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Non-radioactive nucleic acid detection systems
Abstract
Non-radioactive methods to label nucleic acids to be used as probes are being used more often. Non-radioactive detection methods offer several advantages over the usual radioactive methods. Non-radioactive detection methods eliminate the need to deal with the licensing, waste disposal, and safety concerns associated with the use radioactive material. The probes generated are more stable than are probes labeled with 32P. The detection sensitivity of the radioactive and non-radioactive probes is comparable. Non-radioactive detection methods typically require shorter exposure times to detect the hybridization signal [1–5].
... The use of riboprobes complementary to the RNA or DNA ofthe virus was found to be more sensitive than serological assays, labelled cDNA and immunobinding assays due to specificity and the removal of non-hybridised probe to minimise non-specific background (Abad et al., 1992). Non-radioactive methods such as horseradish peroxidase (HRP), dioxigenin-anti-digoxigenin (DIG) and the biotin-streptavidin systems to label nucleic acids that are used as probes are also used to detect plant pathogens (Karcher, 1994). Their use is due to the advantage that the probes generated are stable and require shorter exposure time to detect hybridised material (Karcher, 1994). ...
... Non-radioactive methods such as horseradish peroxidase (HRP), dioxigenin-anti-digoxigenin (DIG) and the biotin-streptavidin systems to label nucleic acids that are used as probes are also used to detect plant pathogens (Karcher, 1994). Their use is due to the advantage that the probes generated are stable and require shorter exposure time to detect hybridised material (Karcher, 1994). ...
... Exposure times to detect the hybridization signal in non-radioactive detection methods are shorter. Overall, non-radioactive probes are cheaper (19). ...
Background
Leishmaniasis represents a major public health concern in tropical and sub-tropical countries. At present, there is no efficacious vaccine against the disease and new control methods are needed. One way to access this important goal is to knock out genes of specific macromolecules to evaluate the effect of deletion on the growth, multiplication, pathogenesis and immunity of the parasite. The aim of this study was to design and clone molecular constructs to knock out N-acetylglucosamine phosphatidylinositol de-N-acetylase (GPI12) gene in Leishmania major.
Methods
For designing and making molecular constructs, we used pLEXSY-neo2 and pLEXSY-hyg2 vectors. The molecular constructs were cloned in E. coli strain Top10. The molecular constructs were transfected by electroporation into L. major in two stages.
Results
The molecular constructs were confirmed by Colony PCR and sequencing. The recombinant strains were isolated by selective antibiotics, after which they were confirmed by PCR, Southern and Western blots.
Conclusion
Recombinant parasites were created and examined for subsequent study. With the use of molecular constructs, it was possible to remove and study gene GPI12 and to achieve a live recombinant Leishmania parasite that maintained the original form of the antigenic parasites. This achievement can be used as an experimental model for vaccine development studies. Further investigations are essential to check this model in a suitable host.
We describe optimized procedures for colorimetrically-detected DNA sequencing with direct blotting electrophoresis. One-step protocols for Sequenase and Klenow enzyme are given. The clapping technique has been adapted to allow convenient casting of very thin gels with an optimal lower gel (transfer) surface. This gives very sharp band patterns, enabling more than 350 bases from a single loading to be read with confidence. The crucial points for direct blotting electrophoresis are discussed. Background problems resulting from unspecific binding of streptavidin to the nylon membranes have been eliminated by the use of high concentrations of SDS in the incubation buffer; and using a single large glass tube for all incubation and washing steps is a very convenient and effective development protocol. Automation of the colorimetric development process is described.
The increasing demand for DNA sequences can be met by replacement of each DNA sample in a device with a mixture of N samples so that the normal throughput is increased by a factor of N. Such a method is described. In order to separate the sequence information at the end of the processing, the DNA molecules of interest are ligated to a set of oligonucleotide "tags" at the beginning. The tagged DNA molecules are pooled, amplified, and chemically fragmented in 96-well plates. The resulting reaction products are fractionated by size on sequencing gels and transferred to nylon membranes. These membranes are then probed as many times as there are types of tags in the original pools, producing, in each cycle of probing, autoradiographs similar to those from standard DNA sequencing methods. Thus, each reaction and gel yields a quantity of data equivalent to that obtained from conventional reactions and gels multiplied by the number of probes used. To date, even after 50 successive probings, the original signal strength and the image quality are retained, an indication that the upper limit for the number of reprobings may be considerably higher.
A series of dATP and dCTP nucleotide analogs have been synthesized which are modified by attachment of aliphatic linkers containing
a functional group to the amino-nitrogen at the hydrogen bonding positions of the bases, that is, at the 6-position of adenine
and the 4-position of cytosine. These nucleotides are incorporated into DNA probes by standard niak-translation protocols.
DNA probes labeled with biotin derivatives of these nucleotides are effectively hybridized to target DNA sequences and can
be detected by a streptavidin and calf intestinal alkaline phosphatase conjugate with a sensitivity (0.25 pg DNA) sufficient
for reproducible and rapid detection of single copy genes in a Southern blot of mammalian DNA.
Also, a procedure has been developed to allow reprobing of nylon filters that have been hybridized with biotinylated probes
and developed with the streptavidin/alkaline phosphatase conjugate and a standard dye system.
A simple and efficient 2.5-h Southern blotting procedure is described that uses 0.4 M NaOH to transfer DNA in a downward direction. The resulting blots give signals that are both sharper and 30% stronger than those obtained by conventional upward-transferred blots.
A simple procedure for the detection of rice RFLPs with non-radioactive probes is described. Rice single-copy DNA was labeled
with non-radioactive digoxigenin-d UTP. When digested total DNA was hybridized with the non-radioactive labeled DNA probes,
RFLPs for rice single-copy DNA could be successfully detected.
One of the most sensitive and widespread colorimetric detection systems in matrix-based application formats is the redox couple 5-bromo-4-chloro3-indolyl phosphate (BCIP)/nitroblue tetrazolium chloride (NBT). It is used for the detection and localization of alkaline phosphatase activity.
Seven nylon membranes were tested in different DNA transfer and post–transfer procedures. The sensitivity of the hybridization signals was increased ten–fold over conventional Southern blot analysis using nitrocellulose, by UV–irradiating DNA blotted to nylon membranes at high salt concentrations. This treatment also improved the sensitivity of the signals with nitrocellulose membranes. UV–irradiation of DNA transferred to nylon membranes in low salt buffers or alkali reduced hybridization efficiency.
A nonradioactive labelling and detection method for plant genomic DNA analysis is compared to the radioactive method. The radioisotopes are replaced by a nucleotide, digoxigenin-11-dUTP, and the signal detection is accomplished by the enzymatic reaction of alkaline phosphatase, conjugated to anti-digoxigenin antibodies, with the chemiluminescent substrate AMPPD (3-(2-spiroadamantane)-4-methoxy-4(3 phosphorytoxy) phenyl-1, 2-dioxetane). The sensitivity of the radioactive and nonradioactive methods are directly compared using identical Southern blots subjected to the radioactive and nonradioactive detection. The advantages of this nonradioactive method are discussed.
An improved protocol for non-radioactive labelling and detection of rice and tomato DNA is described. The procedure includes
the use of polymerase chain reaction for the incorporation of digoxigenin-dUTP in the DNA molecule and the use of a chemiluminescent
compound (AMPPD) for the signal detection.
Biotin-labelled DNA probes, prepared by nick-translation in the presence of biotinylated analogs of TTP, are hybridized to DNA or RNA immobilized on nitrocellulose filters. After removal of residual probe, the filters are incubated for 2--5 min with a preformed complex made with avidin-DH (or streptavidin) and biotinylated polymers of intestinal alkaline phosphatase. The filters are then incubated with a mixture of 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium, which results in the deposition of a purple precipitate at the sites of hybridization. This procedure will detect target sequences in the 1- to 10-pg range after enzyme incubation periods of 1 hr or less. The incubation period can be extended up to 24 hr, if required, to increase the color intensity of the hybridization signal. Furthermore, at high probe concentrations (250--7560 ng/ml), biotin-labeled DNA exhibits lower nonspecific binding to nitrocellulose than does radiolabeled DNA, so hybridization times required for the analysis of unique mammalian gene sequences can be decreased to 1--2 hr. This nonradiographic method of probe detection should be of general utility for genetic studies using Southern, RNA, or dot-blot hybridization protocols.
This paper describes a method of transferring fragments of DNA from agarose gels to cellulose nitrate filters. The fragments can then be hybridized to radioactive RNA and hybrids detected by radioautography or fluorography. The method is illustrated by analyses of restriction fragments complementary to ribosomal RNAs from Escherichia coli and Xenopus laevis, and from several mammals.
A fast and simple protocol for the chemiluminescent detection of digoxigenin-labeled nucleic acids with anti-digoxigenin antibody Fab fragments coupled to alkaline phosphatase and 3-(4-methoxyspiro[1,2-dioxetane-3,2'-tricyclo-[3.3.1.1 (3,7)]decan]-4- yl)phenyl phosphate as substrate is described. The washing and blocking procedure was optimized to yield low background even on positively charged nylon membranes. The sensitivity of the system is equal or better than radioactive methods. Exposure to x-ray or Polaroid film for up to 30 minutes is sufficient for the detection of 70 femtograms of homologous DNA. Human single-copy genes are detected in Southern blots of as low as 0.3 microgram total placental DNA. Blots can be reprobed multiple times very easily. The advantages of the digoxigenin system are high sensitivity, absence of background and ease of reprobing and are illustrated by applications for single-copy gene detection in genomic blots of human DNA, Northern hybridizations to rare mRNA, detection of E. coli genes on blots of genomic digests after pulse field gel electrophoresis, as well as for nonradioactive DNA sequencing blots with digoxigenin-labeled primers.
A method for measuring the amount of a nonradiolabeled DNA probe using four detection substrates is described. In preliminary experiments, digoxygenin-labeled DNA was bound to neutral, nylon membranes and detected with anti-digoxygenin antibodies conjugated to alkaline phosphatase. Four substrates [4-nitrophenyl phosphate, 4-methylumbelliferyl phosphate, AttoPhos, and adamantyl 1, 2-dioxetane phosphate (AMPPD)] were assessed for use in a quantitative hybridization assay. Only AttoPhos and AMPPD were found to have detection limits in the low picogram range and to respond linearly to DNA concentrations ranging from 0 to 1250 pg. In subsequent experiments, a 200-bp DNA probe cloned from the marine bacterium Pseudomonas perfectomarina 23S rRNA gene was hybridized to P. perfectomarina genomic DNA and total RNA. The amount of hybridized probe was determined using AttoPhos. Finally, a digoxygenin-labeled oligonucleotide was probed against genomic DNA. Linearity with respect to DNA concentration was observed using both the 200-bp fragment and the oligonucleotide as probes with a final target detection limit of 166 fg. This study demonstrates the substrate AttoPhos can be used to quantify the amount of nonradiolabeled probe hybridized to target with sufficient sensitivity for very dilute samples, such as environmental samples.
This chapter describes three closely related strategies for the detection of membrane-immobilized DNA, using enzymatically catalyzed chemiluminescence. Several chemiluminescent systems, including the dioxetane system, have been known for some time and have been further developed. However, none of these systems has been able to find general acceptance for routine use. The reason for this is likely to be found in the failure to adequately meet most of the crucial criteria that are needed to develop sensitive, but robust protocols. These criteria are: (1) high quantum yield of the chemiluminescent reaction, (2) high stability of the involved compounds, (3) long lifetime of the reaction, and (4) low complexity of the reaction. Stabilized, enzyme-triggerable 1,2-dioxetanes 2,3 seem to overcome these problems. The chemical and physical properties of such 1,2-dioxetanes have been described in detail in Ref. 1, and therefore, only the principle of the phosphatase/dioxetane reaction will be discussed in this chapter . The protocols described have been successfully tested on dot blots, southern blots, and DNA sequence blots.
A method which allows discrete nucleic acid sequences to be detected with differently colored hybridization signals on the same blot involving only a single hybridization step is described. Nucleic acid probes labeled with digoxigenin, fluorescein, or biotin are hybridized simultaneously to immobilized target nucleic acids. Differential colorimetric detection is carried out in consecutive alkaline phosphatase-based immunoassays with one of three 3-hydroxy-2-naphthoic acid anilide phosphate/diazonium salt combinations as substrate. Each label is visualized by a different color precipitate (green, red, and blue) directly on the membrane. We demonstrate the use of this method in multicolor plasmid mapping, detection of different genomic sequences on a single Southern blot, discrimination of transcription levels in a Northern blot, and colony screening. Advantages and limitations of the method, as well as further applications, are discussed.
The downward alkaline capillary transfer of DNA and RNA from agarose gel to a hybridization membrane was performed using a transfer solution containing 3 M NaCl and 8 mM NaOH. Under mild alkaline conditions, DNA and RNA were completely eluted from the agarose gel and bound to a hybridization membrane within 1 h. On the basis of this new method of transfer a blotting protocol, downward alkaline blotting, was elaborated. It provides a fast and efficient alternative to commonly used Southern and Northern blotting protocols. The downward alkaline blotting of DNA and RNA can be completed in 2.5 and 1.5 h, respectively, and can be used with both plastic and nitrocellulose membranes. In addition, the downward alkaline blotting protocol allows for a hybridization efficiency of DNA and RNA higher than that of the standard blotting protocols performed at neutral pH.
We describe a technique of rapid (within 1-2 h) transfer of DNA and RNA from agarose gels to nitrocellulose or nylon membrane filters. It is characterized by nearly complete elimination of mechanical action on the gel (a thin layer of liquid is placed over the gel and, filtering through the gel into a stack of paper towels beneath, it transfers nucleic acids onto the filter under the gel). This "descending" transfer, as opposed to the widely used "ascending" Southern transfer, reduces the transfer time (to about 1 h) with equal or higher quality of the hybridization signal. The comparison of transfer kinetics by the both methods shows that (a) the Southern transfer of large size DNA fragments proceeds quicker than it has been thought so far and is almost complete within 4 h; (b) the descending transfer has an advantage over the ascending one in the rate of transfer (1-2 h) and its efficiency; and (c) the time of transfer may become a critical parameter upon using a filter with an apparently low retention capacity (Hybond N, Amersham) that is manifested by a decreased signal at longer than optimal transfer times.
A range of nonradioactive nucleic acid labeling and detection systems have been developed that enable the user to label probes directly with enzyme molecules or indirectly with hapten-derivatized nucleotides. Horseradish peroxidase is used for the direct labeling procedures due to the ease of chemical modification and the relative thermal and chemical stability of this enzyme. Horseradish peroxidase has also been conjugated to a high-specificity antifluorescein antibody for detection of hapten (fluorescein)-labeled hybrids. Enhanced chemiluminescence is a light-emitting process optimized for the detection of low levels of horseradish peroxidase on membrane supports. Results are obtained as hard copy images on x-ray film.
A new chemiluminescent 1,2-dioxetane substrate, CSPD, shows improved performance over AMPPD when used in our nonisotopic method for DNA sequencing. CSPD differs from AMPPD by the addition of a chlorine atom to the adamantyl group that limits the amount of aggregation of the dioxetane and its dephosphorylated anion. This results in a shorter time elapsing before reaching steady state light emission when detecting nucleic acids on nylon membrane. An additional advantage of CSPD over AMPPD is that the resolution of imaged DNA bands does not degrade over time. These features of CSPD permit rapid acquisition of high-quality DNA sequence data.
The problem of reading DNA sequence films has been reformulated using an easily implemented, multiplex version of enzymatic DNA sequencing. By utilizing a uniquely tagged primer for each base-specific sequencing reaction, the four reactions can be pooled and electrophoresed in a single lane. This approach has been previously proposed for use with fluorescently labelled probes (1), and is analogous to the principle used in four-dye fluorescence sequencing except that the signals are resolved following electrophoresis (2). After transfer to a nylon membrane, images are obtained separately for each of the four reactions by hybridization using oligonucleotide probes. The images can then be superimposed to reconstitute a complete sequence pattern. In this way the correction of gel distortion effects and accurate band registration are considerably simplified, as each of the four base-specific ladders require very similar corrections. The methods therefore provide the basis for a second generation of more accurate and reliable film reading programs, as well as being useful for conventional multiplex sequencing. Unlike the original multiplex protocol (3), the approach described is suitable for small projects, as multiple cloning vectors are not used. Although more than one vector can be utilized, only a library of fragments cloned into any single phage, phagemid or plasmid vector is actually required, together with a set of tagged oligonucleotide primers.
DNA probes and synthetic oligonucleotides in general present one of the key tools in modern molecular biology research and increasingly also in commercial applications. Along with the many applications that have been developed for and with DNA probes, faster and more sensitive detection methods are being developed. One of the most promising recent developments presents a method based on enzymatically triggered chemiluminescence. Details of this chemistry along with applications in molecular biology and immunology will be discussed and compared to conventional methods.
A highly sensitive method for detecting specific nucleotide sequences was recently developed. The method uses digoxigenin-labeled nucleic acid probes for hybridization to immobilized target nucleic acids. Probes can be labeled by the random-primed method, nick translation, oligonucleotide tailing, cDNA synthesis, photodigoxigenin or SP6/T7/T3 polymerase-mediated transcription. Hybrids are detected by an enzyme-linked immunoassay using an anti-digoxigenin antibody conjugate. Visualization of the bound antibody is accomplished by an enzymatic color reaction, enzymatic chemiluminescent reaction or immunofluorescence, depending on the antibody conjugate and enzymatic substrate used. Here we report the successful application of this technology in the detection of specific cloned DNA in colony and plaque hybridizations, specific detection of a single mRNA species in Northern blots and single-copy gene detection in genomic Southern blots.
We have coupled a chemiluminescent detection method that uses an alkaline phosphatase label to the genomic DNA sequencing protocol of Church and Gilbert [Church, G. M. & Gilbert, W. (1984) Proc. Natl. Acad. Sci. USA 81, 1991-1995]. Images of sequence ladders are obtained on x-ray film with exposure times of less than 30 min, as compared to 40 h required for a similar exposure with a 32P-labeled oligomer. Chemically cleaved DNA from a sequencing gel is transferred to a nylon membrane, and specific sequence ladders are selected by hybridization to DNA oligonucleotides labeled with alkaline phosphatase or with biotin, leading directly or indirectly to deposition of enzyme. If a biotinylated probe is used, an incubation with avidin-alkaline phosphatase conjugate follows. The membrane is soaked in the chemiluminescent substrate (AMPPD) and is exposed to film. Dephosphorylation of AMPPD leads in a two-step pathway to a highly localized emission of visible light. The demonstrated shorter exposure times may improve the efficiency of a serial reprobing strategy such as the multiplex sequencing approach of Church and Kieffer-Higgins [Church, G. M. & Kieffer-Higgins, S. (1988) Science 240, 185-188].
The use of nucleic acid probes directly labeled with horseradish peroxidase for detection of single copy sequences on Southern blots of human genomic DNA by enhanced chemiluminescence is described. Of the target sequences, 6 x 10(5) molecules (1 amol) have been detected on blue sensitive film using exposures of up to 60 min and probes of 0.3-5.1 kb. The chemiluminescent signal quantified using a cooled charge coupled device (CCD) camera is proportional to probe length for DNA probes in the range 50-3571 bases. The enzyme has no significant effect on the stability of a DNA/DNA hybrid formed with a 3571-base probe and target as determined by increasing the stringency of posthybridization washes by decreasing the concentration of a monovalent cation (NaCl) and by a Tm analysis. The kinetics of DNA hybridization have been analyzed by a cooled CCD camera to provide quantitative data. Ten nanograms per milliliter of probe may be used for an overnight hybridization. Southern blots can be reprobed using a DNA probe for the same or a different sequence without the necessity of stripping off the previously bound probe.
The sensitivity, selectivity, and ease of use of nucleic acid probes and the availability of efficient protocols for the isolation and cloning of specific DNA sequences have led to the development of a wide selection of probes for biomedical and clinical applications. In the basic hybridization protocol, a labeled nucleic acid probe is annealed to a complementary DNA or RNA target sequence, which either is in solution or is immobilized on an inert support. The labeled nucleic acid probe is used to determine the presence or absence of the target sequence in the reaction mixture. An effective nonradioactive detection system should recognize the annealed nucleic acid probe with a degree of precision comparable to that obtained in the primary hybridization reaction. The presence of either a free amine or biotin may also be ascertained by chemical analysis. Compounds containing primary amines give positive color development when spotted on thin layer chromatography (TLC) plates and sprayed with ninhydrin. The first approach uses iminobiotin, an analog of biotin in which the ureido group has been replaced with a guanido group. Iminobiotin binds to avidin with a binding constant that increases with increasing pH and nonprotonated iminobiotin binds efficiently to avidin.
A DNA sequencing system based on the use of a novel set of four chain-terminating dideoxynucleotides, each carrying a different
chemically tuned succinylfluorescein dye distinguished by its fluorescent emission is described. Avian myeloblastosis virus
reverse transcriptase is used in a modified dideoxy DNA sequencing protocol to produce a complete set of fluorescence-tagged
fragments in one reaction mixture. These DNA fragments are resolved by polyacrylamide gel electrophoresis in one sequencing
lane and are identified by a fluorescence detection system specifically matched to the emission characteristics of this dye
set. A scanning system allows multiple samples to be run simultaneously and computer-based automatic base sequence identifications
to be made. The sequence analysis of M13 phage DNA made with this system is described.
We have synthesized and studied two 1,2-dioxetane-based chemiluminescent enzyme substrates: 3-(2′-spiroadamantane)-4-methoxy-4-(3″-phosphoryloxy)phenyl-1,2-dioxetane (AMPPD), and, 3-(2′-spiroadamantane)-4-methoxy-4- (3″-β-D′-galactopyrano-yloxy)phenyl-1,2-dioxetane (AMPGD), which can be activated to chemiluminescence at 470 nm by alkaline phosphatase and βD-galactosidase, respectively. In addition, we observed that certain macromolecules enhance the luminescence of AMPPD. For example, the addition of 0.1% bovine serum albumin amplifies the luminescent signal and improves the detection limit for alkaline phosphatase by approximately one order of magnitude under certain conditions. This effect is due to the presence of a hydrophobic microenvironment provided by the enhancer which ‘stabilizes’ the dephosphorylated AMPPD emitter.
Alkaline phosphatase catalysed chemiluminescence from AMPPD is constant for a prolonged period of time. Using AMPPD we were able to detect 0.01 attomole quantities of alkaline phosphatase immobilized on membrane supports and imaged on photographic film and, in solution, measured in a luminometer.
AMPPD and AMPGD offer alternatives to colorimetric and fluorescent subsrates for alkaline phosphatase and β-D-galactosidase labels used in enzyme immunoassays. The simplicity and sensitivity of this chemiluminescent readout allowed the development of rapid clinical assays (e.g. β-hCG, LH, TSH and others).
Chemiluminescent substrates for alkaline phosphatase and beta-galactosidase are now available for use in immuno-assays and DNA hybridization assays.
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 × 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.
We compared the sensitivity of a chemiluminescent substrate 3-(2'-spiroadamantane)-4-methoxy-4-(3"-phosphoryloxy)phenyl- 1,2-dioxetane (AMPPD) and a chromogenic substrate 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium (BCIP/NBT) for detection of an alkaline phosphatase label in a hepatitis B virus "core antigen" DNA (HBVc) probe hybridization assay. Chemiluminescent signal obtained from AMPPD hydrolysis by alkaline phosphatase was detected with Polaroid Instant Black and White Type 612 film. The chemiluminescent assay detected 1.18 x 10(6) copies of HBVc plasmid DNA in 30 min. By comparison, 9.8 x 10(7) copies of DNA could be measured using chromogenic BCIP/NBT substrate within the same incubation time. After further development, the chemiluminescent endpoint permitted detection of 4.39 x 10(4) copies of HBVc plasmid DNA in 2 h.
Analogs of dUTP and UTP that contain a biotin molecule covalently bound to the C-5 position of the pyrimidine ring through an allylamine linker arm have been synthesized. These biotin-labeled nucleotides are efficient substrates for a variety of DNA and RNA polymerases in vitro. Polynucleotides containing low levels of biotin substitution (50 molecules or fewer per kilobase) have denaturation, reassociation, and hybridization characteristics similar to those of unsubstituted controls. Biotin-labeled polynucleotides, both single and double-stranded, are selectively and quantitatively retained on avidin-Sepharose, even after extensive washing with 8 M urea, 6 M guanidine hydrochloride, or 99% formamide. In addition, biotin-labeled polynucleotides can be selectively immunoprecipitated in the presence of antibiotin antibody and Staphylococcus aureus protein A. The unique features of biotin-labeled polynucleotides suggest that they will be useful affinity probes for the detection and isolation of specific DNA and RNA sequences.
The ability to produce specific streptavidin conjugates has been considerably enhanced by using a streptavidin mutant containing a cysteine stretch, in which sulfhydryl groups serve as unique conjugation sites. A streptavidin molecule containing five cysteine residues at its C-terminus, referred to as Stv-28, was efficiently expressed in Escherichia coli, and purified to homogeneity. Purified Stv-28 had full biotin-binding ability and formed a subunit tetramer. Reactive sulfhydryl groups of Stv-28, derived solely from the cysteine stretch, greatly facilitate the specific conjugation of partner molecules to streptavidin by simple sulfhydryl chemistry. In this manner, S-[14C]carboxymethylated streptavidin and a streptavidin-fluorescein conjugate were prepared. These conjugates contain almost twenty [14C]carboxymethyl groups and fluorescein molecules, respectively, per subunit tetramer, indicating that the sulfhydryl groups of the cysteine stretch are fully reactive. More importantly, these conjugates retain full biotin-binding ability and form subunit tetramers, suggesting that the fundamental properties of streptavidin would be unaffected by the conjugation of other partner molecules to the C-terminal cysteine stretch.
A method for efficient nonradioactive labeling of DNA with biotin using random primer extension has been developed. Under the conditions described, a significant amount of DNA synthesis occurs during incorporation of the nonradioactive label, resulting in amplification of the original template DNA. The effect of primer size, substrate concentration, enzyme concentration, and ratio of biotinylated nucleotide to normal nucleotide on the amount of DNA synthesis was determined. Amplifications of 10- to > 300-fold were attained, depending on the starting template concentration. Template may be varied from 1 to 500 ng per reaction. The size of the resulting biotinylated probes is 100-1000 nucleotides with a significant proportion in the 100-300 nucleotide range. The biotinylated probes were used to detect single-copy genes on Southern blot hybridizations and to identify specific loci in metaphase chromosome spreads by in situ hybridization followed by fluorescent detection with streptavidin-fluorescein isothiocyanate. Random primer amplification and labeling provides a convenient method for preparation of biotinylated probes from small amounts of template DNA.
Chemiluminescent detection techniques provide a sensitive, nonradioactive method for DNA sequencing. Standard Sanger dideoxy DNA sequencing reactions are initiated with biotinylated primers, separated by gel electrophoresis, transferred to nylon membrane and detected utilizing chemiluminescent 1,2-dioxetane substrates for alkaline phosphatase. A multiplex-labeling method was developed to permit detection of several overlapping sets of DNA sequence information on a single membrane, thereby increasing the productivity of a single gel electrophoretic separation. Primers labeled with different haptens at the 5' end were used to perform separate sequencing reactions. These were mixed together prior to electrophoresis, and the individual sequencing products sequentially detected using hapten-specific reagents. We incorporated primers labeled with biotin, digoxigenin, 2,4-dinitrophenyl or fluorescein, each consecutively detected with a hapten-specific alkaline phosphatase conjugate and CSPD 1,2-dioxetane chemiluminescent substrate. To further increase the amount of DNA sequence data that can be obtained from a single membrane, a direct transfer electrophoresis apparatus was used for simultaneous separation of the DNA sequencing reactions and membrane transfer. The resulting increased separation of the high molecular weight fragments yields 350-450 bp of readable DNA sequence data from each template. Chemiluminescent detection of overlapping sets of DNA sequencing reactions utilizing multiplex labeling, combined with direct transfer electrophoresis, provides an efficient, nonradioactive method for DNA sequencing.
We have used M13 single-stranded DNA bound by uv to small pieces of nylon membrane for the synthesis of biotinylated single-stranded DNA probes. The labeling method requires a large fragment of DNA polymerase I and random hexanucleotides. There is no need for previous linearization of the template. The clean probe is removed from the membrane by a single wash step. The synthesized probe is completely free of unincorporated precursors. This makes possible the easy control of the reaction of incorporation of biotinylated analogues into the probe by simple staining on the filter, thus allowing evaluation of the efficiency of labeling. The DNA membrane can be stored for reuse. With the procedure described it is possible to biotinylate many DNA fragments in parallel, simultaneously controlling the efficiency of labeling in a time- and cost-saving manner.
A novel system for the detection of polymerase chain reaction (PCR) products has been developed. The system is based on a PCR in which one of the primers is biotinylated and digoxigenin-11-dUTP is incorporated during elongation. Biotinylated PCR products are captured on streptavidin-coated solid supports, and alkaline phosphatase conjugated to anti-digoxigenin antibody is subsequently bound to the incorporated digoxigenin. The detection may be obtained with colorimetric, fluorescent, or luminescent substrates for alkaline phosphatase. The detection system can be performed in microtiter plates allowing easy handling of multiple samples. The total assay time following the PCR is between 1 and 2 h dependent on the type of substrate and the type of solid support applied in the system. Within this period of time the system is capable of detecting 1 template in 29 cycles of PCR.
Increasing the number of readable nucleotides per sequencing reaction reduces the number of template DNA preparations and sequencing reactions; the sequence assembly from individual reactions can be significantly simplified. DNA sequencing requires high detection sensitivity because a typical band contains about 0.02–1 fmol of DNA. These requirements have been met by radioactive, L2 fluorescent, and enzyme-linked detection systems. Enzyme-linked detection offers advantages for both high throughput and occasional needs. For high throughput, automatic film readers and development automatons can be considerably faster and less costly than fluorescent devices. For occasional sequencing, the higher stability of reagents and sequencing reaction products of nonradioactive systems can be helpful; these advantages can be achieved with significantly lower equipment costs for enzyme-linked detection systems than with fluorescent devices. While the membrane-bound sequence patterns produced by direct transfer electrophoresis can be detected by conventional radioactive labeling, they also offer a starting point for other detection methods that require a membrane-bound pattern, such as genomic sequencing, multiplex sequencing, or sequencing with enzyme-linked detection.
A novel method for quantifying chemiluminescent DNA probes is described. The method uses liquid scintillation counting to measure light emission from the alkaline phosphatase-catalyzed breakdown of the substrate PPD (3-(4-methoxyspiro[1,2-dioxetane-3,2'-tricyclo[3.3.1.1 (3.7[decan]- 4-yl)phenyl phosphate) on dot blot preparations. Serial dilutions of either pUC18 DNA or lambda DNA were hybridized with digoxigenin-labeled probes and detected using the method described. Light flux (luminescence) was linearly related to DNA concentration, typically with a coefficient of determination (r2) of 0.9 or better. Due to the stability of alkaline phosphatase and the long-lived luminescence of PPD in the Lumi-phos formulation, repetitive analyses of a given sample can be made for up to 20 h. The method can reliably detect 17 amol of DNA (30 pg pUC18DNA) with a coefficient of variation on replicate samples of 14%. Optimization experiments showed that 7% sodium dodecyl sulfate in the prehybridization and hybridization buffers resulted in the lowest background; the best combination of signal-to-noise ratio and reproducibility was obtained using Bio-Rad Zeta-Probe GT nylon membranes. Direct immersion of samples into a solution of substrate was found to give the most precise results and ensured that substrate limitation at high concentrations of alkaline phosphatase (i.e., higher DNA amounts) did not occur.
A method for simultaneous nonradioactive labeling and amplification of DNA using random primers
- J Mackey
- A Rashtchian
Direct enzyme coupling to oligonucleotide probes for increased sensitivity and simplified nonradioactive detection
- J Mackey
- N Guan
- A Rashtchian
Chemiluminescent detection of multiple copy genes
- S J Karcher
- B W Goodner
- SJ Karcher
Nonisotopic DNA Probe Techniques
- L J Kricka