section 8.5 - detecting nucleic acid · pdf file8.5 detecting nucleic acid ... and determining...

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

8.5 Detecting Nucleic Acid HybridizationThe double-helical structure of nucleic acids, in which one

strand binds specifically to its exact complement, could not havebeen designed to be more useful for the study of biology. Theease of designing assays based on this feature is responsible forthe incredibly fast pace that has characterized molecular biologyresearch since its inception. With the incorporation of fluores-cence technologies, the ability to design multiplex and high-throughput assays has increased the pace still further, to the pointin which sophisticated bioinformatics are required to analyze thehuge outpouring of data. From nucleic acid sequencing to real-time polymerase chain reaction (PCR) assays to microarrays, thenew genomics era owes its development in large part to the devel-opment of fluorescence methodologies. This section discusses theuse of fluorescence- and luminescence-based detection technolo-gies in assays based on hybridization of a nucleic acid fragmentto its complement.

Principles of Fluorescence In Situ Hybridization(FISH)

Fluorescence in situ hybridization (FISH) offers many advan-tages over radioactive and chromogenic methods for localizingand determining the relative abundance of specific nucleic acidsequences in cells, tissue, interphase nuclei and metaphase chro-mosomes. Not only are fluorescence techniques fast and precise,they allow the simultaneous analysis of multiple probes that maybe spatially overlapping.1–6 It is possible to distinguish at leastfour to five different fluorescent signals in a single sample usingtheir excitation and emission properties alone and the appropriateoptical filters, and even more signals using an interferometer andlinear unmixing software. Using defined ratios of two fluorescentlabels per probe (called COBRA for combined binary ratio label-ing) in conjunction with highly discriminating optical filters or aninterferometer and appropriate software, researchers can distin-guish over 40 signals on the same sample 7–11 (Figure 8.72). Incases where optical hardware or image analysis software is lesssophisticated, up to 18 chromosome pairs can be distinguishedusing sequential FISH, in which several rounds of hybridizationare performed on the same sample.12 Chromosome FISH hasbecome extremely important for:

• Gene mapping 6

• Identification of mutations correlated with inherited or somaticgenetic diseases 13–16 (Figure 8.72, Figure 8.73)

• Clinical diagnostics 17–19

• Studies of chromosome and nuclear architecture 20,21

• Identification of viruses and microorganisms within theirnatural environment 22–25

Chromosome paint probes (Figure 8.74) and multicolor band-ing with microdissection probes 26 (Figure 8.75) provide land-marks for specific chromosomes or parts of chromosomes and areused to study a specific region of the genome. Multicolor chro-mosome FISH techniques that cover the entire genome are evenmore powerful, because they allow identification of chromosomallesions without any prior knowledge about their possible location.Entire genome techniques include:

• Spectral karyotyping (SKY) and multiplex FISH (M-FISH), inwhich every chromosome is painted with a different color ormixture of colors 7,14,16,27–31 (Figure 8.72)

• Cross-species color segmenting, in which chromosome paintprobes from other primates are hybridized to human chromo-somes to produce multicolor banding patterns 32 (Figure 8.73)

• Comparative genome hybridization (CGH), in which differen-tially labeled genomic DNA from a test sample and referencesample are simultaneously hybridized to normal human chromo-somes to facilitate detection of deletions and duplications 33,34

FISH in metaphase chromosomes has a resolution of about1 Mbase, but fiber FISH, using stretched chromosomes, hasresolution that can be measured in the hundreds of basepairs.35,36 A discussion of FISH methodology can be found inthe literature.37,38

Figure 8.72 Human metaphase chromosomes hybridized to chromo-some paints. Human metaphase chromosomes from normal humanblood cells (top) or a cancerous cell line (bottom) were hybridized to hu-man chromosome paint probes using SkyPaint (Applied Spectra Imaging).In this method, a combination of dyes is used to label the probes, thespectrum of each pixel is measured and a color assigned based on thespectral signature. Numerous chromosomes with more than one colorreveal the large number of chromosomal translocations and other aber-rations in the cancerous cell line (bottom). Image contributed by AppliedSpectral Imaging (used with permission).

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Molecular Probes offers a wide variety of reagents for thedetection of in situ hybridization signals. These include reagentsand kits for preparing labeled hybridization probes (Section 8.2),secondary detection reagents for detecting labeled probes (Chapter6, Chapter 7), kits and reagents for amplifying signals (Section6.2, Section 6.3) and dyes for counterstaining nuclei or chromo-some spreads (Section 8.6). Molecular Probes’ proprietary fluores-cent dyes are brighter and more photostable than conventionaldyes — including the Cy3 and Cy5 dyes (see the Alexa Fluor 546,Alexa Fluor 555 and Alexa Fluor 647 dyes in Section 1.3) — andprovide the brightest labels for DNA or RNA probes (see AlexaFluor Dyes for Labeling Nucleic Acids in Section 8.2). Our exten-sive dye selection spans the visible spectrum and certain combina-tions of dyes are ideal for multicolor labeling techniques.

Figure 8.73 Karyotype of human metaphase chromosomes using RxFISH.RxFISH painted chromosomes were visualized using Applied Imaging Cyto-Vision. In this method, human metaphase chromosomes are hybridizedwith paint probes made from gibbon chromosomes. The fact that there arevirtually no unpainted regions demonstrates that, while significantly rear-ranged (over 30 translocations), gibbon and human chromosomal DNA iswell conserved from a common ancestor. The top panel shows a normal hu-man karyotype, whereas the bottom panel shows a human karotype with aninversion in chromosome 1. Image supplied by Applied Imaging Corpora-tion (used with permission).

Figure 8.75 Human metaphase chromosomes hybridized to fluorescent probesfrom two overlapping microdissection libraries. Probes specific to chromo-some regions 1p34–35 and 1p36 were labeled using the ULYSIS Oregon Green488 (U-21659) and Alexa Fluor 594 (U-21654) Nucleic Acid Labeling Kits, re-spectively. The chromosomes were counterstained with DAPI (D-1306, D-3571,D-21490). Image contributed by Jingwei Yu, Colorado Genetics Laboratory.

Figure 8.74 A paint probe for chromosome 2 was labeled with the ULYSISAlexa Fluor 546 Nucleic Acid Labeling Kit (U-21652) and hybridized to hu-man metaphase chromosomes. Image provided by Joop Wiegant, LeidenUniversity Medical Center, Leiden, The Netherlands.

Probe Preparation for FISH

Enzymatic Incorporation of ChromaTide-Labeled NucleotidesThe conventional method for labeling FISH probes is to enzy-

matically incorporate a modified nucleotide using a DNA templateand an RNA or DNA polymerase (Figure 8.76). Our ChromaTidedUTP, ChromaTide OBEA-dCTP 39 and ChromaTide UTP nucle-otides (Table 8.5, Table 8.6) are modified with either a fluorophore,a dinitrophenyl group (DNP) or biotin attached on the base (Figure8.38, Figure 8.39, see Legal Notice for ChromaTide UTP anddUTP Nucleotides in Section 8.2). DNP and biotin, as well assome fluorophores, allow for signal amplification through sec-ondary detection techniques (see below). The ChromaTide dUTPnucleotide can be incorporated using a DNA template and stan-

Section 8.5


Figure 8.76 Human metaphase chromosomeshybridized to centromere probes labeled withChromaTide OBEA-dCTP nucleotides. Probes spe-cific to the alpha satellite sequences from humanchromosome 17, chromosome 15 and chromo-some 1 were labeled using nick translation withChromaTide Alexa Fluor 488-7-OBEA-dCTP (green;C-21555), ChromaTide Alexa Fluor 594-7-OBEA-dCTP (red; C-21558) and ChromaTide Alexa Fluor647-12-OBEA-dCTP (pink; C-21559), respectively.The probes were hybridized to metaphase spreadsfrom peripheral blood lymphocyte cultures. Thechromosomes were counterstained with Hoechst33342 (blue) (H-1399, H-3570, H-21492).

Figure 8.78 Labeled paint probes hybridized tohuman metaphase chromosomes. A biotinylatedchromosome 5 probe was detected with AlexaFluor 594 streptavidin (S-11227), and a digoxi-genin-labeled chromosome 2 probe detectedwith mouse anti-digoxigenin in combination withAlexa Fluor 488 goat anti–mouse IgG antibody(A-11001). Image submitted by Joop Wiegant,Leiden University Medical Center, Leiden, TheNetherlands.

dard nick translation, random-primer labeling or PCR techniques.13,31 Some of the Chroma-Tide dUTPs can also be incorporated by reverse transcriptase. Additionally, oligonucle-otides can be end-labeled with a ChromaTide dUTP using terminal deoxynucleotidyl trans-ferase. The ChromaTide OBEA-dCTP nucleotides 39 can be incorporated into a DNA probeusing nick translation and reverse transcription and will most likely be incorporated byother common enzymatic methods as well. The ChromaTide UTP nucleotides can be incor-porated into riboprobes using SP6, T3 or T7 RNA polymerase. Detailed labeling protocolsare available in the product literature.

Aminoallyl dUTP Labeling Using the ARES KitsLabeling with fluorophore-modified nucleotides (as described above) is straightfor-

ward, but does have some drawbacks. The bulky dye molecule on the nucleotide maymake it difficult for the enzyme to incorporate it into DNA or RNA. A protocol opti-mized for one fluorophore may not be optimal for another, chemically different fluoro-phore. To circumvent these problems, it is possible to enzymatically incorporate a lessbulky, amine-modified nucleotide and then label the amine-modified DNA afterwardsusing an amine-reactive reagent. This two-step labeling method (Figure 8.44) is em-ployed in the ARES DNA Labeling Kits (Section 8.2, Table 8.8). Because there is nobulky dye on the nucleotide, the aminoallyl-modified dUTP is incorporated very effi-ciently. In the second step, an excess of an amine-reactive dye is used, which results invery consistent labeling levels, regardless of the dye used in the reaction. The dyes usedin our ARES kits include our bright and photostable Alexa Fluor dyes (see Alexa FluorDyes for Labeling Nucleic Acids in Section 8.2). Although it is possible to label DNA toextremely high levels using this technique, we have optimized the kits to label the DNAto about one dye molecule for every 12–20 bases, which we have determined to be opti-mal for hybridization to metaphase chromosomes (Figure 8.77). The 5-(3-aminoallyl)-2′-deoxyuridine 5′-triphosphate (aminoallyl dUTP, A-21664; Section 8.2) used in the ARESKits and amine-reactive dyes (Chapter 1) are also available separately. In addition, weoffer 5-(3-aminoallyl)uridine 5′-triphosphate 40 (aminoallyl UTP, A-21663), which can beused in combination with our many amine-reactive dyes (Chapter 1) to synthesize dye-labeled RNA probes 41 (Figure 8.40).

Direct Chemical Labeling of Nucleic Acids Using the ULYSIS KitsThe ULYSIS Nucleic Acid Labeling Kits (Section 8.2, Table 8.7) offer an alternative

labeling strategy that eliminates complex enzymatic incorporation protocols altogether.These kits employ a platinum compound that labels the N-7 position of guanine basesdirectly (Figure 8.41) using patented ULS technology developed by KREATECH Bio-technology BV. The reaction is complete in just 15 minutes (Figure 8.42). The method inthe ULYSIS labeling kits can be used to reliably achieve a labeling ratio of about one dyefor every 30–50 bases, which we have found to be optimal for hybridization of ULSplatinum complex–labeled probes to metaphase chromosomes and results in strong stain-ing of hybridization targets (Figure 8.43, Figure 8.74). The optimal labeling ratio issomewhat lower than that for probes labeled on the uridine base. The ULS chemistry hasbeen employed to label probes for metaphase and interphase chromosome FISH,42 multi-

Figure 8.77 Fluorescence in situ hybridization(FISH) mapping of a BAC clone on human meta-phase chromosomes. FISH was performed using aBAC clone labeled using the ARES Alexa Fluor 488DNA Labeling Kit (A-21665). The chromosomeswere counterstained with DAPI (D-1306, D-3571,D-21490). Image provided by Nallasivam Palan-isamy, Cancer Genetics Inc.

Additional ChromaTide nucleotides areunder development at Molecular Probes.Subscribe to our e-mail newsletter([email protected] no message in the body of thee-mail) to receive information aboutthese products as soon as they arereleased.

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color whole genome FISH,8 and CGH.43 The ULYSIS Kits havebeen used for labeling overlapping microdissection probes for usein multicolor chromosome-banding techniques (Figure 8.75).

Labeling OligonucleotidesShort oligonucleotide probes can also be used for FISH,25,44

although a secondary detection method is often required to ampli-fy the signal 45 (see below). However, by using a series of multi-ply labeled oligos to adjacent sequences, oligonucleotide probescan be sufficiently sensitive to detect a single RNA transcript insitu.46 Molecular beacons that are labeled with a fluorophore anda quencher (see below) provide the sensitivity required to detect10 molecules of RNA in a single cell in situ without the need foramplification by PCR.47 A related technique uses a “UniPrimer”universal primer (see below) to detect a single target sequenceafter amplification by in situ PCR or RT-PCR.48

Multiple labels can be added to the 3′-terminus of a probe byusing the enzyme terminal deoxynucleotidyl transferase (TdT) incombination with a labeled ChromaTide dUTP nucleotide (Table8.6). Alternatively, TdT can be used to add an amine-modifieddUTP, such as aminoallyl dUTP (A-21664, Section 8.2), to the 3′-terminus, and the amine subsequently labeled with a dye, haptenor enzyme.

Single or multiple labels can also be added chemically tooligonucleotides or peptide–nucleic acid conjugates (PNA) thathave been modified with an amine or thiol group during synthe-sis. Our amine-reactive succinimidyl esters (Chapter 1) provide asimple and reliable method for labeling amine-modified oligonu-cleotides. The reaction results in a stable amide bond that linksthe fluorophore to the oligonucleotide (Figure 1.2). The AlexaFluor Oligonucleotide Amine Labeling Kits (Section 8.2, Table8.9) make it easy to label amine-modified oligonucleotides withour superior Alexa Fluor dyes (see Alexa Fluor Dyes for LabelingNucleic Acids in Section 8.2). These kits include:

• Three vials of the reactive dye• Buffers• A detailed protocol for labeling 5′-amine-modified oligonucle-

otides and recommendations for conjugate purification

Each kit provides sufficient reagents for three labelings of atleast 50 µg each.

Figure 8.79 Schematic representation of in situ hybridization detectionusing amplification with biotin and a labeled streptavidin.

Chemical labeling of thiolated oligonucleotides can be accom-plished using fluorescent iodoacetamides (Chapter 2).49–56

Signal Amplification for FISH Using SecondaryDetection Reagents

For very low abundance targets, it may be necessary to use asecondary detection strategy to amplify the signal. A commonsecondary detection method uses a dye- or enzyme-labeledstreptavidin to detect a biotinylated probe. It is also possible touse dyes as haptens and amplify the signal using anti-dye anti-bodies. For the highest level of amplification, however, an en-zyme label in combination with a fluorogenic or chemilumines-cent substrate can be employed. Because each enzyme label actsupon many substrate molecules, the resultant signal is greatlyamplified.

Biotin and StreptavidinTable 7.3 and Table 7.17 in Chapter 7 list a wide variety of

antibodies, avidin, streptavidin, NeutrAvidin and CaptAvidinbiotin-binding proteins labeled with fluorophores, haptens, en-zymes, NANOGOLD and Alexa Fluor FluoroNanogold 1.4 nmgold clusters or Captivate ferrofluid magnetic particles. Thefluorescent streptavidin derivatives are important for multicolorfluorescence in situ hybridization applications using biotinylatedprobes (Figure 8.78). Because there are several fluorophoresconjugated to each biotin-binding protein, the signal is potentiallyamplified severalfold over the signal from DNA, RNA or PNAprobes that are directly labeled with fluorophores (Figure 8.79).NeutrAvidin conjugates have been shown to provide improveddetection of single-copy genes in metaphase chromosomespreads.57

Signal Amplification Using Anti-Dye AntibodiesSignal amplification can also be accomplished using a fluoro-

phore or chromophore as a hapten for an anti-dye antibody (Sec-tion 7.4; Table 7.13; Figure 8.80, Figure 8.81). We offer variouslabeled and unlabeled rabbit antibodies to:

• Fluorescein (and Oregon Green dyes)• Alexa Fluor 488 dye

Figure 8.80 Schematic representation of in situ hybridization detectionusing amplification with a dye-labeled anti-dye antibody.

Section 8.5


Figure 8.81 Amplification of FISH signals usingthe Alexa Fluor 488 Signal-Amplification Kit forFluorescein- and Oregon Green Dye–ConjugatedProbes (A-11053). Chromosome spreads wereprepared from the cultured fibroblast cell lineMRC-5 and hybridized with an α-satellite probe la-beled with the Oregon Green 488 dye and specificfor chromosome 17. The probe was labeled usingthe ULYSIS Alexa Fluor 488 Nucleic Acid LabelingKit (U-21659) (top panel). The signal was amplifiedusing the Alexa Fluor 488 conjugate of rabbit anti-fluorescein/Oregon Green antibody (A-11090; alsoavailable in A-11053) (middle panel) and amplifiedonce again using Alexa Fluor 488 goat anti–rabbitIgG antibody (A-11008; also available in A-11053)(bottom panel). Note the significant signal en-hancement with each amplification step.

• Tetramethylrhodamine• Texas Red dye• BODIPY FL dye• Cascade Blue dye• Lucifer yellow• Dansyl• The dinitrophenyl (DNP) and nitrotyrosine haptens

When used in combination with a signal-generating method — for instance, a second-ary antibody to the primary anti-dye antibody, a streptavidin conjugate or an enzymeconjugate in combination with a fluorogenic or chromogenic substrate — anti-dye oranti-hapten antibodies can be used to amplify signals from probes containing those la-bels, to restore fluorescence of partially bleached samples and to discriminate amongprobes labeled with different haptens, including those prepared from several of ourChromaTide nucleotides (Section 8.2; Table 8.5, Table 8.6). The anti-fluorescein anti-bodies also strongly crossreact with our Oregon Green dyes. In addition, we supply theAlexa Fluor 488 Signal-Amplification Kit for easy amplification of fluorescein or OregonGreen signals with an Alexa Fluor 488 dye–conjugated anti-fluorescein/Oregon Greenantibody (A-11053, Section 7.4, Figure 8.81). Preferred haptenylation reagents and theircorresponding anti-hapten antibodies are listed in Table 4.2. Our fluorescent goat anti–rabbit IgG antibody conjugates and the avidin and streptavidin conjugates (Table 7.3,Table 7.17) can be used to further amplify signals from our anti-dye antibodies.

Rabbit and goat polyclonal antibodies and mouse monoclonal antibodies to fluores-cein have been employed to simultaneously detect two different mRNA sequences indouble in situ hybridizations using fluorescein-, Oregon Green dye– or biotin-labeledoligonucleotides.4 The high affinity and specificity of anti-fluorescein/Oregon Greenantibodies makes these dyes excellent haptens for in situ hybridizations and other sec-ondary detection methods.4,58 Researchers have found fluorescein–anti-fluoresceinELISA techniques to display low nonspecific binding and to be similar in sensitivity tobiotin–streptavidin methods.59 Our anti–BODIPY FL preparation has been shown to bindspecifically to BODIPY FL dye–labeled oligonucleotides, where it has been detectedwith an alkaline phosphatase–conjugated anti–rabbit IgG 60 (G-21079, Section 7.3).

Our biotin-XX and fluorescein conjugates of anti-DNP antibodies (A-6435, A-6423;Section 7.4) are especially suitable for detecting hybridization probes labeled with theDNP hapten.6,61 These anti-DNP antibodies are prepared against the DNP–keyhole limpethemocyanin (KLH) conjugate and thus do not crossreact with bovine serum albumin(BSA), which is commonly used as a blocking or carrier molecule in hybridization appli-cations. Anti-DNP antibodies have been used to localize a DNP-labeled DNA probe inHIV-infected cells.62 It has also been reported that human chromosomes can be probedwith equal sensitivities by biotinylated, DNP-modified and digoxigenin-modified cosmidprobes.6 DNP-labeled DNA probes are readily prepared using ChromaTide dinitrophe-nyl-11-dUTP (C-7610, Section 8.2). In addition, the anti-DNP antibodies can be used intyramide signal amplification schemes that use our TSA kits containing DNP-X tyramide(Section 6.2, Table 6.1).

Tyramide Signal Amplification (TSA)The tyramide signal amplification technology — originally referred to as catalyzed

reporter deposition or CARD — is an enzyme-mediated detection method that uses thecatalytic activity of horseradish peroxidase (HRP) to generate high-density labeling of atarget protein or nucleic acid sequence in situ while preserving high spatial resolution ofthe staining pattern.63–67 In this technique, which is described in detail in Section 6.2, aprobe is directly or indirectly labeled with HRP. The HRP enzyme then converts a fluo-rescent or biotinylated tyramide derivative to a highly reactive form that binds covalentlyto nearby residues in proteins and other biomolecules (Figure 8.82). The turnover ofmultiple dye- or hapten-labeled tyramide substrates per peroxidase label results in strongsignal amplification (Figure 6.15, Figure 8.83). The increased sensitivity afforded by thistechnique can be critically important for detecting low abundance DNA or mRNA targetsin FISH.66,68–71 The amplification also makes it possible to use relatively short oligonu-cleotide probes, which can minimize background from nonspecific binding that is some-times seen with longer probes.72 TSA methodology has been used for identifying:

Direct

Amplified once

Amplified twice

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• Bacterial species in environmental samples 24,45

• Loss of heterozygosity in fixed, paraffin-embedded tissue sections 73–75

• Chromosome FISH probes less than 1 kilobase 76

The use of TSA produces fluorescent signals that are sufficiently bright to be seen inthe presence of considerable tissue autofluorescence.45,73 In addition, the optimal probeconcentrations are usually 2- to 10-fold lower for TSA-detected FISH than for conven-tional detection procedures, which can lead to considerable savings on expensive hybrid-ization probes.68

Hybridization probes can be labeled either directly or indirectly with HRP. Mostcommonly, probes are labeled with biotin or a hapten, which is then detected using anHRP conjugate of streptavidin or of an anti-hapten antibody, respectively 76–78 (Figure8.82). Alternatively, oligonucleotides can be directly conjugated to HRP using a bifunc-tional crosslinker 79 (Section 5.2). Direct labeling of nucleic acid probes by HRP canreduce background signals caused by nonspecific binding of HRP conjugates.65,72,79,80

Several probes can be hybridized simultaneously to identify multiple targets. However,signal development using multicolored fluorescent tyramides must be carried out sequen-tially, with a peroxidase inactivation step by dilute acid used between each TSA reactionto prevent crosslabeling.68,77

Molecular Probes’ TSA Kits (Section 6.2, Table 6.1) offer 27 different combinationsof a fluorescent tyramide and an HRP conjugate of either streptavidin or the goat anti–mouse IgG or goat anti–rabbit IgG antibodies. The Alexa Fluor 350, Alexa Fluor 488,Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 647,Pacific Blue and Oregon Green 488 tyramides are nine of our best dyes, with spectra thatcover the entire UV, visible and near-infrared spectrum. Additionally, biotin-XX tyramideand DSB-X biotin are used in six TSA kits in combination with streptavidin HRP, andthree TSA kits use a 2,4-dinitrophenyl tyramide (DNP-X tyramide) as the hapten. TheTSA kits that include DSB-X biotin tyramide permit fully reversible staining of targetsby fluorescent dye– or enzyme-conjugated biotin-binding proteins (Section 7.6). Ourextensive bibliography of TSA applications (T-24831), which contains over 200 referenc-es, should be consulted for detailed information on applications of TSA. Each TSA kitprovides sufficient materials to stain at least 100 slide preparations and includes thefollowing components:

• A fluorescent tyramide, biotin-XX tyramide, DSB-X biotin tyramide or DNP-X tyra-mide (Table 6.1)

• A horseradish peroxidase conjugate of goat anti–mouse IgG antibody, goat anti–rabbitIgG antibody or streptavidin

Figure 8.83 Fluorescence in situ hybridization de-tected by tyramide signal amplification. Chromo-some spreads were prepared from the cultured fi-broblast cell line MRC-5 and hybridized with abiotinylated α-satellite probe specific for chromo-some 17. The probe was generated by nick transla-tion in the presence of ChromaTide biotin-11-dUTP(C-11411). For detection by TSA, hybridized chro-mosome spreads were labeled using TSA Kit #22(T-20932) with HRP-conjugated streptavidin andAlexa Fluor 488 tyramide (upper panel) or withTSA Kit #23 (T-20933) with HRP-conjugatedstreptavidin and Alexa Fluor 546 tyramide (lowerpanel). After counterstaining with DAPI (D-1306,D-3571, D-21490), images were obtained using fil-ters appropriate for DAPI, FITC or TRITC.

Figure 8.82 Schematic representation of mRNA in situ hybridization detection using tyramide signalamplification (TSA). In the presence of horseradish peroxidase (HRP) and hydrogen peroxide, tyra-mide radicals are formed (red box) that can covalently react with nearby residues.

Section 8.5

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• A hydrogen peroxide stock solution• An optimized staining buffer• Blocking reagents• A staining protocol

The biotin-XX tyramide that we use in our TSA kits has an extra 14-atom spacer thatmakes the hapten more accessible (Figure 4.3) than the biotin tyramide used in earlierpublications, many of which are included in our bibliography for TSA Kit #21 (T-20931).DSB-X biotin tyramide and DNP-X tyramide contain a seven-atom spacer between thehapten and the tyramide.

Signal Amplification Using Enzyme-Labeled Fluorescence (ELF) TechnologyOur Enzyme-Labeled Fluorescence (ELF) technology, which is discussed in detail

in Section 6.3, uses our proprietary ELF 97 phosphatase substrate in combination withalkaline phosphatase conjugates to amplify signals. Upon enzymatic cleavage, the weaklyblue-fluorescent ELF 97 phosphatase substrate yields a yellow-green–fluorescent precipi-tate (Figure 8.84) that is significantly brighter than signals achieved either with directlylabeled fluorescent nucleic acid probes or with hapten-labeled probes in combinationwith fluorophore-labeled secondary detection reagents.81–85 Development of the fluores-cent signal is very rapid, occurring in seconds to minutes. The ELF 97 phosphatase sub-strate has been used for detecting in situ hybridization signals in whole-mount zebrafishembryos,84,86,87 sectioned mouse embryo hindlimbs 88 and cultured fibroblasts.84 Thesubstrate was also used to detect the products of reverse-transcription PCR in fixed frozensections to show reversion of a point mutation in the dystrophin gene by gene therapy.89

We have developed and optimized procedures for using the ELF 97 phosphatasesubstrate to detect mRNA in situ hybridization in cells and tissue sections (Figure 8.85).We provide the key reagents for this application in our ELF 97 mRNA In Situ Hybridiza-tion Kits (E-6604, E-6605). The ELF 97 phosphatase substrate can also be used in com-bination with other labeled probes and anti-dye, anti-hapten or secondary antibodies.86–88

The ELF 97 mRNA In Situ Hybridization Kits include:

• A streptavidin–alkaline phosphatase conjugate (in Kit #2, E-6605, only)• ELF wash, blocking and developing buffers• The application-specific ELF 97 phosphatase substrate solution• Hoechst 33342 nucleic acid counterstain• ELF mounting medium

Figure 8.84 Schematic representation of mRNA in situ hybridization detection using the Enzyme-Labeled Fluorescence (ELF) technology (Section 6.3). Alkaline phosphatase converts ELF 97 phos-phate (black triangles) to a brilliant green-fluorescent precipitate (green squares).

ProLong Antifade KitPhotobleaching is the most con-

sistently troublesome practical prob-lem in fluorescence microscopy,degrading the signal-to-noise charac-teristics of images and limiting thetime available for data acquisition.Addition of protective antifade re-agents to the specimen is the moststraightforward practical measure forcounteracting photobleaching, as itdoes not require compromises inexposure time, scan rate, magnifica-tion and other imaging configurationparameters. Among the numerousantifade reagents that have beendeveloped, Molecular Probes’ Pro-Long reagent (see Section 24.1)provides the most consistent perfor-mance in the widest range of experi-mental setups. Outstanding featuresof the ProLong reagent include:

• Compatibility with a wide range ofdifferent dyes and probes

• Uniform effectiveness — theProLong reagent does not distortthe balance of fluorescence signalsin multicolor analysis applications

• A turnkey solution to photobleach-ing problems. No optimization isrequired

• Extended duration of the fluores-cence signal with minimal quench-ing of its instantaneous intensity

• Formation of a dry mountingmedium allowing long-term pres-ervation of specimens with contin-ued antifade potency

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• 50 plastic coverslips• A detailed protocol

The ELF 97 mRNA In Situ Hybridization Kit #2 (E-6605),which contains a streptavidin–alkaline phosphatase conjugate,can be used to detect biotinylated DNA or RNA probes. TheELF 97 mRNA In Situ Hybridization Kit #1 (E-6604), whichdoes not include the streptavidin–alkaline phosphatase conjugate,is designed for use with other alkaline phosphatase conjugates —such as an anti-fluorescein–alkaline phosphatase conjugate(A-21251, A-21252; Section 7.4) — that have been applied todetect DNA or RNA probes conjugated to other haptens such asfluorescein or the Oregon Green dyes. Each kit contains sufficientreagents for 50 slides or coverslips.

Signal Amplification Using Two Rounds of AmplificationAlthough TSA and ELF amplification technologies enhance

FISH signals to a great degree, very low abundance signals canstill elude detection. A strategy using two sequential amplifica-tions has been shown to be useful for visualizing signals that couldnot be seen with only one round of amplification. Sequential TSAand ELF amplification has been used to visualize very low abun-dance mRNA species that could not be seen using either technolo-gy separately.90 In this strategy, a biotinylated probe was amplifiedfirst by streptavidin–HRP in conjunction with biotin tyramide. Thesecond step used streptavidin–alkaline phosphatase, followed byELF 97 phosphate (both components of the ELF 97 mRNA In SituHybridization Kit (E-6605)). In another variation, two rounds ofTSA amplification — the first with a dinitrophenyl tyramide andthe second with an HRP–anti-DNP antibody followed by fluores-cein tyramide — made it possible to detect a FISH signal from asfew as 50–60 HRP-labeled oligonucleotide molecules.72 A particu-larly useful combination uses a first step TSA procedure that lays

Figure 8.85 lacZ mRNA in transformed mouse fibroblasts (CRE BAG 2 cells)hybridized with a singly biotinylated, complementary oligonucleotide. Hybridswere then detected by incubation with a streptavidin–alkaline phosphatase con-jugate in combination with the ELF 97 alkaline phosphatase substrate, both ofwhich are provided in our ELF 97 mRNA In Situ Hybridization Kit (E-6604,E-6605). Cells were counterstained with DAPI (D-1306, D-3571, D-21490) andphotographed using a longpass optical filter appropriate for DAPI.

Figure 8.86 HybriWell hybridization sealing system.

down biotin-XX tyramide at the site of the target, followed by asecond TSA step that uses HRP-labeled streptavidin to deposit afluorescent tyramide (Figure 6.6).

Colorimetric Signal AmplificationThe colorimetric enzyme substrates NBT/BCIP (N-6495,

B-6492, N-6547; Section 9.4) can be used to detect hapten-labeled probes with conjugates of alkaline phosphatase. Thecombination of NBT and BCIP precipitates a dark blue, nonfluo-rescent product at the site of enzymatic activity.

Counterstaining Chromosomes for FISH

Counterstains are used to locate the chromosomes or nuclei inFISH experiments. Molecular Probes provides a spectrum of dyesfor staining hybridized metaphase or interphase chromosomes inFISH assays. For best results, we generally recommend using theless bright blue- or far red-fluorescent dyes as counterstains andthe brighter green- or red-fluorescent dyes to label the probes.The blue-fluorescent nuclear stain DAPI (D-1306, D-3571; Fluo-roPure grade, D-21490; Section 8.6) is commonly used for multi-color chromosome FISH techniques. DAPI provides an excellentcounterstain for green-fluorescent, red-fluorescent and far red-fluorescent probes and also shows a unique banding pattern thathelps to identify the chromosomes in metaphase preparations(Figure 8.75). TO-PRO-3 dye (T-3605, Section 8.6) provides afar red-fluorescent counterstain whose spectra are well separatedfrom those of the common green- and red-fluorescent dyes andfrom tissue autofluorescence. Although staining by the TO-PRO-3 dye can be detected using film or a CCD camera, its fluores-cence cannot be seen with the naked eye, and it is thus best forapplications in which direct visualization of the chromosomes ornuclei is not required to position the slide for analysis.

Gaskets for In Situ Hybridization Experiments

Precut, ready-to-use adhesive gaskets provide low-volumehybridization chambers for in situ hybridization studies. Designedespecially for hybridization experiments, the HybriWell hybrid-ization sealing systems (Figure 8.86) and the higher-volume

Section 8.5


Secure-Seal hybridization chambers (Figure 24.40) isolate single or multiple specimenson a slide. These hybridization gaskets (Table 8.12) have a special adhesive that bonds toglass slides in seconds, creating a watertight seal that holds at high temperatures, but canalso be removed cleanly and easily after hybridization. The hydrophobic surfaces areRNase free and will not trap or bind probes like glass surfaces can. Access ports in thechamber surface allow for the addition or removal of hybridization solutions and areeasily sealed using seal tabs to create leak-proof chambers that eliminate evaporation.Seal tabs are provided with the HybriWell hybridization sealing system and are alsoavailable separately (A-18211) for use with the Secure-Seal chambers. We also provideready-to-use, RNase-free HybriSlip hybridization covers (Figure 24.38), which arehydrophobic plastic coverslips that do not bind to labeled nucleic acids. These coversremain flat, even at high temperatures, to facilitate uniform reagent distribution.

Nucleic Acid Hybridization on Blots and Microarrays

Experiments in which labeled sample nucleic acids are hybridized to nucleic acids onsolid supports, most notably Southern and Northern blots, have been core technologies inthe field of molecular biology. Conventionally, samples were labeled with radioisotopes,hybridized to nucleic acids on the membrane and imaged for long periods with X-rayfilm. With the advent of genomics and the desire for higher-throughput methods of analy-sis, solid-surface hybridization technologies have developed a very high level of sophisti-cation, culminating in the DNA microarray, in which hundreds of thousands of sequencesare spotted in an area of just a few square centimeters on a glass support.91–94 Althoughsome users still employ radioactive labeling for microarray experiments, fluorescentlabeling and detection now dominate the field. Fluorescence provides an important ad-vantage because the majority of microarray experiments rely on comparison of hybridiza-tion signals between two or more samples, and fluorescence detection allows the analysisof multiple samples on the same array, minimizing artifactual variation in signals.

Multicolor fluorescence hybridization on microarrays is especially useful for expressionprofiling, which compares the mRNA levels between two or more samples.95–98 Hybridiza-tion on arrays has been used to identify groups of genes that may be involved in:

Table 8.12 Tools for hybridization experiments.

Cat # Chamber Dimensions Depth UsableVolume

Quantity perPackage

HybriWell hybridization sealing system

H-24720 13 mm diameter 0.25 mm 30 µL 100

H-24721 20 mm diameter 0.15 mm 30 µL 100

H-24723 22 mm × 22 mm 0.15 mm 30–50 µL 100

H-18210 40 mm × 21 mm 0.15 mm 50–100 µL 100

H-24722 40 mm × 22 mm 0.25 mm 180–200 µL 100

Secure-Seal hybridization chambers

S-24734 22 mm × 22 mm 0.8 mm 250 µL 50

S-24730 20 mm diameter 0.8 mm 200 µL 40




HybriSlip hybridization covers

H-18200 22 mm × 22 mm NA NA 500

H-18201 40 mm × 22 mm NA NA 500

H-18202 60 mm × 22 mm NA NA 500

Seal tabs

A-18211 Adhesive seal tab NA NA 400

NA = Not applicable.

Figure 8.88 Comparison of the absorption and flu-orescence emission spectra of the Alexa Fluor 555and Cy3 dyes. Spectra have been normalized to thesame intensity for comparison purposes.

Figure 8.87 DNA microarray hybridized to DNAlabeled using the ARES DNA Labeling Kits. Totalhuman RNA was labeled by reverse transcriptionusing either ARES Alexa Fluor 555 DNA LabelingKit (A-21677) or ARES Alexa Fluor 647 DNA Label-ing Kit (A-21676). Labeled DNA was hybridized toa microarray containing human housekeepinggenes. After hybridization, the array was imagedusing a ScanArray 5000XL microarray scanner(Packard BioScience) using the appropriate lasersand filter sets. The image was pseudocolored sothat red and white areas show the most intensesignal and green and blue areas show the least in-tense signal.

Alexa Fluor 555 Alexa Fluor 647

327

• Tumorigenesis and tumor suppression 99–101

• Apoptosis 102

• Leptin-induced changes in metabolism 103

• Klinefelter’s syndrome 104

• The yeast cell cycle 105

• Drosophila metamorphosis 106

• Drug sensitivity of Mycobacterium tuberculosis 107

• Development of Plasmodium falciparum 108

Microarray-based experiments promise to be useful for drug discovery processes 109,110

and toxicogenomics.111 Microarray technology can also be used for sequencing,112 geno-typing,113,114 detecting DNA copy number changes 115 and gene mapping.116,117

The interpretation of microarray-based experiments relies on the comparison of sig-nals from the labeled samples that have hybridized to the spots of the array. Many vari-ables may contribute to the level of signal detected and, as in any experiment, it is impor-tant to identify and minimize the variables that are artifacts of the experimental process.This section describes Molecular Probes’ products that can help to minimize experimen-tal artifacts so that it is possible to identify and quantitate meaningful signal changes.

Labeling Nucleic Acid Samples for Microarray Experiments

Consistent, Uniform Sample Labeling with ARES DNA Labeling KitsThe use of fluorophore labels rather than radioactive labels makes it possible to mea-

sure several samples on the same array, thereby eliminating variability that can be intro-duced by comparing hybridization results from different arrays. However, labeling DNAwith fluorescent dyes introduces several variables that can make quantitative measure-ments of hybridization efficiency difficult. A large hydrophobic dye attached to a nucle-otide alters the efficiency of enzymatic incorporation. Thus, samples prepared fromlabeled nucleotides may have different levels of labeling, making it difficult to comparelevels of hybridization between samples. Furthermore, variation of the fluorescence yieldwith degree of dye conjugation to the nucleic acid probe — as is observed with Cy3 dye–and Cy5 dye–labeled probes — can significantly reduce the reliability of quantitativemeasures of hybridization-based assays (see Alexa Fluor Dyes for Labeling NucleicAcids in Section 8.2). To minimize this variability, an alternative two-step labeling tech-nique (Table 8.8, Figure 8.44) uses conventional enzymatic incorporation of an amine-modified nucleotide followed by labeling with a reactive fluorescent dye (Section 8.2).The ARES DNA Labeling Kits use this technique to provide reliable and consistentlabeling with our state-of-the-art fluorescent dyes, including our Alexa Fluor 546, AlexaFluor 555 and Alexa Fluor 647 dyes (Figure 8.87), whose nucleic acid conjugates arelikely to perform better than the widely used Cy3 and Cy5 dye conjugates in quantitativemicroarray-based assays. Uniform labeling of the sample is possible because in the firststep of the ARES procedure, there are no bulky dye molecules on the nucleotide to re-strict the enzymatic incorporation. In the second step, the fluorescent labeling is per-formed with an excess of a reactive dye to ensure consistent labeling of the amine-modi-fied DNA. The result is a consistent labeling ratio of approximately one dye molecule per12–20 bases, which is higher than we can obtain with alternative labeling technologies.The ARES DNA Labeling Kits (Section 8.2) are available with many of our best fluoro-phores (Table 8.8), including dyes in the Alexa Fluor series (see Alexa Fluor Dyes forLabeling Nucleic Acids in Section 8.2). Both the Alexa Fluor 546 and Alexa Fluor 555dyes match the Cy3 filter set (Figure 8.88) and the Alexa Fluor 647 dye exactly matchesthe Cy5 filter set (Figure 8.89), while the other dyes in the Alexa Fluor family providechoices matched to other lasers and filter sets. Aminoallyl dUTP (A-21664, Section 8.2)and amine-reactive fluorescent dyes (Chapter 1) are also available separately.

Fast and Easy Direct Labeling with ULYSIS Nucleic Acid Labeling KitsThe ULYSIS kits use a direct chemical labeling method that vastly simplifies nucleic

acid labeling with fluorescent dyes (Section 8.2, Table 8.7), including our Alexa Fluordyes (see Alexa Fluor Dyes for Labeling Nucleic Acids in Section 8.2). These kits usethe platinum-based ULS chemistry developed at KREATECH Biotechnology BV to

Figure 8.89 Comparison of the fluorescence spec-tra of the Alexa Fluor 647 and Cy5 dyes. Spectrahave been normalized to the same intensity forcomparison purposes.

Section 8.5

DNA Arrays: Methodsand Protocols

The development and application ofmicroarray technologies has proceed-

ed rapidly,driven by theneed to analyzethe copiousamounts ofgenetic informa-tion generatedby the HumanGenomeProject. DNAArrays: Meth-

ods and Protocols (D-24835), editedby J.B. Rampal, provides biomedicalresearchers with a forward-looking andstate-of-the-art overview of thesetechniques. In 312 information-packedpages, this benchtop manual providesstep-by-step instructions for arrayprinting, DNA and RNA sample prep-aration, hybridization conditions, signaldetection, probe optimization, datacollection and bioinformatics. Additionaltopics include genotyping, sequencingby hybridization, antisense reagents,and gene expression analysis.


directly label the N-7 position of guanine residues without usingenzymatic incorporation. ULS chemistry has also been used tolabel RNA directly, obviating the need to make cDNA frommRNA samples (Figure 8.90). These kits can be used reliably toachieve a labeling efficiency of about one dye for every 30–50bases, which we have determined is the optimal level for hybrid-ization to dot blots.

Fluorophore-Labeled NucleotidesIt is possible to label nucleic acid samples for microarray-

based experiments using standard enzymatic incorporation ofChromaTide nucleotides via reverse transcription, PCR, nicktranslation or random-hexamer labeling (Figure 8.91). TheChromaTide UTP, ChromaTide OBEA-dCTP 39 and ChromaTidedUTP nucleotides (Section 8.2, Table 8.6, see Legal Notice forChromaTide UTP and dUTP Nucleotides in Section 8.2) areavailable labeled with Alexa Fluor dyes (see Alexa Fluor Dyesfor Labeling Nucleic Acids in Section 8.2), DNP, biotin and otherconventional fluorophores. A careful study of parameters affectingthe quality of microarray data has shown that the incorporation ofAlexa Fluor 546-14-dUTP (C-11401) into a DNA probe by re-verse transcription of total RNA results in a threefold highersignal on microarrays when compared with the signal obtainedfrom reverse transcription of the same RNA sample using a Cy3dye–labeled dUTP.118 Detailed protocols for enzymatic labelingare available in our product literature.

Labeled Random OligodeoxynucleotidesRNA samples can also be labeled by reverse transcription

using fluorophore-labeled random oligonucleotides as primersin combination with unlabeled deoxynucleotide triphosphates.Molecular Probes provides two types of labeled oligodeoxynucle-otides that can be used for this purpose. Our dT18 oligodeoxynu-cleotides are labeled at the 5′-terminus with one of four of ourAlexa Fluor dyes (Table 8.13). The labeled dT18 oligodeoxynucle-otides hybridize to poly(A) tails in RNA samples, providing

Table 8.13 Spectral characteristics of labeled oligonucleotides.

Fluorophore Ex/Em * Panomer 9RandomOligos

Oligo(dT)18

Alexa Fluor 350 345/440 P-21679

Pacific Blue 410/455 P-21678

Alexa Fluor 488 490/520 P-21680 O-21560

Alexa Fluor 546 555/570 P-21681

Alexa Fluor 555 555/565 P-21687 O-21561

Alexa Fluor 594 590/615 P-21682 O-21562

Alexa Fluor 633 630/650 P-21683

Alexa Fluor 647 650/670 P-21686 O-21563

Alexa Fluor 660 660/690 P-21684

Alexa Fluor 680 680/700 P-21685

Biotin NA P-21689

QSY 7 560 † P-21688

* Approximate excitation (Ex) and fluorescence emission (Em) maxima, in nm.† Approximate absorption maximum, in nm. NA = Not applicable.

primers for reverse transcription. Our Panomer 9 random-sequenceoligodeoxynucleotides are covalently labeled on the 5′-terminuswith one of our proprietary fluorescent dyes, with a nonfluorescentQSY 7 quencher dye or with biotin (Table 8.13). The Panomer 9oligonucleotides are also useful as primers for synthesizing labeledDNA via Klenow DNA polymerase or reverse transcriptase. Inthese reactions, the primer provides the detectable label, whereasunlabeled nucleotides are incorporated by the enzyme. This label-ing strategy ensures efficient and unbiased incorporation of nucle-otides because the bulky dye molecule does not interfere withnucleotide incorporation. However, because the resulting DNAfragments contain only a single label, the labeling efficiency willtypically be lower than that achieved by incorporating fluorophore-or hapten-labeled nucleotides.

Using Labeled Oligonucleotides as Biosensors on Solid SupportsMolecular beacons 119 are dual-labeled oligonucleotides that

become fluorescent upon binding to their complementary sequence(Figure 8.101, see below). These probes were originally employedfor solution-based hybridization assays, such as real-time PCR, butthey can also be used on solid supports. By linking a biotin mole-cule to the oligonucleotide, the molecular beacon can be attached toa streptavidin-coated surface, such as a glass slide or optical fibersurface.120,121 Multiple molecular beacons attached to a surface atdefined locations provide an array that can identify specific se-quences in complex samples without the need to label the samples.

Secondary Detection for Signal Amplification

Detecting Biotin Labels with Fluorescent Streptavidin ConjugatesFor very low amounts of sample or low abundance sequences,

it may be necessary to use a secondary detection strategy to amplifythe signal. A common secondary detection method uses streptavidinto amplify a sample labeled with a biotin-11-dUTP 102 (C-11411,Section 8.2). Labeled streptavidins are available that are conjugatedto Alexa Fluor dyes, fluorescent phycobiliproteins and many otherfluorescent dyes (Section 7.6, Table 7.17). The phycobiliproteins,phycoerythrin and allophycocyanin (Section 6.4), are extremelybright fluorescent proteins that can be visualized with filter setsstandard on most microarray readers 102 and are commonly used forsignal amplification in microarray experiments (Figure 6.39). Thesuperior Alexa Fluor dyes provide bright and photostable fluorophorelabels that span the spectrum (Figure 1.14, Figure 1.21, Figure 1.30;see The Alexa Fluor Dye Series — Peak Performance Across theVisible Spectrum in Section 1.3).

Alternative secondary detection methods include detection ofDNP with a labeled anti-DNP antibody (Section 7.4) or detection ofdyes, such as fluorescein or the Oregon Green 488 dye, with alabeled anti-dye antibody (Section 7.4). Although they were opti-mized for staining cells or tissues, our Alexa Fluor Signal Amplifi-cation Kits (Section 7.3, Table 7.8) may potentially be adapted foramplification of microarray signals.

Signal Amplification with the ELF 97 Phosphatase SubstrateOur Enzyme-Labeled Fluorescence (ELF) technology, which is

discussed in Section 6.3, uses our patented ELF 97 phosphatasesubstrate to amplify alkaline phosphatase–generated signals. Uponenzymatic cleavage, the weakly blue-fluorescent ELF 97 phos-phatase substrate yields a bright yellow-green–fluorescent precipi-

329

tate (Figure 8.84). Because one phosphatase enzyme can act uponmany substrate molecules, the signal is greatly amplified. TheELF 97 mRNA In Situ Hybridization Kits (E-6604, E-6605),which include the ELF 97 phosphatase substrate, have beenused to detect hybridization signals on oligonucleotide arrays.122

Samples labeled with biotin were incubated with a streptavidin–alkaline phosphatase conjugate and then detected with theELF 97 phosphatase substrate.

Chemiluminescent Detection on BlotsChemiluminescent enzyme substrates provide a sensitive alter-

native to radioactive labeling for Southern and Northern blots andfor macroarrays on nitrocellulose or nylon membranes. MolecularProbes offers the BOLD APB chemiluminescent substrate 123

(B-21901), an alkaline phosphatase substrate developed for appli-cations on nitrocellulose or nylon.123 To use this detection meth-od, the nucleic acid probe is first labeled with biotin using theChromaTide biotin dUTP (C-11411). After hybridization of thebiotinylated probe to the blot, the blot is incubated with an alka-line phosphatase conjugate of streptavidin (S-921), followed bythe BOLD APB substrate. Alternatively, the probe can be labeledwith a dye, such as fluorescein or the DNP hapten, and thendetected with an anti-dye antibody conjugated to alkaline phos-phatase (Table 7.13). Many other combinations of haptens andantibodies can also be used (Section 7.4).

The BOLD APB substrate is based on a 1,2-dioxetane mole-cule that emits bright chemiluminescence upon reaction withalkaline phosphatase.123 The substrate is provided as a ready-to-use solution that requires no mixing, making it extremely easy touse — there is no need for special blockers or enhancers that arerequired for use of other chemiluminescent substrates. The sig-

Figure 8.91 A microarray hybridized to Alexa Fluor546 dye–labeled cDNA. cDNA was labeled by re-verse transcription from Vibrio cholerae total RNAusing ChromaTide Alexa Fluor 546-14-dUTP(C-11404). Labeled cDNA was then hybridized to aV. cholerae O1 El Tor microarray. The array wasimaged with a ScanArray 5000XL scanner (Pack-ard BioScience). Contributed by Kimberly Chongand Gary Schoolnik, Stanford University.

Figure 8.92 Microarray expression analysis usingan Alexa Fluor 546 dye–labeled dendrimer. Com-plementary DNA (cDNA) from 500 ng of totalmouse liver RNA was prepared by reverse tran-scription using specially designed primers. Theprimers were designed such that a dendrimer cap-ture sequence was attached to the 5′-end of anoligo(dT) sequence. The cDNA was then hybridizedto three DNA dendrimers labeled with the AlexaFluor 546 dye such that approximately 125 AlexaFluor 546 dyes were coupled to the dendrimers.The labeled cDNA was then hybridized to a mi-croarray. The arrays were scanned using a Scan-Array 3000 scanner (Packard BioScience). Imagecontributed by Robert Getts, Genisphere.

Figure 8.90 ULYSIS reagent–labeled RNA hybrid-ized to a microarray. Poly(A)+ RNA samples fromthe spleen of an irradiated or unirradiated mousewere labeled using the Alexa Fluor 594 ULSreagent or Alexa Fluor 546 ULS reagent, respec-tively. The labeled samples were mixed and hybrid-ized to a cDNA microarray. Image contributed byRahul Mitra (Baylor College of Medicine) and MiniKapoor, Thomas H. Burrows, and Rachel Grier (MDAnderson Cancer Center).

nal-to-noise ratio is exceptionally high, allowing for sensitivitythat is potentially several times that of fluorescence techniques.The BOLD APB chemiluminescent substrate emits a signal thatincreases in intensity for two hours and then remains constant forat least six more hours, allowing plenty of time for the multipleexposures that may be necessary for optimal detection sensitivity.In contrast to fluorescent reagents, chemiluminescent reagents donot require an excitation light source; the energy from the chemi-cal reaction generates light. The chemiluminescent signal can bedetected by directly exposing the blot to X-ray film or by using ascanning instrument designed for chemiluminescence. TheBOLD APB chemiluminescent substrate is available in a 25 mLready-to-use solution, sufficient to stain 25 minigel blots.

Other Types of Signal AmplificationDendrimers 119 are complexes of labeled oligonucleotides built

up from several layers of overlapping oligonucleotide sequenc-es.124 The outer layer comprises two types of oligonucleotides,one that contains a capture sequence complementary to the se-quence of interest, and the other is labeled with a fluorescent dye.Because there are so many labeled oligonucleotides in this layer(about 250), the dendrimer exhibits a greatly amplified fluores-cent signal compared to a single fluorescent dye. For microarray-based applications, labeled samples are generated by reversetranscription using primers tailed with the complement to thecapture sequence.125 The cDNA samples are then labeled byhybridization of the dendrimer capture sequence to the comple-mentary sequence on the primer. This amplification techniquemakes it possible to use very small amounts of sample in thehybridization reaction 125 (Figure 8.92).

Section 8.5

The BOLD APB chemiluminescent alkaline phosphatase substrate provide superiorperformance for detection of both proteins and nucleic acids on blots.


Figure 8.93 DNA microarrays stained with nucleic acid stains for quality con-trol. DNA microarrays were stained with dilutions of either SYBR Green II dye(green, S-7568, S-7564, S-7586), POPO-3 dye (orange, P-3584) or SYTO 59dye (magenta, S-11341) in aqueous buffer. The microarrays were imaged on aScanArray 5000XL microarray scanner (Packard BioScience) using the appro-priate lasers and filters. Staining reveals the variable amounts of DNA spottedonto the different microarrays.

Figure 8.94 Panomer 9 oligodeoxynucleotides for quality control of microarrayspotting. Three microarrays were made using three different spotting protocols.Each microarray was then hybridized to a Panomer 9 oligonucleotide, conjugatedwith either Alexa Fluor 488 dye (top) or Alexa Fluor 546 dye (middle and bottom),washed and imaged using a ScanArray 5000XL microarray scanner (Packard Bio-Science). One representative spot from each slide was selected for comparisonpurposes. The spots were analyzed using Metamorph software (Universal Imag-ing, Inc.) and the data presented as a 3-dimensional graph with high-intensity ar-eas as peaks and low-intensity areas as valleys. For further clarification, thegraphs are color coded so that the highest intensity areas are red and the lowestintensity areas are blue. The comparison shows that the Panomer 9 oligodeoxy-nucleotides are ideal for microanalysis of spot morphology on DNA microarrays.

Techniques for Blot and Microarray Quality Controland Normalization

Whether using Northern blots, macroarrays on membranes ormicroarrays on glass slides, it is important to normalize the signalto the amount of nucleic acid on the solid support. Differences inpurification, loading and transfer may create differences in RNAlevels on blots, and even with sophisticated robotics, direct spot-ting techniques for creating arrays of nucleic acids on solid sup-ports vary widely in reproducibility (Figure 8.93, Figure 8.94).

Our experience is that the uniformity of nucleic acid arrays de-pends to a significant extent on its production; some commercial-ly available arrays have poor uniformity (Figure 8.94). At thevery least, a method to qualitatively determine the amount ofnucleic acid on a support is desirable for quality control purposes.Quantitative data that can be used for signal normalization is evenmore useful, making it possible to validate perceived changes inRNA expression between samples, despite differences in thenucleic acid levels on the solid support. Often, hybridization of a“constitutively expressed” RNA sequence is used for normaliza-tion on Northern blots. However, it is sometimes discovered thatthe level of such RNA sequences is not constant through chang-ing physiological states of a cell or tissue. Direct measurement ofthe levels of nucleic acid spotted on the slide provides a reliablemethod for normalization and can also be used to assess theamount of nucleic remaining on a support after stripping off aprobe and before reusing the blot or array.

SYBR DX DNA Blot StainIn addition to providing a means of normalizing hybridization

signals, staining denatured DNA or RNA directly on filter mem-branes after blotting protocols provides for more accurate com-parison of the sample to molecular weight markers and eliminatesguesswork about transfer efficiency. However, direct staining onblotting membranes has not been widely used because the mostcommon methods for detecting denatured DNA or RNA — ethid-ium bromide 126 or methylene blue staining — give rise to highbackground fluorescence. Silver staining or gold staining fol-lowed by silver enhancement provides 10- to 100-fold bettersensitivity than ethidium bromide but is expensive, time-consum-ing and tedious. Also, because of the affinity of gold for sulfur,only agarose gels containing less than 0.1% sulfate are suitablefor use with gold staining; higher amounts of sulfate invariablyresult in unacceptably high background signals.

By contrast, our patented SYBR DX DNA blot stain (S-7550)provides a rapid and easy method for fluorescent staining ofdenatured DNA and RNA on blotting membranes, with sensitivitysuperior to that of any other nucleic acid stain we have tested.Direct staining of immobilized nucleic acids following blottinggives an indication of the efficiency of transfer from the gel to themembrane; thus, there is no need to re-examine the gel for residu-al DNA. After transfer, DNA is fixed to the membrane by UVcrosslinking or baking in a vacuum oven, as usual. Then, the blotis simply incubated with the diluted SYBR DX stain for as littleas ten minutes and photographed. Unlike silver-enhanced goldlabeling, no blocking steps are required. We have found that it ispossible to detect 200 pg/band of denatured M13 RF DNA fol-lowing Southern transfer to a nylon membrane using the SYBRDX DNA blot stain with 254 nm epi-illumination, a SYBRGreen/Gold gel stain photographic filter (S-7569) and Polaroid667 black-and-white print film (Figure 8.95). The sensitivity isreduced somewhat when using 300 nm transillumination due tolight scattering through the nylon membrane. However, in bothcases, the sensitivity is at least 10-fold better than that obtainedwith ethidium bromide in parallel experiments. Blot staining withthe SYBR DX dye can also be analyzed with commercially avail-able CCD documentation systems and laser-based scannersequipped with appropriate filters.

Both neutral and positively charged nylon membranes can beused with the SYBR DX DNA blot stain, and staining is fully

331

compatible with subsequent hybridization and colorimetric detection of alkaline phos-phatase–coupled probes using the chromogenic enzyme substrates nitro blue tetrazolium(NBT, N-6495) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP, B-6492, N-6547;Section 10.3). Blots that have been hybridized and detected by radioactive, chemilumi-nescent or colorimetric reagents can also be stained with the SYBR DX dye, providedthat the membrane is not blocked with ssDNA.

The SYBR DX stain has been employed to measure trace amounts of contaminantDNA in drinking water.127 In a broad-spectrum assay for bacterial contamination, sam-ples were simply spotted or vacuum-blotted onto nylon membranes and stained with theSYBR DX dye. Detection of DNA was quantitative down to about 50 ng when a fluores-cence-based microplate reader was used.

The SYBR DX DNA blot stain (S-7550) is provided as a 1000-fold concentrate inDMSO. This amount of dye is sufficient for staining about 50–100 blots, and the stainingsolution can be reused at least three times with little loss of staining intensity.

Nucleic Acid Stains for MicroarraysThe simplest technique for comparing the amounts of DNA spotted onto arrays is to

use a fluorescent nucleic acid stain. Molecular Probes’ proprietary nucleic acid stainsexhibit a strong fluorescence signal when bound to nucleic acids, providing an easy andeffective method for measuring the amount of nucleic acids on solid supports (Figure8.93). Several nucleic acid stains that have been used effectively for microarrays are listedin Table 8.14. Procedure for Staining Microarrays with SYBR Green II Dye shows thesimple technique employed for this experiment. Other dyes in these families (Section 8.1)should also prove useful in this application. When staining nucleic acids on solid supportswith nucleic acid stains, it is important to choose a dye that matches the light sources andfilter sets available in the image analysis system or the array reader. For instance, the

Figure 8.95 DNA stained with the SYBR DX DNAblot stain (S-7550) following Southern transfer.M13mp19 RF DNA was digested with HindIII andBgl II restriction enzymes, creating fragments of6548 bp and 701 bp. Ten samples of the digestedDNA were prepared by serial twofold dilution andapplied to an agarose gel for separation by electro-phoresis. The samples ranged in total DNA contentfrom 100 ng to 0.2 ng. The DNA from the gel wasthen transferred to a Hybond-N+ positively chargednylon membrane (Amersham Life Science, Inc.) bySouthern blotting, stained with SYBR DX DNA blotstain, visualized with 254 nm epi-illumination andthen photographed through a SYBR photographicfilter (S-7569) using Polaroid 667 black-and-whiteprint film. Not all of the bands visible in the originalphotograph are visible in this reproduction.

Table 8.14 Nucleic acid stains used for microarrayquality control.

Nucleic Acid Stain Ex/Em * Cat #

SYBR Green II stain 492/513 S-7564S-7568S-7586

POPO-3 dye 534/570 P-3584

SYTO 59 dye 622/645 S-11341

SYTO 61 dye 628/645 S-11343

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

Procedure for Staining Microarrays withSYBR Green II Dye

TECHNICAL NOTE

Procedure for staining microarrays with nucleic acid stains. SYBR Green II RNA gelstain (S-7564, S-7568, S-7586) provides a quick and easy test to determine whether DNAhas been uniformly spotted onto a microarray.1

• Dilute the SYBR Green II stain 10,000-fold in TBEbuffer (45 mM Tris-borate, 1 mM EDTA, pH 8.0).

• Cover the microarray with the diluted stain andincubate at room temperature for 2–3 minutes.

• Wash the microarray 3–4 times with TBE buffer.• Spin-dry the microarray for 1–2 minutes.• Scan the microarray using filter sets appropriate

for the Alexa Fluor 488 dye or fluorescein (FITC).SYBR Green II dye has a fluorescence excitationmaximum at 494 nm and a fluorescence emissionmaximum at 521 nm.

To wash the stain off of the slide, the slide isincubated at room temperature for one hour in asolution of 0.1% SDS, 10 mM Tris, 1 mM EDTA, pH7.5. After drying the slides, they can be used inhybridization experiments.

References

1. Biotechniques 29, 78 (2000).

Section 8.5

Microarray QualityThere is a huge difference in the

quality of commercial microarrays andmicroarray spotters, resulting in con-siderable variations in spotted arrays.Thus, careful quality control of com-mercial microarrays and arrayingequipment is essential.


POPO-3 dye has a maximum fluorescence excitation at 534 nmand a maximum fluorescence emission at 570 nm, which reason-ably match the filter sets for the Alexa Fluor 546 dye, Alexa Fluor555 dye or Cy3 dyes. When testing dyes to use for normalizationof signals, it is important to carefully test several dilutions of thedye to determine the one that gives the most linear response overthe range of DNA in the spot. It is also important to determine anyeffects of the dye on subsequent hybridization.

Panomer Random-Sequence OligonucleotidesFluorescently labeled random-sequence oligonucleotides

provide an alternative method for assessing the level of nucleicacids immobilized on solid supports. This method assays thecapability of spotted DNA to hybridize, making it possible todetermine if hybridization efficiency varies across the array. OurPanomer 9 random oligodeoxynucleotides (Table 8.13) are idealfor this application (Figure 8.96). These 9-base, random-sequenceoligodeoxynucleotides are covalently labeled on the 5′-end with afluorescent dye. The variety of available fluorescent dyes makesit possible to use any fluorescence channel of interest and to

Figure 8.96 DNA microarray hybridized to Panomer 9 random oligodeoxynucleo-tides. A DNA microarray slide was hybridized sequentially with one of threedifferent Panomer 9 random oligodeoxynucleotides at room temperature fortwo minutes. After each hybridization, the slide was washed first in 2× SSC and0.2% SDS and then in 1× SSC. After drying, the slide was imaged on a ScanArray5000XL microarray reader (Packard BioScience), using appropriate lasers and fil-ter sets. After imaging, the Panomer 9 oligodeoxynucleotide was stripped fromthe microarray by incubation in deionized water for one minute at room tempera-ture and the microarray was hybridized to another Panomer 9 oligodeoxynu-cleotide. From left to right, the images show the array hybridized to Panomer 9oligodeoxynucleotides labeled with the Alexa Fluor 488 dye (P-21680), AlexaFluor 546 dye (P-21681) and Alexa Fluor 647 dye (P-21686). Each image hasbeen pseudocolored to indicate the different dyes. The hybridization results re-veal the variable amounts of DNA spotted onto this microarray.

Figure 8.97 Microarray spotted with the Alexa Fluor 546 (P-21681) and AlexaFluor 647 (P-21686) Panomer 9 random oligodeoxyribonucleotides. Equalamounts of two Panomer 9 random oligodeoxyribonucleotides were spotted di-rectly onto microscope slides and the fluorescence documented using two dif-ferent channels of a ScanArray 5000XL microarray reader (Packard Bio-Science). The Alexa Fluor 546 Panomer 9 random oligodeoxyribonucleotidesignal was completely separated from the Alexa Fluor 647 Panomer 9 randomoligodeoxyribonucleotide signal.

compare relative signal intensities per spot in several differentchannels (Figure 8.97). It is also possible to use the Panomer 9oligodeoxynucleotides for quality control of spotting techniques(Figure 8.98) or to assay the stability of DNA spots after the arrayis subjected to washing, boiling, hybridization or other conditions(Figure 8.96).

Sample Quantitation

Measuring the amount of nucleic acid in the sample helps toensure success and consistency in microarray printing or samplelabeling. The PicoGreen dsDNA Quantitation Kit and reagent(P-7581, P-7589, P-11495, P-11496; Section 8.3) and RiboGreenRNA Quantitation Kit and reagent (R-11490, R-11491; Section8.3) make it easy to quantitate large numbers of samples ofdsDNA or RNA, respectively. The one-step procedures requireonly a 5-minute incubation and are easily adaptable to high-throughput automation. The assays are orders of magnitude moresensitive than UV absorbance (A260) measurements, so only mini-mal amounts of sample are required. Furthermore, in contrast toUV absorbance measurements, where proteins and free ribonucle-otides in the mixture interfere with accurate quantitation, thePicoGreen and RiboGreen reagents measure only polymeric nu-cleic acids. The assays can be used to quantitate yields from PCRamplification, RNA purification or cDNA synthesis reactions.

Hybridization Chambers for Microarrays

Uniform distribution of the labeled sample is essential for effi-cient and even hybridization across the array. Precut, ready-to-useadhesive gaskets help to optimize fluid dynamics in the hybridiza-tion reaction. The 22 mm × 22 mm Secure-Seal hybridizationchambers (Figure 8.99) are ideal for microarray-based hybridiza-tion experiments. When the chambers are partially filled and placedon a rocker, they provide a surface-to-volume ratio that facilitatesuniform hybridization. They adhere directly to glass slides, creatinga watertight seal that is temperature resistant, but can also be re-moved cleanly and easily after the hybridization. The hydrophobicsurfaces are RNase free and will not trap or bind probes like glass

333

Figure 8.99 Secure-Seal gasket for use with mi-croarray hybridizations.

Figure 8.100 HybriSlip covers for hybridization.

surfaces can. Access ports in the chamber surface allow for the addition or removal ofsolutions and are easily sealed using adhesive seal-tabs (A-18211) to create leak-proofchambers that eliminate evaporation. For very small volume samples, the lower-volumeHybriWell Sealing Systems can be used (Table 8.12, Figure 8.86). As an alternative, wealso provide HybriSlip hybridization covers, which are hydrophobic plastic coverslips thatdo not bind to labeled nucleic acids (Table 8.12, Figure 8.100). The covers remain flat,even at high temperatures, to facilitate uniform reagent distribution.

Solution-Based Hybridization Assays

Assays based on measurement of a specific hybridization product formed in solutionbecome very powerful if the hybridization product can be measured directly in thereaction tube, without any separation steps. Fluorescence technology using dyes selec-tive for dsDNA or using fluorescence resonance energy transfer (FRET, see Section 1.3)methods make it possible to perform such homogeneous assays with very high sensitivi-ty and very high throughput, making them valuable screening tools. This type of meth-odology is particularly useful for real-time PCR assays but can be used in other solu-tion-based hybridization assays as well.

Real-Time Quantitative PCR Using FRET and Quenching TechniquesPCR products can be quantitated during the linear portion of the amplification reac-

tions, allowing accurate quantitation of templates. Several fluorescence-based methodsexist for real-time quantitation of PCR products, including the use of nucleic acid dyesand FRET techniques that use fluorescently labeled primers or molecular beacons. Inaddition to providing a more streamlined assay, single-tube methods minimize thepossibility for cross-contamination between PCR reactions.

Fluorescence resonance energy transfer (FRET, see Section 1.3) refers to a processby which energy is transferred from one dye molecule (the donor) to another (the accep-tor) without the emission of a photon. If the acceptor dye is a fluorophore, the energymay be emitted as fluorescence that is characteristic of the acceptor dye; otherwise, theenergy is dissipated and the fluorescence quenched. FRET technology has been used inseveral ways to develop homogeneous and real-time hybridization-based assays.

A molecular beacon is made up of an oligonucleotide with a fluorescent dye at-tached to one end and a quencher (nonfluorescent acceptor dye, such as our QSY 7 orQSY 9 dye) attached to the other. The sequence is designed so that the oligonucleotide

Figure 8.98 Panomer 9 oligodeoxynucleotides spot-ted onto a microarray. Panomer 9 Alexa Fluor 555random oligodeoxynucleotide (P-21687) and Pano-mer 9 Alexa Fluor 647 random oligodeoxynucleotide(P-21686) were spotted onto a glass slide in com-plementary patterns. The fluorescence signals weredetected on a ScanArray 5000XL microarray reader(Packard BioScience) using either the “Cy3” or“Cy5” channels. The digital files were pseudocoloredand digitally overlaid.

Figure 8.101 Schematic representation of molecular beacons. In the hairpin loop structure, thequencher (black circle) forms a nonfluorescent complex with the fluorophore (green circle). Upon hy-bridization of the molecular beacon to a complementary sequence, the fluorophore and quencher areseparated, restoring the fluorescence.

Section 8.5

See the description of all of ourGrace Products for microscopy inSection 24.3.


forms a hairpin loop, which brings the fluorescent dye andquencher together 128 (Figure 8.101). In this configuration, thefluorescence is nearly completely quenched. The loop portion ofthe hairpin is complementary to the sequence of interest in theassay. Upon hybridization to the sequence, the hairpin unfolds,separating the fluorescent dye from the quencher (Figure 8.101).Thus, a fluorescent signal indicates hybridization of the molecu-lar beacon to the sequence of interest.

Molecular beacons 119 can be constructed by using an oligonu-cleotide modified with an amine group on one end and a thiolgroup on the other end. Amine-reactive (Chapter 1) and thiol-reactive (Chapter 2) dyes can be covalently bound to the amineand thiol modifications, respectively. Dabcyl (D-2245, Section1.8) is a very efficient quencher, reducing the fluorescence emis-sion of most fluorophores by over 99%,129 making it possible todetect as few as 10 complementary sequences out of 100,000available molecules.130 Dabcyl has been reported to act as a uni-versal quencher, dissipating the fluorescence emission of anyfluorophore, regardless of its fluorescence emission profile.129 Inaddition, the quenching efficiencies are much higher than usuallyreported for FRET. Because this pattern does not follow the rulesof classical FRET, it has been suggested that the quenching by thedabcyl dye occurs by another mechanism, possibly by the forma-tion of a nonfluorescent complex comprising both dyes.129 TheQSY 7, QSY 9, QSY 22 and QSY 35 quenchers (Section 1.6,Section 1.8, Section 2.2, Section 3.3) have much stronger absor-bance than the dabcyl quencher at visible wavelengths (Figure1.66) and they efficiently quench a broad assortment of fluores-cent donors, including fluoresceins, the Oregon Green 488 andOregon Green 514 dyes, the Alexa Fluor 488 and Alexa Fluor 532dyes and other similar fluorophores (Table 1.8) and may provevery useful in molecular beacon probe design.

NANOGOLD 1.4 nm gold clusters, which can be attached toappropriately derivatized oligonucleotides with NANOGOLDmono(sulfosuccinimidyl ester) (N-20130, Figure 1.84, Section1.6) or NANOGOLD monomaleimide (N-20345, Figure 2.17,Section 2.2), have been shown to be a particularly efficientquencher in molecular beacon probes, with reported sensitivityenhancements up to 100-fold over the dabcyl-labeled quencherand eightfold greater sensitivity in detection of single-base mis-matches.131

Molecular beacons can be used in either end-point or real-timePCR to detect the synthesis of a specific amplicon. Because thefluorescence signal is based on hybridization to a complementarysequence, molecular beacons provide much greater specificitythan gel-based analysis. Depending on the design, a molecularbeacon is capable of distinguishing differences of a single basepair,129,132 short tandem repeat markers 133 or specific single-nucleotide polymorphisms in a complex sample.134 Furthermore,the difference in stability between the stem–loop structure and thestructure of the oligonucleotide bound to the target ensures thatthe molecular beacons bind their targets more specifically than doconventional FRET probes.135 Multiple molecular beacons, bear-ing different fluorophores, can be used simultaneously in thesame PCR reaction for multiplex analysis.130,133,136–138

The use of “wavelength-shifting” molecular beacons 119 makesmultiplex analysis even more versatile. In this design, there aretwo fluorophores on one end — a “harvester” that can transferenergy to the “emitter” at the same end via FRET and a quencherat the other end of the probe 139 (Figure 8.102). In the hairpinformation, the “harvester” is quenched; upon hybridization to thetarget, the “harvester” transfers energy to the “emitter.” By usingdifferent FRET pairs with the same “harvester” fluorophore andvarying “emitter” fluorophores, one can design multiple molecu-lar beacons that have different fluorescence emissions whenexcited by the same wavelength.

Molecular beacon–based PCR assays have been used for manyapplications, including to:

• Distinguish CC chemokine receptor genotypes 136,140

• Detect and quantitate the levels of multiple viruses in clinicalsamples 130

• Detect specific alleles of c-Ki-Ras in stool samples 134

• Identify human papillomavirus (HPV) particles in tissuespecimens 141

• Identify drug-resistant variants of Mycobacteriumtuberculosis 142,143

• Discriminate between different species of Candida 144

• Identify different bacterial species in environmental or foodsamples 145,146

Reverse transcription followed by real-time PCR with molecu-lar beacons can be used to measure RNA transcript levels.147,148

Molecular beacons have also been used for real-time monitoringof RNA-based amplification reactions using an RNA poly-merase.149 Our SYBR Green I nucleic acid stain has also beenextensively used to follow real-time PCR (Section 8.3).

The TaqMan assay 119 uses FRET to monitor PCR reactions inreal time.150 A non-extendable oligonucleotide probe (the detec-tion probe) complementary to the sequence of interest is labeledwith a donor fluorophore at one end and an acceptor fluorophoreat the other (Figure 8.103). FRET between the two fluorophores

Figure 8.102 Schematic representation of wavelength-shifting molecularbeacons. The molecular beacon has two fluorophores on one end — a“harvester” (green circle) and an “emitter” (red circle) — and a quench-er on the other end (black circle). In the hairpin loop structure, thequencher forms a nonfluorescent complex with the harvester. Upon hy-bridization of the molecular beacon to a complementary sequence,quenching of the harvester fluorophore is relieved, and it transfers ener-gy (via FRET) to the emitter, which emits fluorescence.

335

Figure 8.103 Schematic representation of real-time PCR with TaqMan primers. In the intact TaqManprobe, energy is transferred (via FRET) from the short-wavelength fluorophore on one end (green cir-cle) to the long-wavelength fluorophore on the other end (red circle), quenching the short-wave-length fluorescence. After hybridization, the probe is susceptible to degradation by the endonucleaseactivity of a processing Taq polymerase. Upon degradation, FRET is interrupted, increasing the fluo-rescence from the short-wavelength fluorophore and decreasing the fluorescence from the long-wavelength fluorophore.

results in quenching of the donor’s fluorescence. During the hybridization steps, thisquenched probe, along with flanking PCR primers, hybridizes to the sequence of interest.When Taq polymerase encounters the detection probe during the extension steps, it usesits 5′-endonuclease activity to cleave it, separating the fluorophores and relieving thequenching of the donor. Real-time measurement of donor fluorescence allows accuratequantitation of the amplicon. Multiplex measurements of as many as seven colors havebeen achieved through the use of multiple detection probes.151,152 The TaqMan assay canprovide similar discrimination to molecular beacon–based assays, with each assay exhib-iting superior characteristics in particular situations.153 The TaqMan assay has been usedfor SNP detection 152 and quantitation of enterovirus RNA in urban sludge samples,154

and it has been adapted for quantitative detection of nucleic acid methylation 155 and fordetection of reverse transcriptase activity.156

In a modification to the molecular beacon design, Scorpion 119 primers incorporate thehairpin directly into the primer.157,158 The hairpin loop is complementary to the ampliconand performs a “flip” as it releases from the hairpin to hybridize to the newly formedamplicon. The faster kinetics of this intramolecular hybridization, compared to the bimo-lecular reaction with molecular beacons, allows identification of the amplicon usingfewer cycle numbers.

UniPrimer 119 technology (also called Sunrise or Amplifluor) also uses a hairpinquencher design, but one that can be used to detect any sequence of interest amplified ina PCR reaction.159 In the PCR reaction, one of the primers is designed with a 5′-tailidentical to a 3′-tail on the “universal” labeled hairpin primer (Figure 8.104). After thesecond cycle, the tail on the PCR primer and its complement are both part of the ampli-con sequence. Thus, in the third cycle, the universal primer can hybridize to the ampliconto start DNA synthesis. By the fourth cycle, synthesis in the opposite direction extendsthrough the universal primer, unfolding the hairpin and relieving the quenching. The

Section 8.5

Send UsMore Great Images

Our customers provide many of thesuperb fluorescence images for ourpublications. Submit an image of aMolecular Probes product in actionand we’ll send you a colorful T-shirt. Ifwe use the image in any of ourpublications, we’ll also send you acredit certificate (US$200 or €250)good toward the purchase of any ofour products.

To submit images by e-mail —Attach the high-resolution digitalimage to an e-mail message. Include adescription of the specimens andexperimental techniques in the body ofthe message and send it to:

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To submit images by mail — Weaccept either slides or high-resolutiondigital images on 3.5" floppy, Zip orJaz discs, or on CD-ROM. If the slideis encased in glass, please use apadded mailer (glass tends to shatterin standard envelopes). Include adescription of the specimens andexperimental techniques along with thesubmission and mail to:

Molecular Probes, Inc.Attention: Publications Department29851 Willow Creek RoadEugene, OR 97402 USA


advantage of this design is that it can be used to detect any de-sired sequence. UniPrimer technology has been used for SNPgenotyping,160 identification of point mutations,161 detection ofprostate-specific antigen cDNA,159 a closed-tube telomeric repeatamplification protocol (TRAP) 162 and in situ PCR and RT-PCR.48

Other Solution-Based Hybridization Assays Using FRET andQuenching Techniques

Specific DNA or RNA molecules have been quantitated insolution by measuring the degree of quenching observed uponhybridization with a BODIPY FL dye–labeled oligonucleotide.163

The oligonucleotide contained a BODIPY FL dye–modifiedcytosine at its 5′-end, and its fluorescence was quenched uponbinding to the complementary guanine in the target DNA. Therate of quenching was reportedly proportional to the amount oftarget DNA. This technique can be applied to quantitative DNAdetection in solution, as well as to real-time quantitative PCR.

Single-stranded RNA molecules from a plant viral genomehave been detected using FRET techniques in conjunction with aset of fluorescently labeled DNA probes that hybridize next toeach other on the target RNA.164 In this study, the BODIPY493/503 dye was chosen as the donor fluorophore because of itsnarrow excitation and emission spectra, which minimizes back-

ground fluorescence. When hybridized near Cy5 dye–labeledacceptor oligonucleotides on target RNA, donor oligonucleotidesthat were double-labeled with the BODIPY 493/503 dye showedan unexpected enhancement of FRET signals as compared withsingle-labeled donor probes. These double-labeled donor probesalso exhibited less fluorescence when not hybridized on the targetRNA, probably due to self-quenching of the BODIPY 493/503fluorophores.

Other Solution-Based Hybridization Assays Using FluorescentNucleic Acid Stains

The use of melting curve analysis with the SYBR Green Inucleic acid stain has proven to be an ideal method for character-izing amplicons in a PCR reaction, as described in Section 8.3.This method can also be used to streamline the restriction-lengthfragment polymorphism (RFLP) analysis, where SNPs and othermutations are detected based on changes in fragment sizes pro-duced by restriction endonucleases. Conventionally, fragment sizeis determined by agarose gel electrophoresis. However, becausemelting temperature depends on fragment length, the fragmentsize can also be detected by measuring the melting temperature.In this way, this simple and accurate assay can be performed in asingle tube, enormously increasing throughput possibilities.165

Figure 8.104 Schematic representation of real-time PCR with UniPrimers.In the first round of amplification, the reverse primer, containing a specialsequence tag, primes synthesis along the template. In the second round, theforward primer primes synthesis that extends through the special sequencetag, forming a complementary sequence to the tag. In the third round, the

UniPrimer hybridizes to this complementary sequence via the special se-quence tag. The hairpin structure of the UniPrimer ensures that the quench-er (black circle) suppresses the fluorescence of the fluorophore (green cir-cle). Finally, in the fourth round, synthesis extends through the hairpin loop,relieving the quenching of the fluorophore.

337

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found useful in the practice of a variety of patents.Although we indicate the usefulness of ourmaterials and methods for the practice of thirdparty technology, we emphasize that purchase ofour products does not include a license to practiceany third party patent, unless a license under sucha patent is clearly indicated in our product litera-ture. 120. Anal Biochem 283, 56 (2000);121. Chem Eur J 6, 1107 (2000); 122. Biotech-niques 30, 368 (2001); 123. BOLD is a trademarkof Serologicals Corporation; manufactured forMolecular Probes by Serologicals Corporation;patent pending. 124. J Theor Biol 187, 273(1997); 125. Physiol Genomics 3, 93 (2000);126. J Neurosci Methods 42, 211 (1992); 127. JEnviron Health 60, 14 (1997); 128. Nat Biotech-nol 14, 303 (1996); 129. Nat Biotechnol 16, 49(1998); 130. Proc Natl Acad Sci U S A 96, 6394(1999); 131. Nat Biotechnol 19, 365 (2001);132. Clin Chem 44, 482 (1998); 133. Biotech-niques 29, 1296 (2000); 134. Proc Natl Acad SciU S A 96, 9236 (1999); 135. Proc Natl Acad SciU S A 96, 6171 (1999); 136. Proc Natl Acad SciU S A 96, 12004 (1999); 137. Genet Anal 14, 151(1999); 138. Science 279, 1228 (1998); 139. NatBiotechnol 18, 1191 (2000); 140. AIDS 14, 483(2000); 141. J Virol Methods 89, 29 (2000);142. Antimicrob Agents Chemother 44, 103(2000); 143. Nat Biotechnol 16, 359 (1998);144. J Clin Microbiol 38, 2829 (2000); 145. ApplEnviron Microbiol 63, 1143 (1997); 146. J FoodProt 63, 855 (2000); 147. Cancer Res 60, 1711(2000); 148. Cytokine 11, 1031 (1999); 149. Nu-cleic Acids Res 26, 2150 (1998); 150. GenomeRes 6, 986 (1996); 151. Biotechniques 27, 1116(1999); 152. Biotechniques 27, 342 (1999);153. Biotechniques 28, 732 (2000); 154. Biotech-niques 29, 88 (2000); 155. Nucleic Acids Res 28,E32 (2000); 156. Biotechniques 25, 972 (1998);157. Nat Biotechnol 17, 804 (1999); 158. NucleicAcids Res 28, 3752 (2000); 159. Nucleic AcidsRes 25, 2516 (1997); 160. Genome Res 11, 163(2001); 161. Mol Diagn 3, 217 (1998); 162. Bio-techniques 26, 552 (1999); 163. Nucleic AcidsRes 29, E34 (2001); 164. Nucleic Acids Res 28,E107 (2000); 165. Biotechniques 30, 358 (2001).

Section 8.5

CoverWell Incubation Chamber GasketsCoverWell incubation chamber gaskets are silicone

gaskets with a clear plastic cover that are expresslydesigned for immunocytochemistry and in situ hybrid-ization. The gasket is simply pressed onto a wet or drymicroscope slide to form a watertight chamber thatholds reactants in place and prevents evaporation. Thechambers improve the uniformity and sensitivity ofstaining by enclosing a large sample area while minimizing the reagent volume required.The incubation chamber gaskets are easily removed and reapplied for multiple-step proce-dures. These chamber gaskets are heat resistant, autoclavable and nuclease free. Cover-Well incubation chamber gaskets are available with circular (13 mm diameter, 0.2 mmdeep, C-18155; 13 mm diameter,0.5 mm deep, C-18156) or rectangular (40 mm × 22 mm,0.2 mm deep, C-18150; 40 mm × 22 mm, 0.5 mm deep, C-18151) recesses.


Cat # Product Name Unit SizeA-18211 Adhesive seal-tab, for HybriWell™ hybridization sealing system *set of 400* ..................................................................................................... 1 setA-21663 5-(3-aminoallyl)uridine 5′-triphosphate, trisodium salt (aminoallyl UTP) *2 mM in TE* ...................................................................................... 500 µLB-21901 BOLD™ APB chemiluminescent substrate *for membrane-based alkaline phosphatase detection* *25 minigel blots* ...................................... 25 mLE-6604 ELF® 97 mRNA In Situ Hybridization Kit #1 *50 assays* ..................................................................................................................................... 1 kitE-6605 ELF® 97 mRNA In Situ Hybridization Kit #2 *with streptavidin, alkaline phosphatase conjugate* *50 assays* ................................................... 1 kitH-18200 HybriSlip™ hybridization cover, 22 mm x 22 mm *RNase free* *set of 500* ...................................................................................................... 1 setH-18201 HybriSlip™ hybridization cover, 40 mm x 22 mm *RNase free* *set of 500* ...................................................................................................... 1 setH-18202 HybriSlip™ hybridization cover, 60 mm x 22 mm *RNase free* *set of 500* ...................................................................................................... 1 setH-24720 HybriWell™ hybridization sealing system, 13 mm diameter chamber, 0.25 mm deep *set of 100* ..................................................................... 1 setH-24721 HybriWell™ hybridization sealing system, 20 mm diameter chamber, 0.15 mm deep *set of 100* ..................................................................... 1 setH-24723 HybriWell™ hybridization sealing system, 22 mm × 22 mm chamber, 0.15 mm deep *set of 100* .................................................................... 1 setH-18210 HybriWell™ hybridization sealing system, 40 mm × 21 mm chamber, 0.15 mm deep *set of 100* .................................................................... 1 setH-24722 HybriWell™ hybridization sealing system, 40 mm × 22 mm chamber, 0.25 mm deep *set of 100* .................................................................... 1 setP-21679 Panomer™ 9 random oligodeoxynucleotide, Alexa Fluor® 350 conjugate ............................................................................................................ 10 nmolP-21680 Panomer™ 9 random oligodeoxynucleotide, Alexa Fluor® 488 conjugate ............................................................................................................ 10 nmolP-21681 Panomer™ 9 random oligodeoxynucleotide, Alexa Fluor® 546 conjugate ............................................................................................................ 10 nmolP-21687 Panomer™ 9 random oligodeoxynucleotide, Alexa Fluor® 555 conjugate ............................................................................................................ 10 nmolP-21682 Panomer™ 9 random oligodeoxynucleotide, Alexa Fluor® 594 conjugate ............................................................................................................ 10 nmolP-21683 Panomer™ 9 random oligodeoxynucleotide, Alexa Fluor® 633 conjugate ............................................................................................................ 10 nmolP-21686 Panomer™ 9 random oligodeoxynucleotide, Alexa Fluor® 647 conjugate ............................................................................................................ 10 nmolP-21684 Panomer™ 9 random oligodeoxynucleotide, Alexa Fluor® 660 conjugate ............................................................................................................ 10 nmolP-21685 Panomer™ 9 random oligodeoxynucleotide, Alexa Fluor® 680 conjugate ............................................................................................................ 10 nmolP-21689 Panomer™ 9 random oligodeoxynucleotide, biotin-XX conjugate ........................................................................................................................ 10 nmolP-21678 Panomer™ 9 random oligodeoxynucleotide, Pacific Blue™ conjugate .................................................................................................................. 10 nmolP-21688 Panomer™ 9 random oligodeoxynucleotide, QSY® 7 conjugate ........................................................................................................................... 10 nmolS-24732 Secure-Seal™ hybridization chamber gasket, eight chambers, 9 mm diameter, 0.8 mm deep *set of 20* .......................................................... 1 setS-24733 Secure-Seal™ hybridization chamber gasket, eight chambers, 9 mm diameter, 1.3 mm deep *set of 20* .......................................................... 1 setS-24730 Secure-Seal™ hybridization chamber gasket, one chamber, 20 mm diameter, 0.8 mm deep *set of 40* ............................................................ 1 setS-24731 Secure-Seal™ hybridization chamber gasket, one chamber, 20 mm diameter, 1.3 mm deep *set of 40* ............................................................ 1 setS-24734 Secure-Seal™ hybridization chamber gasket, one chamber, 22 mm × 22 mm, 0.8 mm deep *set of 50* ........................................................... 1 setS-7550 SYBR® DX DNA blot stain *1000X concentrate in DMSO* ................................................................................................................................... 1 mL

8.6 Nuclear and ChromosomeCounterstaining and Nissl Stains

The use of nucleic acid stains to visualize nuclei and chromosomes and for chromo-some banding is discussed in this section. Nucleic acid stains and related products foranalyzing cell cycle, measuring cell proliferation and detecting apoptotic and dead cellsare discussed in Chapter 15. The counterstains described in this section are compatiblewith a wide range of cytological labeling techniques, including direct or indirect anti-body-based detection methods, in situ hybridization and detection of specific cellularstructures with fluorescent probes such as our mitochondrion-selective MitoTracker(Section 12.2, Table 12.1) and F-actin–selective phalloidin (Section 11.1, Table 11.1)probes. These counterstains can also serve to fluorescently label cells for analysis inmulticolor imaging experiments and several of these stains find specific application asNissl stains in neuronal cells. Although particularly known for its unique nucleic acidstains, Molecular Probes is also the world’s primary manufacturer of high-quality DAPI,propidium iodide and the “Hoechst” dyes.

Nuclear Counterstaining of Fixed Cells and Tissues

Blue-Fluorescent CounterstainsDAPI (D-1306, D-3571; FluoroPure grade, D-21490) is the classic nuclear and chro-

mosome counterstain, used for years to identify nuclei and show chromosome-bandingpatterns. DAPI binds selectively to dsDNA and thus shows little to no background stain-

Figure 8.105 Mouse intestine cryosection showingbasement membranes labeled with our anti-fibro-nectin antibody (A-21316) and the Alexa Fluor 488goat anti–chicken IgG antibody (A-11039, green).Goblet cells and crypt cells were labeled with AlexaFluor 594 wheat germ agglutinin (W-11262, red).The microvillar brush border and smooth musclelayers were visualized with Alexa Fluor 680 phalloi-din (A-22286, pseudocolored purple). The sectionwas counterstained with DAPI (D-1306, D-3571,D-21490, blue).

Product List — 8.5 Detecting Nucleic Acid Hybridization

section 8.5 - detecting nucleic acid · pdf file8.5 detecting nucleic acid ... and determining...

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