Evaluation of non-radioactive labelling and detection of deoxyribonucleic acids: Part one: chemiluminescent methods

The growth of analytical methods for the detection of nucleic acid from various biological samples reflects recent advances in biotechnology development especially in the areas of genetic, infections and cancer diagnosis. The target DNA is detected by hybridization techniques derived from Southern's blotting. However such assays, based on the use of ³²P labelled DNA probes, bring with them the associated problems of handling radioactive materials. In order to overcome these difficulties, a number of chemiluminescent detection methods have recently been developed.These new, alternative probe labelling procedures and chemiluminescent detection methods are easy to use in routine assays performed in research laboratories as well as for medical applications, and can reach the level of sensitivity found in classical radiolabelling techniques.The techniques investigated include peroxydase, biotin 16-dUTP or digoxigenin 11-dUTP probe labelling. The target DNAs are transferred onto nitrocellulose or nylon membranes and further fixed by heat or UV crosslinking. Specific hybridization on the target DNA is finally revealed by the use of chemiluminescent substrates. For all these techniques the detection limit is 10 aM (attomol) of a 561 bp target DNA. However for the probes labelled with peroxydase and with digoxigenin the detection limit drops to 1.0 aM of the target DNA. In the present paper we shall compare several of these DNA labelling and detection procedures and show that the detection threshold can vary by as much as a factor of 20 from method to method. This is the first time that various chemiluminescent methods for label and detection of DNA are compared and evaluated in order to determine the best protocol.     

Detection protocols for biotinylated probes: optimization using multistep techniques.

Recent studies using biotinylated in situ hybridization (ISH) have utilized a wide range of detection protocols for the biotinylated hybrids, leading to conflicting reports in the literature regarding sensitivity. In this study we compared 11 different detection protocols for biotinylated ISH using a measles virus-specific RNA probe on formalin-fixed, paraffin-embedded central nervous system tissue infected with measles virus. Maximum sensitivity was achieved with five-step detection protocols incorporating the use of a monoclonal antibody to biotin. Single-step detection protocols were found to be insensitive, as shown by their failure to detect viral nucleic acid in infected white-matter cells. Only by increasing the number of steps in the detection protocols were these infected cells demonstrable. Unless pre-hybridization, hybridization, and detection protocols are optimized, the results obtained in pathogenicity studies using ISH could be misinterpreted, leading to false conclusions about nucleic acid distribution. This also applies to the ever-increasing use of ISH for diagnostic purposes.     

生物素肼化学标记和DIG标记cDNA探针检测柑桔裂皮病类病毒

分别用生物素肼化学标记和ＤＩＧ标记我国柑桔裂皮病类病毒（ＣＥＶｄ）全长克隆ｃＤＮＡ,用以上探讨对不同来源的的核酸样品进行斑点杂交。两种探针可检出病柑桔总核酸的最低含量久为４００ｎｇ／斑点和８０ｎｇ／斑点;生物素肼标记ＣＥＶｄ－ＤＮＡ探讨针,可检出ＣＥＶｄ－ｃＤＮＡ的最低含量约为１０ｐｇ／斑点,研制的ＣＥＶｄ检测试剂盒能检测出发病和隐性带毒苗木中的ＣＥＶｄ,灵敏、特异、简便、快速。试剂盒已使用于检测     

Use of bioluminescence in nucleic acid hybridization reactions

Luminescence reactions can be used to detect specific nucleic acid sequences hybridized with a nucleic probe. Different labels such as cytidine sulphone, fluorescein, and biotin can be incorporated into DNA or oligonucleotide molecules and detected by antibody or avidin conjugates coupled to glucose-6P dehydrogenase. On supports such as nitrocellulose filters, sensitivity is not greatly increased using luminescence, but detection is rapid and easy to perform using polaroid film. Moreover, hybridization can be performed with different labelled probes on the same sample. In solution, luminescence can be used to monitor sandwich reactions. The method is less sensitive than detection on filters but can easily be automated. The performance of these assays can be increased considerably by enzymatic amplification of the target catalysed by a thermostable polymerase.     

Non-radioactive labeling and detection of nucleic acids. III. Applications of the digoxigenin system

The digoxigenin-based non-radioactive DNA labeling and detection system was applied in various hybridization protocols using digoxigenin-labeled probes obtained by enzymatic incorporation of Dig-[11]-dUTP. In genomic blots single-copy genes (human tissue-type plasminogen activator, constant part of immunoglobulin kappa light chain) can be detected with only 0.5 to 5 micrograms human DNA depending on the type of probe and the length of the hybridizing region. Due to its high sensitivity and specificity, the digoxigenin system is also appropriate for colony-, plaque-, and in situ hybridizations with metaphase chromosome spreads and fixed cells. Especially in the latter applications it is of great advantage, that with the digoxigenin system any significant background or unspecific side reactions with biological materials are avoided.     

Non-radioactive hybridization probes prepared by the chemical labelling of DNA and RNA with a novel reagent, photobiotin.

A photo-activatable analogue of biotin, N-(4-azido-2-nitrophenyl)-N'-(N-d-biotinyl-3-aminopropyl)-N'-methyl-1,3- propanediamine (photobiotin), has been synthesized and used for the rapid and reliable preparation of large amounts of stable, non-radioactive, biotin-labelled DNA and RNA hybridization probes. Upon brief irradiation with visible light, photobiotin formed stable linkages with single- and double-stranded nucleic acids yielding probes which were purified from excess reagent by 2-butanol extraction and ethanol precipitation. Using single-stranded phage M13 DNA probes chemically labelled with one biotin per 100-400 residues and dot-blot hybridization reactions on nitrocellulose, as little as 0.5 pg (6 X 10(-18) mol) of target DNA was detected colorimetrically by avidin or streptavidin complexes with acid or alkaline phosphatase from three commercial sources. The sensitivity of detection of target RNA in dot-blots and Northern blots was equivalent to that obtained with 32p-labelled DNA probes. Photobiotin was also used for the labelling of proteins with biotin.     

Design and testing of ITS probes for distinguishing Phaeocystis species

Oligonucleotide probes provide rapid, easy identification of species difficult to identify by more traditional means, e.g. light microscopy. Phaeocystis is a difficult genus to identify as a unicell and thus presents a good candidate for probe development. Hybridisation using PCR products or cell lysates rather than whole cells as targets provides access to more highly variable regions of the genome, such as non-coding spacer regions of the ribosomal operon for detection of fine scale molecular variation not available in the coding regions. The ITS region is an excellent region of the genome to discriminate between Phaeocystis species. PCR amplified ITS-1 sequence from one Phaeocystis antarctica strain was labelled with digoxigenin and hybridised to total nucleic acids from 35 Phaeocystis strains, the prymnesiophyte Emiliania huxleyi, the diatom Cylindrotheca closterium, the rhodophyte Phycodrys austrogeorgia, the phaeophyte Desmarestia aculeata and the chlorophyte Acrosiphonia arcta. Strong signals were observed in all cold water species, i.e. Phaeocystis antarctica and Phaeocystis pouchetii, whereas other warm-water Phaeocystis spp. were not labelled or only weakly labelled with the ITS-1 probe. No hybridisations were observed in all other genera. A short oligonucleotide probe for all cold-water Phaeocystis spp. and for Phaeocystis pouchetii was designed from the ITS-1. Both probes were labelled with digoxigenin and tested in a dot blot analysis against PCR products from 23 Phaeocystis species. Only the two cold water species or Phaeocystis pouchetii were, respectively, labelled by their specific probe.     

Multicolour whole-mount in situ hybridization to Drosophila embryos

 We report an extended whole-mount in situ hybridization procedure for Drosophila embryos. By using probes labelled with digoxigenin, fluorescein and biotin, respectively, this protocol allows the detection in three colours of RNAs derived from three different genes. Hybridized probes are detected by consecutive staining with appropriate alkaline phosphatase conjugates using different chromogenic substrate combinations, and serial removal of the antibody conjugates by low pH washes. Received: 7 May 1996/Accepted: 7 July 1996     

High-density hapten labeling and HRP conjugation of oligonucleotides for use as in situ hybridization probes to detect mRNA targets in cells and tissues.

Oligonucleotides that carry a detectable label can be used to probe for mRNA targets in in situ hybridization experiments. Oligonucleotide probes (OPs) have several advantages over cDNA probes and riboprobes. These include the easy synthesis of large quantities of probe, superior penetration of probe into cells and tissues, and the ability to design gene- or allele-specific probes. One significant disadvantage of OPs is poor sensitivity, in part due to the constraints of adding and subsequently detecting multiple labels per oligonucleotide. In this study, we compared OPs labeled with multiple detectable haptens (such as biotin, digoxigenin, or fluorescein) to those directly conjugated with horseradish peroxidase (HRP). We used branching phosphoramidites to add from two to 64 haptens per OP and show that in cells, 16-32 haptens per OP give the best detection sensitivity for mRNA targets. OPs were also made by directly conjugating the same oligonucleotide sequences to HRP. In general, the HRP-conjugated OPs were more sensitive than the multihapten versions of the same sequence. Both probe designs work well both on cells and on formaldehyde-fixed, paraffin-embedded tissues. We also show that a cocktail of OPs further increases sensitivity and that OPs can be designed to detect specific members of a gene family. This work demonstrates that multihapten-labeled and HRP-conjugated OPs are sensitive and specific and can make superior in situ hybridization probes for both research and diagnostic applications.     

Chromosomal in situ hybridization with double-labeled DNA: signal amplification at the probe level.

A double-labeling approach was applied to nonisotopic in situ hybridization with individual cosmid and plasmid clones, using digoxigenin or biotin as label and a combination of two separate enzymatic labeling methods. Probe labeling was achieved by nick translation, followed by tailing of the probe by terminal deoxynucleotidyl transferase. The double-labeling method, in conjunction with an improved detection protocol, provides for a higher signal intensity than that obtainable with single-labeled probes.     

Monoadduct forming photochemical reagents for labeling nucleic acids for hybridization.

We describe the synthesis of three angelicin derivatives which can be used for labeling nucleic acids with biotin. These compounds were used to label nucleic acids in the presence of lysed cell constituents. The resulting labelled nucleic acids show hybridization to a genus specific probe for E. coli. The relative comparison of sensitivity indicates that a polyamine linker is better than a polyethylene oxide linker between the biotin and angelicin moieties.     

Using non-radioactive probes on plants: a few examples

Due to costs in using and disposing of radiochemicals and to health considerations, we have been developing applications which include non-isotopic detection of DNA and proteins using chemiluminescence. Our major interests are in the detection of viral nucleic acids and in the analysis of transgenic plants. Generally, probes were labelled with digoxigenin, either by the random priming method or by PCR, and then detected with CSPD or CDP-Star. We routinely use a tissue blotting protocol for diagnosing TYLCV, a plant virus becoming a pest in the Mediterranean region. Test results were comparable with those using the same radiolabelled probe. When total nucleic acids are extracted from the plant samples and used in dot-blot or Southern blot assays, viral DNAs are promptly detected by chemiluminescence. In transgenic plants, chemiluminescence was used to detect the transgene on genomic Southern blots, the transgenic mRNAs on Northern blots, and the transgenic protein on Western blots. In Southern and Northern blots, the quality of the results obtained was usually satisfactory, but not as good as with a radiolabelled probe, the main problem being the signal-to-background ratio. Our goal is now to improve the quality of results in demanding applications such as genomic Southern blots, by reducing the background on membranes. © 1998 John Wiley & Sons, Ltd.     

Chemically synthesized non-radioactive biotinylated long-chain nucleic acid hybridization probes.

A new method for the chemical labelling of nucleic acid with biotin to produce non-radioactive probes has been developed. NN'-Bis-(3-aminopropyl)butane-1,4-diamine (spermine) and long-chain diamino compounds (diaminohexane, diaminodecane and diaminododecane) were linked covalently to biotin and the resultant conjugates were attached to nucleic acid by using a cross-linking reagent (glutaraldehyde or diepoxyoctane). Iodoacetylation and biotinylation of the long-chain diamino compounds produced modified biotinylated conjugates that can be linked to DNA without the use of a cross-linking reagent. These types of probes attach one biotin molecule to each linker arm of spermine, diamino and iodoacetylated amino derivatives. Such probes have long linker arms separating the biotin moiety from the hybridization sites of the nucleic acid. These probes can detect 10 pg of target DNA by dot-blot hybridization.     

两种DNA探针杂交检测结核分支杆菌方法的研究

为改进结核杆菌DNA探针的特异性与实用性,研制了以生物素标记的两种对结核分支杆菌特异的DNA探针:一个5’端标记的20bp的寡核苷酸探针和一个采用PCR方法合成的188bp长链探针。两种探针分别与结核分支杆菌的全染色体DNA,以及基因组上IS6110序列的一段317bp的PCR扩增产物进行斑点杂交,以碱性磷酸酶(AP)催化的染色反应检测,测试了两个探针的敏感性和特异性。系统地比较研究了两种探针杂交检测条件:探针的浓度选择,杂交温度与洗膜温度的选择,以及杂交与洗膜温度对检测的敏感性与特异性的影响。寡核苷酸探针和188bp探针杂交检测纯化结核分支杆菌基因组DNA的敏感性分别为100ng与6ng,杂交检测PCR产物的敏感性分别是400pg与50pg。两探针的最佳杂交浓度均为40～160ng/ml,最佳杂交温度分别是42℃与68℃,最佳洗膜温度分别是60℃与60～68℃之间。两种探针均仅与结核分支杆菌及BCG有杂交信号,而与其它受试分支杆菌及非分支杆菌杂交结果都呈阴性。它们的特异性都很强,但188bp探针的敏感性约是寡核苷酸探针的7～16倍,而且188bp探针检测本底较低,是检测结核分支杆菌的较佳选择     

Design of a sandwich-mode amperometric biosensor for detection of PML/RARα fusion gene using locked nucleic acids on gold electrode

In this study, a novel DNA electrochemical probe (locked nucleic acid, LNA) was designed and involved in constructing an electrochemical DNA biosensor for detection of promyelocytic leukemia/retinoic acid receptor alpha (PML/RARα) fusion gene in acute promyelocytic leukemia for the first time. This biosensor was based on a 'sandwich' sensing mode, which involved a pair of LNA probes (capture probe immobilized at electrode surface and biotinyl reporter probe as an affinity tag for streptavidin-horseradish peroxidase (streptavidin-HRP). Since biotin can be connected with streptavidin-HRP, this biosensor offered an enzymatically amplified electrochemical current signal for the detection of target DNA. In the simple hybridization system, DNA fragment with its complementary DNA fragment was evidenced by amperometric detection, with a detection limit of 74 fM and a linear response range of 0.1-10 pM for synthetic PML/RARα fusion gene in acute promyelocytic leukemia (APL). Otherwise, the biosensor showed an excellent specificity to distinguish the complementary sequence and different mismatch sequences. The new pattern also exhibited high sensitivity and selectivity in mixed hybridization system.     

High sensitivity detection of HPV-16 in SiHa and CaSki cells utilizing FISH enhanced by TSA

 Detection of integrated human papillomavirus type 16 (HPV-16) DNA in SiHa and CaSki cells was used as a model system to demonstrate sensitivity and resolution of a well defined target. Using 293- to 1987-base polymerase chain reaction (PCR)-synthesized probes to the E6 and E7 open reading frames of HPV-16, several fluorescent in situ hybridization (FISH) detection methods, enhanced with tyramide signal amplification (TSA), were compared. The synthetic probes were biotin labeled by a nick translation method and the hybridized probes were detected by various fluorescent TSA methods using cyanine 3 tyramide, biotinyl tyramide and a biotin TSA Plus reagent. High sensitivity detection in SiHa cells was demonstrated using a 619-base probe to detect two single copies of integrated HPV-16 DNA. In CaSki cells, which contain up to 600 copies of HPV-16 DNA, a 293-base probe was used for detection. The results of these comparisons show that with refinement of TSA methods and reagents, increasing levels of high sensitivity detection can be achieved and that these methods allow subnuclear localization as well. Accepted: 20 June 1997     

32P- and biotin-labelled in vitro transcribed cRNA probes for the detection of potato spindle tuber viroid and chrysanthemum stunt viroid

Replacing nick-translated DNA probes by in vitro transcribed complementary RNA (cRNA) probes considerably increased the sensitivity of dot-blot detection tests of potato spindle tuber viroid and chrysanthemum stunt viroid. As compared to the limit of detection of 5–10 pg of viroid obtained with ³²P-labelled DNA probes, cRNA probes allow the detection of less than 1 pg of pure viroid. When labelled with biotin by incorporation of biotin-labelled ribonucleotides, the cRNA probes have a limit of detection of approximately 5 pg of purified viroid.     

DNA hybridization detection in a microfluidic channel using two fluorescently labelled nucleic acid probes

A conceptually new technique for fast DNA detection has been developed. Here, we report a fast and sensitive online fluorescence resonance energy transfer (FRET) detection technique for label-free target DNA. This method is based on changes in the FRET signal resulting from the sequence-specific hybridization between two fluorescently labelled nucleic acid probes and target DNA in a PDMS microfluidic channel. Confocal laser-induced microscopy has been used for the detection of fluorescence signal changes. In the present study, DNA hybridizations could be detected without PCR amplification because the sensitivity of confocal laser-induced fluorescence detection is very high. Two probe DNA oligomers (5'-CTGAT TAGAG AGAGAA-TAMRA-3' and 5'-TET-ATGTC TGAGC TGCAGG-3') and target DNA (3'-GACTA ATCTC TCTCT TACAG GCACT ACAGA CTCGA CGTCC-5') were introduced into the channel by a microsyringe pump, and they were efficiently mixed by passing through the alligator teeth-shaped PDMS microfluidic channel. Here, the nucleic acid probes were terminally labelled with the fluorescent dyes, tetrafluororescein (TET) and tetramethyl-6-carboxyrhodamine (TAMRA), respectively. According to our confocal fluorescence measurements, the limit of detection of the target DNA is estimated to be 1.0 x 10(-6) to 1.0 x 10(-7)M. Our result demonstrates that this analytical technique is a promising diagnostic tool that can be applied to the real-time analysis of DNA targets in the solution phase.