Rapid identification of hybridization probes for chromosomal walking

The presence of repeated elements in restriction fragments used as hybridization probes for chromosomal walking poses a major obstacle to the success of this gene-cloning strategy. This report describes a simple and rapid means of identifying restriction fragments devoid of repeated sequences and therefore useful as hybridization probes for chromosomal walking. Restriction fragments derived from a genomic DNA clone are Southern blotted and hybridized to nick-translated total genomic [32P]DNA. Fragments of the genomic clone that contain high abundance sequences (i.e., repeated elements) hybridize strongly to their nick-translated counterparts, which, due to their high copy number, comprise a significant proportion of the total genomic DNA probe. Conversely, fragments containing single-copy or low-abundance sequences do not hybridize, as their nick-translated counterparts are poorly represented in the total genomic DNA probe. These latter fragments, by virtue of their low-abundance sequences, are well suited as probes for chromosomal walking. Ensuring the absence of repeated elements in restriction fragments prior to their purification and utilization as chromosomal walking probes results in marked savings of time, effort and materials.     

Nonradioactive nucleic acid detection by enhanced chemiluminescence using probes directly labeled with horseradish peroxidase

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.     

Array-MAPH: a methodology for the detection of locus copy-number changes in complex genomes

High-throughput genome-wide screening methods to detect subtle genomic imbalances are extremely important for diagnostic genetics and genomics. Here, we provide a detailed protocol for a microarray-based technique, applying the principle of multiplex amplifiable probe hybridization (MAPH). Methodology and software have been developed for designing unique PCR-amplifiable sequences (400-600 bp) covering any genomic region of interest. These sequences are amplified, cloned and spotted onto arrays (targets). A mixture of the same sequences (probes) is hybridized to genomic DNA immobilized on a membrane. Bound probes are recovered and quantitatively amplified by PCR, labeled and hybridized to the array. The procedure can be completed in 4-5 working days, excluding microarray preparation. Unlike array-comparative genomic hybridization (array-CGH), test DNA of specifically reduced complexity is hybridized to an array of identical small amplifiable target sequences, resulting in increased hybridization specificity and higher potential for increasing resolution. Array-MAPH can be used for detection of small-scale copy-number changes in complex genomes, leading to genotype-phenotype correlations and the discovery of new genes.     

Comparison of three nonradioactive and a radioactive DNA probe for the detection of target DNA by DNA hybridization

The specificity and sensitivity of three methods for the preparation and detection of nonradioactive probe DNA (biotin-nick translation, biotin-photolabel, and antigen-chemical linkage) were evaluated and compared with a nick-translated³²P-labeled DNA probe in DNA hybridization studies. The DNA probes were prepared from a restriction fragment (HindIII-3) from bacteriophage P1 DNA, and target DNA consisted of purified phage P1 DNA or P1 prophage DNA in lysogens ofEscherichia coli. A probe concentration of 50 ng/ml resulted in clear detection with the three nonradioactiveHindIII-3 DNA probes, whereas the specificity of the³²P-HindIII-3 DNA probe was satisfactory at a concentration of 25 ng/ml. However, the detection of false positives was greater with the³²P-labeled probe. The sensitivity of the radiolabeled DNA probe was marginally greater than that of the nonradioactive probes in dot blot hybridizations with purified phage P1 DNA. However, when the preparation time, ease of use, safety, duration of storage, and expense were compared for the four methods of labeling, the nonradiolabeled probes were generally superior to the radiolabeled probe.     

Non-radioactive detection of oligonucleotide probes by photochemical amplification of dioxetanes.

We describe a new method of non-radioactive labelling and detection of oligonucleotide probes. The approach is based on a simple chemical principle. Oligonucleotides labelled with methylene blue (a photosensitizer) are hybridized on a membrane to immobilized DNA target sequences. After hybridization and stringency washing 2(-)[3-(hydroxyphenyl)methoxymethylene] adamantane is added to the membrane and the membrane is irradiated with a tungsten lamp light source through a cut-off filter. Thermally stable dioxetanes are amplified during irradiation at the positions of the labelled probe. These amplified dioxetanes are detected using chemically triggered chemiluminescent decay. Signals are recorded on commercial X-ray film. Detection is possible immediately after the last washing step and a hard copy of the blot is obtained within 1 h. Dependent on the level of the target sequences, the sensitivity of the method allows detection of 0.3 pg single-stranded M13mp18(+) plasmid DNA in dot blots and 75 pg in Southern blots. Additional immunological reaction steps and washing steps with blocking reagents and buffers are avoided. Furthermore, expensive reagents and equipment for physical detection are not necessary. The method might be particularly useful for fast routine analysis in forensic and medical applications. The synthesis of the olefin, conditions of hybridization and the protocol of detection are described in detail.     

Southern blot analysis with synthetic oligonucleotides. Application to prostatic protein genes.

1. An ethanol precipitation procedure was developed to purify radiolabeled DNA and oligonucleotide probes to be used in Southern blots. 2. The radiolabeled probes produced strong hybridization signals on a clear background on Southern blot analysis of single gene copies even after 5 days of exposure on X-ray films. 3. An oligonucleotide probe complementary to human glandular kallikrein-1 coding region (amino acids 161-167) detected a single DNA fragment after digestion with Bam H1, Hind III or Pst 1. 4. Another oligonucleotide probe coding for the same region of human prostate-specific antigen detected 3 DNA fragments on Southern blots by contrast to a 1.5 kb full length cDNA probe which detected the presence of only one strong hybridization signal. 5. Oligonucleotide probes appear to be excellent tools for gene mapping. Their sensitivity, specificity and limitations can be compared to the one of monoclonal antibodies used in epitope mapping of proteins.     

An alternative nonradioactive method for labeling DNA using biotin

An alternative nonradioactive labeling method and a highly sensitive technique for detecting specific DNA sequences are described. The labeling method requires the "Klenow" fragment of DNA polymerase I and random hexanucleotides (synthesized or naturally extracted) as a primer for the production of highly sensitive DNA probes. The system has three main steps: (i) labeling of DNA with biotinylated 11-dUTP; (ii) detection of biotinylated DNA by a one-step procedure with streptavidin-alkaline phosphatase complex; (iii) blocking of background with Tween 20. Twenty attograms (2 X 10(-17) g) of pBR322 plasmid DNA was detected by dot-blot hybridization. Upon Southern blot hybridization, 7.4 fg (7.4 X 10(-15) g) of pBR322 HindIII DNA was detected using the biotinylated pBR322 plasmid DNA probe; 40.8 ag and 7.4 fg of lambda HindIII DNA were detected with the biotinylated whole lambda DNA probe by dot and Southern blot hybridization, respectively. Specific bands were also detected with the biotinylated argininosuccinolyase probe upon Northern blotting of mouse poly(A+) RNA. Further applications for in situ hybridization are also described.     

Preparation of fluorescent-labeled DNA and its use as a probe in molecular hybridization

A method of the fluorescent-labeled DNA preparation for visualization of the complementary nucleotide sequences has been developed. Polynucleotide probes were alkylated randomly by 4-(N-methylamino-N-2-chloroethyl)-benzylamine followed by modification with such fluorochromes as dansyl chloride or fluorescein isothiocyanate (FITC). It was found that the FITC but not dansyl-labeled polynucleotides could serve as efficient probes when about 4% of nitrogen bases were modified. The conditions minimizing the loss of the alkylated bases from DNA were determined. The procedure for hybridization with FITC-labeled DNA as a probe is described, concentration of DNA probe being about 4 ng/mm2 of the nitrocellulose filter.     

Sequence organization of feline leukemia virus DNA in infected cells

A restriction site map has been deduced of unintegrated and integrated FeLV viral DNA found in human RD cells after experimental infection with the Gardner-Arnstein strain of FeLV. Restriction fragments were ordered by single and double enzyme digests followed by Southern transfer (1) and hybridization with 32P-labeled viral cDNA probes. The restriction map was oriented with respect to the 5' and 3' ends of viral RNA by using a 3' specific hybridization probe. The major form of unintegrated viral DNA found was a 8.7 kb linear DNA molecule bearing a 450 bp direct long terminal redundancy (LTR) derived from both 5' and 3' viral RNA sequences. Minor, circular forms, 8.7 kb and 8.2 kb in length were also detected, the larger one probably containing two adjacent copies of the LTR and the smaller one containing one comtaining one copy of the LTR. Integrated copies of FeLV are colinear with the unintegrated linear form and contain the KpnI and SmaI sites found in each LTR.     

A rapid non-radioactive procedure for plaque hybridization using biotinylated probes prepared by random primed labeling

A simple, non-radioactive method has been developed for the rapid screening of phage libraries. In the present study, nanogram amounts of a small restriction fragment (135 bp) were biotinylated via random primed labeling and used to probe cDNA libraries using a modified plaque hybridization protocol. The high backgrounds that are seen typically with avidin/biotin-based methods for plaque hybridization were eliminated by incubation of filters with one of several different proteases prior to hybridization. A comparison of several detection systems indicated that streptavidin conjugated to calf intestinal alkaline phosphatase (AP) was the most sensitive, yielding signals comparable to those obtained with 32P-labeled probes. The times required for phage growth and pre-hybridization were reduced substantially, permitting a convenient one-day screening protocol. Nitrocellulose filters gave the best signal to noise ratio, although "streaking" of plaque DNA was observed occasionally; this problem can be overcome by using nylon-based membranes, which allows exact visualization of the positive plaques. The method was highly reliable; 29 out of 33 putative clones retested positive and the authenticity of these was confirmed by DNA sequence analysis. The random primed biotinylation procedure has been utilized successfully with several different cDNA fragments and has proven useful for other hybridization-based methods (Northern and Southern blots), without the problems associated with the use of radiolabeled probes.     

Isolation of Rhizobium loti Strain-Specific DNA Sequences by Subtraction Hybridization

Mixed-phase (heterogeneous) and single-phase (homogeneous) DNA subtraction-hybridization methods were used to isolate specific DNA probes for closely related Rhizobium loti strains. In the heterogeneous method, DNA from the prospective probe strain was repeatedly hybridized to a mixture of DNA from cross-hybridizing strains (subtracter DNA) which was immobilized on an epoxy-activated cellulose matrix. Probe strain sequences which shared homology with the matrix-bound subtracter DNA hybridized to it, leaving unique probe strain sequences in the mobile phase. In the homogeneous method, probe strain sequences were hybridized in solution to biotinylated, mercurated subtracter DNA. Biotinylated, mercurated subtracer DNA and probe strain sequences hybridized to it were removed by two-step affinity chromatography on streptavidin-agarose and thiol-Sepharose. The specificity of the sequences remaining after subtraction hybridization by both methods was assessed and compared by colony hybridization with R. loti strains. Both methods allowed the rapid isolation of strain-specific DNA fragments which were suitable for use as probes.     

Isolation of bovine Y-derived sequence: potential use in embryo sexing.

To obtain bovine Y-derived probes, we have constructed a bovine plasmid library enriched for Y-specific DNA sequences by the deletion enrichment method. The resulting clones were analyzed by hybridization to Southern blots of male and female genomic DNA. From 200 clones tested, two (BC1.2 and BC1.34) were entirely male specific, six gave a male-female differential hybridization pattern, and the remaining reacted similarly with male and female DNA. Interspecies somatic cell hybrid studies and chromosomal in situ hybridization confirmed that the BC1.2 sequence was derived from the Y chromosome. This 54-bp fragment is present at about 2000-2500 copies in the bovine male genome. No polymorphism was revealed with any of the restriction enzymes used, suggesting enzyme site conservation within blocks of repeats. Evolutionary study has shown that the BC1.2 sequence is conserved within Bos and Bison genera and remains male specific. The male specificity and repeated nature of the BC1.2 sequence have enabled us to use it as a molecular probe for sex determination on small numbers of cells by in situ hybridization.     

Chemiluminescent detection of sequential DNA hybridizations to high-density, filter-arrayed cDNA libraries: a subtraction method for novel gene discovery.

A chemiluminescent approach for sequential DNA hybridizations to high-density filter arrays of cDNAs, using a biotin-based random priming method followed by a streptavidin/alkaline phosphatase/CDP-Star detection protocol, is presented. The method has been applied to the Brugia malayi genome project, wherein cDNA libraries, cosmid and bacterial artificial chromosome (BAC) libraries have been gridded at high density onto nylon filters for subsequent analysis by hybridization. Individual probes and pools of rRNA probes, ribosomal protein probes and expressed sequence tag probes show correct specificity and high signal-to-noise ratios even after ten rounds of hybridization, detection, stripping of the probes from the membranes and rehybridization with additional probe sets. This approach provides a subtraction method that leads to a reduction in redundant DNA sequencing, thus increasing the rate of novel gene discovery. The method is also applicable for detecting target sequences, which are present in one or only a few copies per cell; it has proven useful for physical mapping of BAC and cosmid high-density filter arrays, wherein multiple probes have been hybridized at one time (multiplexed) and subsequently "deplexed" into individual components for specific probe localizations.     

Southern blotting

This protocol describes a basic method to perform the Southern blot. Blotting allows the detection of specific molecules among a mixture separated by gel electrophoresis. Molecules are transferred from the gel to a porous membrane by capillary action using absorbent paper to soak solution through the gel and the membrane. For DNA, specific sequences are detected in the membrane by molecular hybridization with labeled nucleic acid probes. The original method, on which this protocol is based, used labeled RNAs to detect specific DNA fragments in genomic DNA that had been digested with restriction endonucleases. This protocol can be completed in 1-5 d and is inexpensive to carry out, as it requires only basic laboratory equipment.     

生物素标记寡核苷酸探针探测扩增的病理性突变珠蛋白基因

用末端转移酶催化生物素核苷酸底物(Biotin-ll-dUTP)共价连接在合成的寡核苷酸3’羟基末端,从而合成了两种寡核苷酸探针(β~T_(41-42)及β~A_(41-42))。用它们分别与克隆化扩增的正常和突变的β—珠蛋白基因片段杂变。结果表明该探针都具有与~(32)P探针相似的特异性,其杂交的灵敏度为2—3pg(特异序列)。进而将探测HbS基因的正常和异常两种寡核苷酸19聚体(β~A_6和β~S_6)用~(32)P和生物素分别标记;将HbS杂合子病人的白细胞DNA经聚合酶链反应(PCR)法扩增,并以含正常β—珠蛋白基因的DNA片段作对照,与两种探针分别进行斑点杂交。所得结果完全一致;Hbs杂合子DNA对正常和异常探针都显出杂交信号,而正常DNA只与β~A探针显杂交信号。     

Pig genome analysis: differential distribution of SINE and LINE sequences is less pronounced than in the human and mouse genomes

The distribution of SINE and LINE sequences in the pig genome was examined by fluorescence in situ hybridization (FISH), interspersed repeat PCR, and restriction analysis of high molecular weight DNA. FISH revealed a largely uniform hybridization to the euchromatic chromosome regions with both interspersed repeats, although a bias toward the G-bands was observed for the LINE probe. Southern blots of inter-SINE and inter-LINE PCR products showed strong hybridization to LINE and SINE probes, respectively. High molecular weight DNA derived from a pig |m~ hamster hybrid cell line was cut with a panel of G + C and A + T rich rare cutter restriction enzymes, then run on a pulsed field gel and Southern blotted. Sequential hybridization with SINE and LINE probes showed that SINE hybridization was to relatively low molecular weight fragments with the G + C rich enzymes, whereas the LINE probe gave hybridization to significantly larger fragments produced by these enzymes. DNA samples digested with A + T rich enzymes gave essentially similar patterns with SINE and LINE probes. We conclude that the pattern of differential distribution of SINEs and LINEs, which has been described in man and mouse, does exist in the pig but is much less pronounced. Received: 25 April 1995 / Accepted: 1 September 1995     

Nonisotopic detection of RNA in an enzyme immunoassay using a monoclonal antibody against DNA-RNA hybrids

A sensitive nonisotopic solution hybridization assay for detection of RNA is described and characterized using a pSP65 plasmid model system. The assay procedure is based on a hybridization reaction in solution between a biotinylated DNA probe and a target RNA. The biotin-labeled hybrids are captured on a microtiter plate coated with an antibody to biotin. Bound DNA-RNA hybrids are detected by an immunoreaction with an enzyme-labeled monoclonal antibody specifically directed against DNA-RNA heteropolymers and the hybrids are quantitatively measured with the addition of a fluorogenic substrate. Optimal conditions under which to perform the assay were hybridization time, 1000 min; temperature, 75 degrees C; probe concentration, 0.2 microgram/ml; extent of probe biotinylation, 6.7%; buffer stringency, 2x SSC. A bisulfite-modified DNA probe was compared to nick-translated probes synthesized with reporter groups of different lengths (bio-11-dUTP or bio-19-dUTP). All probes could detect 10 pg/ml of target RNA. The presence of nonhomologous DNA or RNA sequences reduced the sensitivity of RNA detection by one half-log to 32 pg/ml (1.6 pg/assay).     

Improved hybridization assays employing tailed oligonucleotide probes: a direct comparison with 5'-end-labeled oligonucleotide probes and nick-translated plasmid probes

Terminal deoxynucleotidyl transferase was used to add labeled dAMP residues to the 3' end of oligonucleotide probes that hybridize to the 5' end of the neomycin phosphotransferase II gene. Southern hybridization conditions were described in which the sensitivity per unit of exposure time was about 30-fold greater for the tailed probe as compared to the 5'-end-labeled probe. The tailed oligonucleotide probe had the sensitivity per unit of exposure time comparable to that of a nick-translated probe of high specific activity: in 3 h of autoradiographic exposure both easily detected an amount of target equivalent to a single-copy gene in 10 micrograms of human DNA. The thermal dissociation profiles of 5'-end-labeled and tailed oligonucleotide probes were virtually identical and the tailed oligonucleotide probe was as allele specific as the 5'-end-labeled oligonucleotide probe. The useful lifetime of a 32P-tailed probe was about 1-2 weeks. Finally, by adding 50 35S-labeled nucleotides to the 3' end, we prepared a stable oligonucleotide probe with a sensitivity per unit of exposure time comparable to that of the unstable 5'-32P-labeled oligonucleotide probe.     

Identification of defined sequences in domain V of E. coli 23S rRNA in the 50S subunit accessible for hybridization with complementary oligodeoxyribonucleotides.

The accessibility of specific sequences in domain V of E. coli 23s rRNA in the 50S subunit to complementary oligodeoxyribonucleotides (cDNA) has been investigated. The apparent percentage of subunits engaged in complex formation was determined by incubation of radiolabeled cDNA probe with 50S subunits, followed by nitrocellulose membrane filtration of the reaction mixtures and measurement of the bound radiolabeled cDNA probes by liquid scintillation counting of the filters. The site(s) of hybridization were determined by digestion of the RNA in the RNA/DNA heteroduplex by RNase H. The results of this study indicated that single-stranded sequences, 2058-2062, 2448-2454, 2467-2483, and 2497-2505 were available for hybridization to cDNA probes. Bases 2489-2496, which have been postulated to be base paired with 2455-2461 were also accessible for hybridization.