labeled oligonucleotides and nucleic acids and using them in nucleic acid sequencing are described in this section. Section 8.5 describes use of labeled nucleic acids as hybridization reagents for microarrays, FISH and real-time PCR assays. Section 8.5 also includes a discussion of our important ELF and TSA technology for amplifying FISH signals.

ChromaTide Nucleotides

Molecular Probes offers a series of uridine triphosphates (UTP, Table 8.6) and deoxyuridine or deoxycytidine triphosphates (dUTP, OBEA-dCTP; Table 8.7) conjugated to an extensive selection of fluorophores and haptens, including several that incorporate our superior Alexa Fluor dyes (Product Highlight: Alexa Fluor Dyes for Labeling Nucleic Acids). These ChromaTide nucleotides are useful for generating labeled nucleic acids for molecular biology and molecular cytogenetics applications, including chromosome and mRNA FISH experiments (), gene expression studies and mutation detection on arrays and microarrays (Figure 8.100), and in situ PCR and RT-PCR. The ChromaTide dinitrophenyl (DNP)-11-dUTP (C7610MP) is useful for signal amplification in FISH and microarrays and for detecting probes hybridized to blots (Section 8.5). Our extensive selection of fluorescent labels provides the ideal tools for multicolor techniques such as spectral karyotyping, multilocus FISH analysis, "chromosome painting" and comparative genome hybridization (Section 8.5).

Structures of the ChromaTide Nucleotides

The ChromaTide UTP and dUTP nucleotides are modified at the C-5 position of UTP or dUTP via a unique aminoalkynyl linker (Figure 8.38). The C-5 position of UTP and dUTP is not involved in Watson–Crick base-pairing and so interferes little with probe hybridization. The aminoalkynyl linker between the fluorophore and the nucleotide in the ChromaTide UTP and dUTP nucleotides is designed to reduce the fluorophore's interaction with enzymes or target binding sites. In addition to this four-atom bridge, several of these nucleotides contain a seven- to 10-atom spacer that further separates the dye from the base. The number in the product's name (e.g., the "12" in ChromaTide fluorescein-12-dUTP) indicates the net length of the spacer in atoms. Longer spacers typically result in brighter conjugates and increased hapten accessibility for secondary detection reagents.

The ChromaTide OBEA-deoxycytidine triphosphates (OBEA-dCTP, Table 8.7) are modified at the N-4 position of cytosine using a patented 2-aminoethoxyethyl (OBEA) linker (Figure 8.39). The Alexa Fluor 546 and Alexa Fluor 647 OBEA-dCTP conjugates (C21555, C21559) also have a built-in spacer that reduces possible steric interference caused by the presence of the dye.

Fluorescent ChromaTide Nucleotides

The spectral diversity of our ChromaTide dUTP and ChromaTide OBEA-dCTP nucleotides (Table 8.7) and of the ChromaTide UTP nucleotides (Table 8.6) gives researchers significant flexibility in choosing a label that is compatible with a particular optical detection system or multicolor experiment (ChromaTide Labeled Nucleotides, Fluorescence Excitation and Emission Spectra for ChromaTide Nucleotides). Probes made from the fluorescent ChromaTide nucleotides can be imaged directly; alternatively, some fluorophores can be used as a hapten for signal amplification, as described in Section 8.5. In many cases, the TSA (Section 6.2) or ELF technologies (Section 6.3) can be used to significantly amplify the signal of dye-labeled hybridization probes in cells and tissues and on microarrays (Section 8.5). Combination of the TSA and ELF technologies promises to yield the most sensitive detection of in situ hybridization that is currently possible. The Alexa Fluor conjugates of UTP, OBEA-dCTP and dUTP provide fluorophore labels with demonstrably superior fluorescence properties, as compared with conventional dyes (Product Highlight: Alexa Fluor Dyes for Labeling Nucleic Acids). The Alexa Fluor 488, Alexa Fluor 568 and Alexa Fluor 594 nucleotides are spectrally similar to fluorescein, Lissamine rhodamine B and Texas Red conjugates, respectively, but the Alexa Fluor conjugates exhibit superior spectral and chemical properties. ChromaTide OBEA-dCTP nucleotides have been prepared 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, Texas Red and Cy5 dyes, respectively (Product Highlight: Alexa Fluor Dyes for Labeling Nucleic Acids).

The ChromaTide Alexa Fluor dUTP and ChromaTide Alexa Fluor OBEA-dCTP nucleotides are highly water soluble, as are DNA probes that contain them. Thus, Alexa Fluor dye–labeled DNA probes do not aggregate or precipitate, even in high-salt hybridization solutions. Fluorescence of the Alexa Fluor conjugates is not pH sensitive in the range used for hybridization or microscopy mounting media. Additionally, the enhanced photostability of these conjugates makes them ideal for imaging applications.

We also have available the Oregon Green 488, Rhodamine Green and Texas Red conjugates of dUTP (C7630, C7629, C7631, C7608). When compared with the corresponding fluorescein conjugates (C7603, C7604), the Oregon Green 488 and Rhodamine Green conjugates have similar fluorescence spectra but superior photostability (Section 1.5). Texas Red-12-dUTP (C7631) has an emission spectrum in solution that is narrower and about 25% more intense than that of Texas Red-5-dUTP (C7608). For certain multicolor applications, we recommend conjugates of the BODIPY dyes because they have narrow emission bandwidths with minimal spectral overlap. The BODIPY 630/650-14-dUTP (C11395) and BODIPY 650/665-14-dUTP (C11396) are well suited to excitation by the 633 nm spectral line of the He–Ne laser and the 647 nm spectral line of the Ar–Kr laser, respectively. Oregon Green 488-5-dUTP has been microinjected into unfertilized oocytes to follow DNA synthesis in oocytes following fertilization. Other microinjected fluorescent nucleotides have been utilized to follow the dynamics of chromosome formation and cell proliferation in live cells.

ChromaTide Dinitrophenyl (DNP)-11-dUTP

Our ChromaTide dinitrophenyl-11-dUTP (DNP-11-dUTP, C7610MP) can be incorporated into DNA probes using a variety of enzymatic techniques (Table 8.7, Methods for Enzymatic Incorporation of ChromaTide dUTPs), providing a hapten that can be combined with fluorophores, biotin or other haptens in double-labeling experiments. The DNP hapten can be detected with our rabbit anti–DNP-KLH antibody, which is available unlabeled (A6430, Section 7.4) or labeled with the Alexa Fluor 488 dye (A11097, Section 7.4) or fluorescein (A6423, Section 7.4).

Using ChromaTide Nucleotides in Enzymatic Labeling Methods

The ChromaTide nucleotides can be incorporated into DNA and RNA using conventional enzymatic labeling techniques (Table 8.6, Table 8.7). Protocols for many of these techniques are provided with the ChromaTide nucleotides (ChromaTide Labeled Nucleotides, Methods for Enzymatic Incorporation of ChromaTide dUTPs, Methods for Enzymatic Incorporation of ChromaTide UTPs). Enzymes that we have used successfully include:

• Taq polymerase in polymerase chain reaction (PCR) assays (Note: we have observed that the long-wavelength BODIPY dye conjugates (C11395, C11396) do not serve as Taq polymerase substrates and appear to inhibit the Taq polymerase reaction.)
• DNA polymerase I in nick-translation and primer-extension assays
• Klenow polymerase in random-primer labeling
• 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 8.6 and Table 8.7 for information on applications of individual ChromaTide nucleotides.

ChromaTide nucleotides have also been used in the TUNEL assay for detecting DNA fragmentation in apoptotic cells (Section 15.5, ). Microinjected fluorescent nucleotides are incorporated into cellular nucleic acids where they assemble into chromosomes and persist through cell replication.

Amine-Modified Nucleotides

Unlabeled and Labeled aha-dUTP and aha-dCTP

5-Aminohexylacrylamido-dUTP (aha-dUTP) and 5-aminohexylacrylamido-dCTP (aha-dCTP) can be used to produce amine-modified DNA by conventional enzymatic incorporation methods such as reverse transcription, nick translation, random primed labeling or PCR, and they are incorporated more efficiently into DNA than are aminoallyl deoxynucleotides. The amine-modified DNA can then be labeled with any amine-reactive dye or hapten (described in Chapter 1). This two-step technique consistently results in a uniform and high degree of DNA labeling that is difficult to obtain by other methods. The protocols provided with the aha-dUTP and aha-dCTP yield a labeling efficiency of ~5–8 dyes per 100 bases, which we have found to be optimal for fluorescence in situ hybridization (FISH), dot blot hybridization and especially microarray applications, in which the consistency of labeling between samples is critical for accurate interpretation of results. The aha-dUTP and aha-dCTP nucleotides are available as 500 µL of a 2 mM solution (A32760, A32768) or as 50 µL of a 50 mM solution (A32761, A32769) in 10 mM Tris, 1 mM EDTA, pH 7.5 (TE). Molecular Probes also provides a wide variety of amine-reactive reagents for labeling amine-modified DNA, including succinimidyl esters of our Alexa Fluor dyes, conventional fluorophores, biotin and dinitrophenyl (DNP) (Chapter 1).

The labeled aha-dUTP and aha-dCTP nucleotides can be used to generate labeled nucleic acid hybridization probes for many molecular biology and molecular cytogenetics applications, including multicolor techniques. These nucleotides are modified at the C-5 position of uridine and cytosine, respectively, with a unique hexylacrylamide linker, which serves as a spacer between the nucleotide and the dye or hapten (). This spacer reduces interactions between the nucleotide and the dye or hapten, producing brighter conjugates and increased hapten accessibility for secondary detection reagents. The Alexa Fluor 555 aha-dUTP and aha-dCTP nucleotides (A32762, A32770), with excitation/emission maxima of 555/570 nm, and the Alexa Fluor 647 aha-dUTP and aha-dCTP nucleotides (A32763, A32771), with excitation/emission maxima of 650/670 nm, respectively, are compatible with commonly used microarray scanners. These fluorescent nucleotides provide greater signal correlation (R²) values than do the spectrally similar Cy3 and Cy5 dye pair, thereby improving the resolution of two-color microarray gene expression assays. The exceptionally bright and photostable Alexa Fluor dyes are also essentially insensitive to pH and are highly water soluble.

We also offer biotin aha-dUTP and aha-dCTP (B32766, B32772) and fluorescein aha-dUTP and aha-dCTP (F32767, F32773) nucleotides, which can be used to generate nucleic acid probes that can be detected with streptavidin conjugates (Section 7.6, Table 7.23) or labeled anti-fluorescein antibodies (Section 7.4), respectively. Nucleic acid probes labeled with biotin have generally been the most common nonisotopic probes used in hybridization techniques. Biotinylated probes are readily detected with fluorophore or enzyme conjugates of avidins or streptavidins (Section 8.5), providing amplification of the signal (Figure 8.87). Biotin can also be detected with anti-biotin antibodies, which we provide unconjugated (A11242, Section 7.4), or conjugated to the bright green-fluorescent Alexa Fluor 488 dye or the intensely red-fluorescent Alexa Fluor 594 dye (A31801, A31800; Section 7.4). The signal from biotin-labeled hybridization probes can be considerably amplified, while retaining excellent spatial resolution, by combination with Enzyme-Labeled Fluorescence (ELF) technology (Section 8.5, ) or tyramide signal amplification (TSA) technology (Section 8.5, Figure 8.92). In addition, biotinylated nucleic acids can be adsorbed onto streptavidin agarose or CaptAvidin agarose (S951, C21386; Section 7.6; Figure 7.96), bound to the streptavidin conjugate of Captivate ferrofluid superparamagnetic particles (C21476, Section 7.6) or detected with NANOGOLD or Alexa Fluor FluoroNanogold streptavidin (N24918, A24926, A24927; Section 7.6).

The aha-dUTP nucleotides have been used in two-color microarray assays, Southern and Northern blots, colony and plaque hybridizations, DNA sequencing, primer extension, DNA and RNA amplification and bead-based separation techniques. In these applications, the labeled samples are generally detected with enzyme conjugates of streptavidin or anti-fluorescein antibody in conjunction with fluorescent, chemiluminescent or colorimetric substrates such as those employed in our Tyramide Signal Amplification (TSA) Kits (Section 6.2, Table 6.1).

Aminoallyl UTP and Aminoallyl dUTP

Aminoallyl UTP (5-(3-aminoallyl)uridine 5'-triphosphate, A21663) and aminoallyl dUTP (5-aminoallyl-2'-deoxyuridine 5'-triphosphate; 2 mM in TE, A21664; 50 mM in TE, A32764) can be incorporated into RNA and DNA, respectively, using conventional enzymatic incorporation techniques, as described above for the ChromaTide UTP, OBEA-dCTP and dUTP nucleotides. The resulting amine-modified nucleic acid can then be labeled using the amine-reactive dyes and other reagents that are described in Chapter 1. Lacking bulky dye groups, the aminoallyl-modified nucleotides can be incorporated to extremely high and consistent levels. Subsequent reaction of the amine-modified nucleic acid with an excess of amine-reactive reagent achieves correspondingly high and consistent labeling efficiency, regardless of the labeling reagent chosen. We typically obtain labeling efficiencies of ~5–8 dyes per 100 bases. This two-step labeling method also eliminates the need to optimize an enzymatic reaction to accommodate different dye-modified nucleotides, which may incorporate at very different rates. This labeling method is ideal for both FISH probes (Figure 8.86, ) and microarray-based experiments (). Aminoallyl dUTP labeling can be achieved easily using our convenient ARES DNA Labeling Kits (see below), which provide aminoallyl dUTP, premeasured aliquots of our best reactive dyes and carefully tested protocols.

Alexa Fluor Amine-Reactive Dye Decapacks for Labeling Amine-Modified DNA and RNA

For labeling amine-modified DNA or RNA probes in microarray-based experiments, we offer four of our outstanding amine-reactive Alexa Fluor dyes conveniently packaged in 10 single-use vials and rigorously tested for the ability to efficiently label aminoallyl-modified DNA — the Alexa Fluor 488 reactive dye decapack (A32750), the Alexa Fluor 555 reactive dye decapack (A32756), the Alexa Fluor 594 reactive dye decapack (A32751), the Alexa Fluor 647 reactive dye decapack (A32757) and a set containing both the Alexa Fluor 555 and Alexa Fluor 647 reactive dye decapacks (A32755) for two-color experiments. These specially packaged amine-reactive dyes can be used in conjunction with our aminohexylacrylamido-dUTP (aha-dUTP, A32760), aminoallyl dUTP or aminoallyl UTP (A21664, A21663) nucleotides or with commercially available aminoallyl nucleotide–based nucleic acid labeling kits. With excitation/emission maxima of 495/519 nm, 555/565 nm, 590/617 nm and 650/668 nm, respectively, the Alexa Fluor 488, Alexa Fluor 555, Alexa Fluor 594 and Alexa Fluor 647 succinimidyl esters match the most popular wavelength channels used to scan microarrays. Furthermore, the Alexa Fluor 555/Alexa Fluor 647 dye pair have been shown to display higher signal correlation coefficients than the Cy3/Cy5 dye pair in two-color DNA microarray assays.

5-Bromo-2'-Deoxyuridine, 5-Bromo-dUTP (BrdUTP) and 5-Bromo-UTP (BrUTP)

Cells can naturally incorporate the thymidine analog 5-bromo-2'-deoxyuridine (BrdU, B23151) into their DNA during cell division, making this nucleoside analog an excellent marker of both cell cycle and cell proliferation. Analysis of incorporated BrdU can be either by direct detection with an antibody to BrdU-modified DNA or by modification of the fluorescence of a nucleic acid stain. For instance, the fluorescence of TO-PRO-3 and LDS 751 is considerably enhanced by the presence of BrdU in DNA, whereas that of the Hoechst dyes is specifically quenched. 5-Bromo-2'-deoxyuridine 5'-triphosphate (BrdUTP, B21550) is commonly used in TUNEL-based methods to detect proliferating or apoptotic cells, as in the ABSOLUTE-S SBIP Cell Proliferation Assay Kit (A23150, Section 15.4, ) and the Apo-BrdU TUNEL Assay Kit (A23210, Section 15.5). In addition, this nucleotide is a substrate for reverse transcriptase and has been used in a sensitive nonisotopic assay for detection of HIV-1–associated reverse transcriptase activity. Similarly, the corresponding brominated ribonucleotide, 5-Bromouridine 5'-triphosphate (BrUTP, B21551) is an excellent substrate for RNA polymerase and has been used to monitor nucleolar transcription in situ.

BrdUTP, a component of the Apo-BrdU TUNEL Assay Kit (A23210, Section 15.5), is readily incorporated into apoptotic cells by terminal deoxynucleotidyl transferase (TdT), and is apparently 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 for nucleic acid labeling and detection in the ABSOLUTE-S SBIP Cell Proliferation Assay Kit (A23150, Section 15.4). BrdUTP can also be used to detect excision and repair of strand breaks in UV light–damaged DNA in cells. BrdUTP is a substrate for reverse transcriptase and Klenow polymerase, and has been used in a sensitive nonisotopic assay for detecting HIV-1–associated reverse transcriptase activity. Nucleic acids containing halogenated bases can be photocrosslinked to proteins with which they interact. BrUTP and BrdUTP can serve as low-cost building blocks for nucleic acid probes, in the manner of the fluorescent ChromaTide nucleotides (see below), with detection by labeled anti-BrdU antibodies (Section 15.4). BrUTP is reported to be a better substrate for RNA polymerase than is UTP itself. BrUTP that has been microinjected into cells is incorporated into RNA of a nucleolar compartment. For an especially efficient and low-cost method of producing large quantities of DNA probe, BrdUTP can be incorporated into DNA by cells or plasmid-containing bacteria grown in the presence of BrdU.

Molecular Probes offers anti-BrdU mouse monoclonal antibodies conjugated with several of our superior Alexa Fluor dyes (Section 15.4). Because incorporation of BrdU and the related BrdUTP into DNA is specific, use of the labeled anti-BrdU antibody permits unequivocal detection of DNA in cells. Also, our fluorescently labeled anti-BrdU antibody crossreacts with ribonucleic acids that have incorporated bromouridine or BrUTP, thus permitting the only method of specifically detecting transcribed RNA in cells with a fluorescent dye.

ULYSIS Nucleic Acid Labeling Kits

ULYSIS Nucleic Acid Labeling Kits (Table 8.8) combine some of Molecular Probes' best fluorescent dyes with the versatile, patented Universal Linkage System (ULS) platinum-based chemistry developed at KREATECH Diagnostics, resulting in a simple, fail-safe method for producing bright, fluorophore-labeled hybridization probes. The ULS labeling technique directly labels nucleic acids without the need for enzymatic incorporation of modified nucleotides. The ULS method is based on the use of a platinum–dye complex, patented by KREATECH Biotechnology BV, that forms a stable adduct with the N-7 position of guanine and, to a lesser extent, adenine bases in DNA, RNA, peptide–nucleic acid conjugates (PNA) and oligonucleotides (Figure 8.42). The labeling reaction requires only 15 minutes, and separation of the labeled nucleic acids from the unreacted ULS complex can be accomplished through use of a simple spin-column procedure (Figure 8.43). DNA longer than ~1000 base pairs requires a 10-minute DNase digestion before labeling, which both optimizes labeling and fragments the probe for efficient hybridization.

The ULYSIS Kits allow researchers to label DNA with a wide variety of our exceptionally bright and photostable Alexa Fluor dyes (Product Highlight: Alexa Fluor Dyes for Labeling Nucleic Acids) and the Oregon Green 488 dye (Table 8.8). Probes labeled using the ULYSIS Kits are stable indefinitely and hybridize effectively to target DNA. The ULS method has been used to prepare labeled probes for dot, Southern, and Northern blot analysis, RNA and DNA in situ hybridization, multicolor fluorescence in situ hybridization (FISH, Section 8.5; Figure 8.44, , ), comparative genome hybridization (CGH) and microarray analysis (). We maintain a bibliography of references for the ULS technology that use our ULS reagents and those previously described (Bibliography for U24832). Combination of the Oregon Green 488 dye or Alexa Fluor 488 dye with antibodies to these dyes (Section 7.4) permits detection by chemiluminescent, chromogenic or fluorogenic enzyme-linked methods — including signal amplification schemes that use the TSA and ELF technologies (Section 8.5) or the BOLD APB chemiluminescent substrate for alkaline phosphatase (B21901, Section 8.4).

Each ULYSIS Nucleic Acid Labeling Kit provides:

• 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 hybridization probes optimized for chromosome in situ hybridization and dot-blot hybridization (ULYSIS Nucleic Acid Labeling Kits)

Sufficient materials are supplied in each kit for 20 labelings of 1 µg DNA each.

ARES DNA Labeling Kits

ARES DNA Labeling Kits (Table 8.9) provide a versatile two-step method for labeling DNA with fluorescent dyes (Figure 8.45). This method achieves uniformity and consistency of labeling that is difficult to obtain with conventional enzymatic incorporation of labeled nucleotides. In the first step, an amine-modified nucleotide, 5-(3-aminoallyl)-dUTP, is enzymatically incorporated into DNA. 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 reverse transcription or nick translation, for which we provide the protocols (ARES DNA Labeling Kits); other enzymatic methods are also likely to be compatible. In the second step, the amine-modified DNA is chemically labeled using an amine-reactive fluorescent dye. This chemical reaction varies little in its efficiency from dye to dye, so that it is possible to use any combination of the ARES Kits, with their broad selection of the brightest and most photostable dyes, and obtain consistent labeling for every DNA sample. The labeling protocols provided generally result in about one dye per 12–20 bases, which we have determined to be optimal for FISH and dot-blot hybridization. Nucleic acids labeled using this method are ideal for FISH () or microarray experiments ().

The ARES Kits are supplied with some of our best fluorescent dyes (Table 8.9). The Alexa Fluor dyes (Section 1.3) have properties superior to conventional dyes for labeling nucleic acids (Product Highlight: Alexa Fluor Dyes for Labeling Nucleic Acids). The Oregon Green 488 dye is a modified fluorescein with reduced pH sensitivity and higher photostability (Figure 1.12, ). The signal of nucleic acids labeled with this dye can be amplified using our anti-fluorescein/Oregon Green antibodies, as described in Section 8.5.

Each ARES DNA Labeling Kit provides:

• 5-(3-Aminoallyl)-dUTP
• Amine-reactive fluorescent dye and an appropriate solvent
• Sodium bicarbonate
• Nuclease-free H₂O
• A detailed protocol for labeling DNA using reverse transcriptase or nick translation (Product Information Sheet)

Sufficient materials are supplied for 5–10 labelings, each containing 1–5 µg DNA. The 5-(3-aminoallyl)-dUTP (A21664, ) and succinimidyl ester dyes are also available as stand-alone reagents. Enzymatic incorporation of 5-(3-aminoallyl)-dUTP (Aminoallyl dUTP) permits incorporation of almost any amine-reactive dye in Chapter 1 into nucleic acids.

Labeled Oligonucleotides

DNA can also be labeled from RNA templates by reverse transcription using fluorophore-labeled random oligonucleotide primers and unlabeled deoxynucleotide triphosphates. Molecular Probes provides two types of labeled oligodeoxynucleotides that can be used for this purpose. Our dT₁₈ oligodeoxynucleotides (O21561, O21562, O21563) are labeled at the 5'-terminus with one of three of our popular Alexa Fluor dyes (Table 8.18). The labeled dT₁₈ oligodeoxynucleotides hybridize to poly(A) tails in RNA samples, providing primers for reverse transcription or hybridization probes for poly(A)-terminated mRNA in cell- and solution assays. Our Panomer 9 random-sequence oligodeoxynucleotides (Section 8.5) are covalently labeled on the 5'-terminus with one of our proprietary fluorescent dyes, with a nonfluorescent QSY 7 quencher dye () or with biotin (Table 8.18). The Panomer 9 oligonucleotides are also useful as primers for synthesizing labeled DNA via Klenow DNA polymerase or reverse transcriptase. In these reactions, the primer provides the fluorescent label, whereas unlabeled nucleotides are incorporated by the enzyme. This labeling strategy ensures efficient and unbiased incorporation of nucleotides, because the bulky dye molecule does not interfere with nucleotide incorporation. However, because the synthesized probe contains only a single fluorophore, the labeling efficiency will typically be lower than that achieved by incorporating fluorophore-labeled nucleotides.

Labeling Amine- and Thiol-Modified Oligonucleotides

Amine or thiol groups can be incorporated into a chemically synthesized oligonucleotide. These groups can then be directly conjugated to an amine-reactive (Chapter 1) or thiol-reactive (Chapter 2) fluorophore or hapten (Figure 8.49). Fluorophore-labeled oligonucleotides are extensively used as primers for sequencing or PCR reactions (see below). Double-labeled oligonucleotides are used to produce fluorescence resonance energy transfer (FRET) (Technical Focus: Fluorescence Resonance Energy Transfer (FRET)) or quenched reporters for real-time PCR assays (Section 8.5). Labeled oligonucleotides can also be used as probes for fluorescence in situ hybridization (Section 8.5).

Alexa Fluor Oligonucleotide Amine Labeling Kits

The Alexa Fluor Oligonucleotide Amine Labeling Kits (Table 8.10) provide the reagents required for labeling synthetic oligonucleotides that have amine groups incorporated at their 5'- or 3'-terminus. Our outstanding Alexa Fluor dyes (