Probe-target Complex Research Articles

Blot Overlays with 32P-Labeled Fusion Proteins

Proteins labeled with 32P can be used as sensitive "prime" in blot overlays to detect binding proteins or domains. Small G-protein Ras can bind GTP with extremely high affinity (Kd ∼ 10−11 - 10−12 M) in the presence of Mg2+. We have taken advantage of this property of Ras to develop a vector that expresses proteins of interest such as glutathione S-transferase (GST)/Ras fusion proteins for noncovalent labeling with [γ-32P]GTP. The labeling efficiency of this method is >60% and involves a single short incubation step. We have previously identified several binding proteins for the second SH3 domain of the adaptor Nck using this method. Here we illustrate the overlay method using the GST/Ras system and compare results with the SH3 domain labeled by phosphorylation with [γ-32P]ATP. Both methods are similarly specific and sensitive; however, we show that signals are dependent primarily on GST-mediated probe dimerization. These dimeric probes allow a more stable probe-target complex similar to immunoglobulin interactions, thus significantly improving the sensitivity of the technique.

Direct cloning by covalent attachment of probe DNA to target DNA.

A novel cloning procedure which makes use of covalent attachment of probe DNA to specific target DNA is reported. We show that specific gene fragments found in complex genomes such as the human genome can be cloned directly from a pool of genomic DNA with very high efficiency. This direct cloning method totally eliminates certain steps in current cloning procedures such as construction of DNA libraries and colony (plaque) hybridization. The resulting process has made cloning methods simpler and more time efficient, while achieving high cloning efficiency due to the stable nature of the probe-target DNA complex through covalent bonding. Most importantly, since clones are directly obtained from a pool of genomic DNA, the isolated clones are considered to be faithful copies of the original genes. This has apparently solved the problem of isolating clones with misincorporated bases or chimeric DNA, both of which are often encountered in cloning processes using PCR or other methods involving in vitro DNA synthesis.

Photocleavable aminotag phosphoramidites for 5'-termini DNA/RNA labeling.

We report the design and evaluation of two non-nucleosidic photocleavable aminotag phosphor-amidites. These reagents introduce a photocleavable amino group on the 5'-terminal phosphate of synthetic oligonucleotides. The 5' photocleavable amino group enables introduction of a variety of amine-reactive markers onto synthetic oligonucleotides as well as immobilization on activated solid supports. The photocleavable bond on the 5'-phosphate can then be selectively cleaved by near-UV illumination, thereby enabling release of the marker or detachment of the oligonucleotide from a solid support. The preparation of photocleavable conjugates with biotin, digoxigenin and tetramethylrhodamine are described. In the case of biotin, a conjugate was used in a high sensitivity hybridization assay as a photocleavable probe for a complementary sequence immobilized on beads. It is also demonstrated that the 5'-PC-amino group can be used as an affinity tag for photocleavage-mediated affinity purification and phosphorylation of synthetic oligonucleotides in conjunction with activated supports. Such 5'-PC-amino labeled oligonucleotides should be useful in a variety of applications in molecular biology including multiple non-radioactive probing of DNA/RNA blots, affinity isolation and purification of nucleic acids binding proteins, diagnostic assays requiring release of the probe-target complex or specific marker, cassette mutagenesis and PCR. They will also enable the spatially-addressable photorelease of the probe-target complexes or marker molecules for diagnostic purposes.

RecA-Assisted Rapid Enrichment of Specific Clones from Model DNA Libraries

An approach to library screening is being developed, in which the desired clone is "fished" out of a mixture of all the recombinants in a library with a RecA-coated probe. In the current embodiment of this method, we used as a probe the (+) strand of an M13 phage containing a fragment of the human albumin gene and a (dA)49 stretch. We screened a library of two plasmids, one containing the same albumin fragment as the probe, and one heterologous to the probe in 50-100 fold molar excess. The plasmids were linearized. Probe and library were reacted in the presence of RecA, the mixture was loaded onto an oligo(dT) column, which retained the probe-target complex by base-repairing to the dAs of the probe, the uncaptured plasmids were washed, and the probe-target complex was released from the column, religated and propagated into E. coli. Recovery of the homologous target was 15-28%, and enrichment for the homologous plasmid was 200 to 400-fold. This approach may provide a general method for expedited DNA library screening.

Chemiluminescence dot blot hybridization assay for detection of B19 parvovirus DNA in human sera

A chemiluminescence dot blot hybridization assay was used for the detection of B19 parvovirus DNA in human sera by using digoxigenin-labeled probes. The probes were revealed immunoenzymatically by use of anti-digoxigenin Fab fragments conjugated with alkaline phosphatase. The chemiluminescence signal was obtained by reacting the labeled probe-target complex with an enzyme-triggerable dioxetane substrate. The emitted photons were detected with instant photographic films. In the search for B19 parvovirus DNA, 2,808 serum samples were analyzed.

A Colorimetric DNA Hybridization Method for the Detection of Escherichia coli in Foods

A colorimetric DNA hybridization assay has been developed for the rapid detection of Escherichia coli in foods. The method employs two oligonucleotide probes which are specific for the 16S ribosomal RNA of E. coli. Probes are added to lysates of test cultures and allowed to hybridize to target rRNA if present. The probe-target complex is captured via hybridization to a polystyrene dipstick. The immobilized target is detected using an antibody-horseradish peroxidase conjugate which binds to the immobilized probe-target complex. The probe-target-antibody complex generates a colorimetric signal when exposed to a substrate/chromogen mixture. A total of 233 E. coli isolates representing typical, toxigenic, invasive, hemorrhagic serotype 0157:H7, and other pathogenic strains all resulted in a positive assay signal. Dose-response experiments indicate the sensitivity of the assay is approximately 1 × 106 CFU/ml. Specificity of the assay was determined by testing 207 strains of non-E. coli species at 109 CFU/ml. All of the non-E. coli organisms tested were negative with the exception of Escherichia fergusonii and Shigella species. A total of 345 enriched samples including inoculated, uninoculated, and naturally-contaminated foods was tested for the presence of E. coli by the hybridization assay and a conventional cultural method. The false-negative rate for the hybridization assay was 1.2%. By comparison, the false-negative rate for the culture method in these studies was 23.4%. Based on these data, the DNA hybridization method is significantly more accurate than conventional methods for the detection of E. coli in foods.

A rapid chemiluminescent DNA hybridization assay for the detection of Chlamydia trachomatis

With an estimated 3-4 million new cases per year, human infections from Chlamydia trachomatis are probably the most prevalent sexually transmitted disease (STD) in the United States. Diagnosis of Chlamydia is usually conducted by tissue culture methods. Direct immunofluorescence and ELISA tests have become available, but there remains a need for a test with better specificity and sensitivity. In response to this need, we have developed a rapid DNA hybridization assay using synthetic oligonucleotide probes to detect the presence of the Chlamydia trachomatis specific 7.4 kb plasmid. The assay involves solution phase hybridization of unlabelled probes, rapid capture of the probe-target duplex onto a microtitre dish surface, a new signal amplification technique that employs chemically cross-linked oligonucleotides, and an alkaline phosphatase labelled probe. Signal is obtained by reacting the labelled probe-target complex with an enzyme triggerable dioxetane substrate. Detection of the chemiluminescent output is performed either with a luminometer or by exposure to instant film. All 15 serovars of Chlamydia trachomatis react positively, while organisms known to co-inhabit the human urogenital tract react negatively.

The specificity of pilin DNA sequences for the detection of pathogenic Neisseria

A nucleic acid hybridization assay for the detection of the pilin gene of Neisseria gonorrhoeae has been devised. The method involves solution hybridization of pilin specific synthetic oligonucleotide probes to genomic DNA in crude cell lysates. This is followed by capture of the probe-target complex onto a microtitre dish well, signal amplification and labelling based on horseradish peroxidase conjugated to oligonucleotides. Detection is achieved with a chemiluminescent enzyme substrate. With a detection limit of about 20,000 cells, the 4-h assay is as sensitive as a radioactive dot-blot method. Over 150 strains of Neisseria gonorrhoeae collected from a variety of sources were detected with the assay. Several N. meningitidis serogroups were also found to react positively. No reactivity was observed with non-pathogenic Neisseria spp. or with other known pathogenic or normal microbial inhabitants of the human urogenital tract.

Rapid chemiluminescent nucleic acid assays for detection of TEM-1 beta-lactamase-mediated penicillin resistance in Neisseria gonorrhoeae and other bacteria.

Two new assays for the detection of TEM-1 beta-lactamase-mediated bacterial penicillin resistance were developed that involve the use of specific nucleic acid hybridization. Both techniques are based on a solution-phase hybridization of oligonucleotide probes to the target DNA sequence, solid-phase capture of the probe-target complex, and an amplified chemiluminescent labeling method. One configuration of hybridization probes detected the presence of TEM-1 in Neisseria gonorrhoeae (45 strains), Haemophilus spp., Escherichia coli, Shigella sonnei and Salmonella typhi. A second configuration (TEM-1NH) detected TEM-1 beta-lactamase-mediated penicillin resistance only in N. gonorrhoeae (97 strains) and Haemophilus (6 strains) isolates in which TEM-1 is inserted in a pFA7-type plasmid. Both methods were 100 times more sensitive than a commercially available colorimetric beta-lactamase activity test and approximately 5 times more sensitive than radioisotopic dot blot screening for the gene. The assays are particularly well suited to the analysis of large numbers of samples, can be performed in a total of 4 h, and are sensitive to 10(4) to 10(5) CFU.

Solution hybridization of crosslinkable DNA oligonucleotides to bacteriophage M13 DNA: Effect of secondary structure on hybridization kinetics and equilibria

Several DNA oligonucleotides have been photochemically modified with the furocoumarin 4′-hydroxymethyl-4,5′,8-trimethylpsoralen (HMT) such that each contained a single HMT furan side monoadduct to thymidine at a unique 5′ TpA 3′ sequence. When these oligonucleotides were hybridized to their respective complements, the HMT adduct could be driven to form an interstrand crosslink by irradiation of the hybrid with 360 nm light. The ability to crosslink probe-target complexes has allowed us to determine the kinetics and the extent of hybridization in solution between these oligonucleotides and their complementary sequences in single-stranded bacteriophage M13 DNA. Our data indicate that these parameters are strongly influenced by the existence of local as well as global secondary structure in the viral DNA. During hybridization, rearrangement of this secondary structure so as to expose the target sequence can be rate-limiting. Upon attainment of equilibrium, only a portion of the target sequence may be hybridized to the probe with the remainder involved in intrastrand base-pairing. Using crosslinkable oligonucleotide probes hybridized and irradiated near the melting temperature of the respective probe-target complex one can partially overcome these secondary structure effects.

Reverse Southern hybridization.

A DNA oligomer 25 nucleotides long which contained an HMT (4'-hydroxymethyl-4,5', 8-trimethylpsoralen) furan side monoadduct to thymidine at a 5'-TpA-3' site was used as a probe for the polylinker sequence present in single-stranded M13 mp19 DNA and in double-stranded pUC 19 DNA. Hybridization and photofixation were carried out simultaneously in solution under conditions approximating the melting temperature of the probe-target hybrid. Use of probe concentrations greater than 10(-8) M permitted hybridization times of a few minutes. Irradiation with near ultraviolet light converted the HMT monoadduct present in hybrid complexes into an interstrand crosslink. Efficient photofixation removed hybrid from the equilibrium distribution and resulted in the formation of additional probe-target complex. After removal of excess probe by centrifugation through a semi-permeable membrane (Centricon-30), samples were electrophoresed through an alkaline agarose gel which was analyzed by autoradiography. When using an HMT-modified 25-mer probe end-labeled with 3,000 Ci/mmole 32P, 0.015 ng (3.8 X 10(6) copies) of M13 DNA could be detected. With this same probe 10 micrograms of denatured human DNA (corresponding to 3.0 X 10(6) copies) did not give a signal.