Combining ligation reaction and capillary gel electrophoresis to obtain reliable long DNA probes.

New DNA amplification methods are continuously developed for sensitive detection and quantification of specific DNA target sequences for, e.g. clinical, environmental or food applications. These new applications often require the use of long DNA oligonucleotides as probes for target sequences hybridization. Depending on the molecular technique, the length of DNA probes ranges from 40 to 450 nucleotides, solid-phase chemical synthesis being the strategy generally used for their production. However, the fidelity of chemical synthesis of DNA decreases for larger DNA probes. Defects in the oligonucleotide sequence result in the loss of hybridization efficiency, affecting the sensitivity and selectivity of the amplification method. In this work, an enzymatic procedure has been developed as an alternative to solid-phase chemical synthesis for the production of long oligonucleotides. The enzymatic procedure for probe production was based on ligation of short DNA sequences. Long DNA probes were obtained from smaller oligonucleotides together with a short sequence that acts as bridge stabilizing the molecular complex for DNA ligation. The ligation reactions were monitored by capillary gel electrophoresis with laser-induced fluorescence detection (CGE-LIF) using a bare fused-silica capillary. The capillary gel electrophoresis-LIF method demonstrated to be very useful and informative for the characterization of the ligation reaction, providing important information about the nature of some impurities, as well as for the fine optimization of the ligation conditions (i.e. ligation cycles, oligonucleotide and enzyme concentration). As a result, the yield and quality of the ligation product were highly improved. The in-lab prepared DNA probes were used in a novel multiplex ligation-dependent genome amplification (MLGA) method for the detection of genetically modified maize in samples. The great possibilities of the whole approach were demonstrated by the specific and sensitive detection of transgenic maize at percentages lower than 1%.

Automated One-Double-Z Pair BaseScope™ for CircRNA In Situ Hybridization.

Circular RNAs (circRNAs) are single-stranded RNA, typically exons, connected head to tail by back-splicing. The functions of circRNAs include binding of microRNA, regulation of transcription, regulation of alternative splicing, and modulation of immune response. As for other RNA transcripts their levels vary during development and may also become deregulated during disease progression. Different from linear RNAs, the circRNAs are not susceptible to traditional exonuclease activity and therefore more stable in tissues and blood. This makes the circRNAs an attractive new group of potential biomarkers. Specific detection of circRNAs in situ is challenged by the need to discriminate bona fide circRNAs from the linear precursor forms and splice variants that contain largely overlapping sequences. Knowing the sequence around the splice junction site makes the branched DNA probe technology, BaseScope, suitable for selective detection of unique circRNAs. Here, we present the automated application of BaseScope with a one-double-Z pair probe set designed for the junction of circHIPK3.

A real-time Taqman method for hepatitis C virus genotyping and methods for further subtyping of isolates.

The HCV genome is highly heterogeneous; more and more genotypes, each with several distinct subtypes, are being identified around the world. Knowledge of genotype is important for planning of treatment regimes, whereas subtype identification is useful in epidemiological studies and outbreak investigation. We describe HCV genotyping and subtyping assays, based on real-time PCR, that are sensitive, specific, and reliable. These assays provide fast, accurate, and convenient methods for HCV genotyping/subtyping to support clinical practice.

Electrophoretic mobility shift assays for the analysis of DNA-protein interactions.

Electromobility shift assay is a simple, efficient, and rapid method for the study of specific DNA-protein interactions. It relies on the reduction in the electrophoretic mobility conferred to a DNA fragment by an interacting protein. The technique is suitable to qualitative, quantitative, and kinetic analyses. It can also be used to analyze conformational changes.

Anti-counterfeiting DNA molecular tagging of pharmaceutical excipients: An evaluation of lactose containing tablets.

The licensed pharmaceutical industry and regulators use many approaches to control counterfeiting, but it remains a very difficult task to differentiate between counterfeit and real products. Moreover, there is a lack of techniques available for providing a batch specific molecular bar code for tablets that has the required traceability, specificity and sensitivity to be fit for purpose. The aim of this study was to evaluate DNA molecular tags as a potential anti-counterfeiting technology in tablets. Lactose tablets (400 mg) were used as a model to investigate incorporation DNA molecular tag into a solid dosage form: DNA authentication was carried out on an Applied DNA SigNify® qPCR instrument. Tablet batches were subjected to accelerated stability conditions (40 °C and 75% RH) for up to 6 months. All batches passed the monograph specifications of the British Pharmacopoeia (hardness, friability and mass uniformity) throughout the storage period. In all of recovery plots, the number of cycles required for DNA detection (Cq values) increased as a function of storage time, which indicated a reduction in tag levels, but it should be noted for all storage experiments the tag was clearly detected. It would appear that DNA molecular tags could feasibly be applied within the pharmaceutical development cycle when a new solid dosage form is brought to the market so as to mitigate the risk and dangers of counterfeiting.

Fluorescence In Situ Hybridization and Rehybridization Using Bacterial Artificial Chromosome Probes.

Fluorescence in situ hybridization (FISH) method enables in situ genetic analysis of both metaphase and interphase cells from different types of material, including cell lines, cell smears, and fresh and paraffin-embedded tissue. Despite the growing number of commercially available FISH probes, still for large number of gene loci or chromosomal regions commercial probes are not available. Here we describe a simple method for generating FISH probes using bacterial artificial chromosomes (BAC). Due to genome-wide coverage of BAC clones, there are almost unlimited possibilities for the analysis of any genomic regions using BAC FISH probes.

Somatic mutation and transcriptional deregulation of myc in endemic Burkitt's lymphoma disease: heptamer-nonamer recognition mistakes?

We examined the structure and expression of the myc protooncogene in DNA extracted from a primary (uncultured) endemic Burkitt's lymphoma sample designated eBL3. Dot and Northern (RNA) blot analyses demonstrated extreme levels of myc RNA in the eBL3 sample. Nearly complete sequence data of the altered myc locus isolated from eBL3 DNA demonstrated extensive mutations (duplications, insertions, and deletions) in critical myc regulatory regions. Taken together, the data support the idea that myc transcriptional deregulation in Burkitt's lymphoma disease may be a consequence of the position and number of mutations produced within and around the myc locus. Furthermore, the myc exon-1-intron-1 hypermutable PvuII site is part of a potential heptamer-nonamer recognition sequence, suggesting a mechanism for mutation in endemic Burkitt's lymphoma disease.

Identification of an anonymous RFLP DNA probe through multiple arbitrary amplicon profiling and its use for strain differentiation in a field isolate of cellulose-degrading Aspergillus niger.

The genome of Aspergillus niger (MPS-002) was subjected to RAPD fingerprinting using none different random oligonucleotide primers. A 0.7 Kb PCR amplicon, generated by primer-3 could be used as a RFLP probe to differentiate A. niger (ATCC 16880) from A. niger (MPS-002). The probe revealed DNA polymorphism internal to two different EcoRI recognition sequences spaced apart at a distance of 0.4 Kb within a 4.0 Kb EcoRI fragment of the genome of both the strains. Localized genome mapping analysis further revealed that the 0.7 Kb RFLP probe was positioned at a distance of 2.7 Kb and 0.6 Kb from the two ends of a 4.0 Kb EcoRI fragment, respectively within the genome of the two strains of A. niger.

Murine transforming growth factor-beta 2 cDNA sequence and expression in adult tissues and embryos.

Murine transforming growth factor-beta 2 (TGF-beta 2) cDNAs were isolated from cDNA libraries derived from a differentiated murine embryonic carcinoma cell line, PCC3. The composite cDNA sequence is 4267 nucleotides long, including a 1217 nucleotides 5'-untranslated sequence, and encodes a murine TGF-beta 2 precursor of 414 amino acids with 96% identity to its human counterpart. Several consensus polyadenylation sequences are present in the 1807 nucleotides 3'-untranslated sequence. Five TGF-beta 2 mRNA species are observed in the developing mouse fetus and they show different patterns of expression during development. TGF-beta 2 mRNA expression was also examined in adult mouse tissues, in which four of the five RNA species were observed. TGF-beta 2 mRNAs were present in all adult mouse tissues examined, except liver, and was most abundant in placenta, the male submaxillary gland and lung. The patterns of expression suggest a physiological role for TGF-beta 2 both in embryonic development and in the maintenance of adult tissues.

Structure of 3'-downstream segment of the human smooth muscle (aortic-type) alpha-actin-encoding gene and isolation of the specific DNA probe.

We isolated the 3'-downstream part of the human aortic smooth muscle alpha-actin (SM alpha A)-encoding gene and determined the nucleotide sequence, including the ninth (last) exon and 3'-untranslated (UT) region. From the comparison of the human 3'-UT region with rat and chicken 3'-UT regions, its homology is lower than those in 3'-UT regions of other actin isoforms such as cardiac alpha-actin and cytoskeletal beta-actin. Therefore, by using the 3'-UT region of the human SM alpha A gene as an actin isoform-specific probe, this gene was detected as a single copy only in the human genome, which expressed the 1.7-kb RNA transcript in an aortic tissue-specific manner.

The use of a DNA probe for the differentiation of rodent malaria strains and species.

A clone, PCsv4, derived from a partial genomic library of the rodent malaria Plasmodium chabaudi chabaudi AS strain, contains an insert which when used as a probe at low stringency in Southern blotting of genomic DNA from a variety of strains, subspecies and species of rodent malaria parasites, results in a pattern of hybridisation which is specific to each of the DNA samples used, thus providing an accurate method to define a rodent malaria line. The insert only hybridises to DNA derived from malaria parasites of rodent species. The insert also hybridises to a small number of RNA transcripts.

Evaluation of dual-labeled fluorescent DNA probe purity versus performance in real-time PCR.

Real-time PCR technology using dual-labeled fluorescent oligonucleotide probes allows for sensitive, specific, and quantitative determination of mRNA or DNA targets. Historically, dual-labeled probes have been the most expensive reagent in real-time PCR because of the postsynthesis high-performance liquid chromatography (HPLC) and/or gel purification steps required due to limitations in traditional synthesis chemistry. The recent availability of quencher reagents that allow the 3' quencher incorporation as part of the on-machine synthesis has presented the possibility that probes, when carefully synthesized, may be used without extensive postsynthesis purification. This would substantially reduce cost, making the synthesis of dual-labeled fluorescent probes affordable to any DNA synthesis laboratory. The Nucleic Acids Research Group (NARG) of the Association of Biomolecular Resource Facilities (ABRF) (Santa Fe, NM, USA) tested the hypothesis that now any DNA synthesis laboratory is capable of making quality dual-labeled fluorescent probes suitable for real-time PCRs without the need for postsynthesis purification. Members of the DNA synthesis community synthesized dual-labeled human beta-actin probes and submitted them for quality and functional analysis. We found that probes that were at least 20% pure had the same efficiency as those near 100% purity, but the sensitivity of the assay was reduced as the level of purity decreased.

Evaluation of DNA probe removal from nylon membrane.

Genetic fingerprinting is one of the most challenging applications of any hybridization membranes. Forensic DNA fingerprinting typically uses samples in the range of 100-400 ng of genomic DNA. To ensure the ability to successfully reprobe the samples, it is imperative that repeated stripping of sample DNA be minimized while stripping of the probe DNA be maximized. By using standard dilutions of K562 cell line, we compared the following three stripping techniques: NaOH at 25 degrees C, formamide (HCONH2) at 65 degrees C and 0.1 x standard saline citrate and 0.1% sodium dodecyl sulfate at 95 degrees C (high-temperature stripping). The largest amount of genomic DNA was stripped from the membrane with NaOH, with the other two techniques removing less. Formamide and high-temperature procedures resulted in a loss of approximately 5-10 ng of DNA per strip. In contrast, the NaOH resulted in a loss of approximately 10-20 ng per strip.

Generation of vector-insensitive dig-labeled probes from large cosmid library inserts using PCR.

We have devised a general method for producing vector-insensitive probes from clones in which insert DNA (ca. 40 kb) could not be amplified in one piece nor be excised from the vector sequence. The method involves PCR and vector-specific primers in combination with restriction digestion and ligation. It yields specific PCR products that could subsequently be labeled using DIG-11-dUTP in a single-cycle PCR. In colony blot hybridization, the probes were specific for the insert DNA from which the probe was derived and, importantly, did not detect vector sequences.

Identification of salivary Lactobacillus rhamnosus species by DNA profiling and a specific probe.

The Lactobacillus genus has been shown to be associated with the dental carious process, but little is known about the species related to the decay, although Lactobacillus rhamnosus is suspected to be the most implicated species. Conventional identification methods based on biochemical criteria lead to ambiguous results, since the Lactobacillus species found in saliva are phenotypically close. To clarify the role of this genus in the evolution of carious disease, this work aimed to find a rapid and reliable method for identifying the L. rhamnosus species. Methods based on hybridization with DNA probes and DNA amplification by PCR were used. The dominant salivary Lactobacillus species (reference strains from the ATCC) were selected for this purpose as well as some wild strains isolated from children's saliva. DNA profiling using semirandom polymorphic DNA amplification (semi-RAPD) generated specific patterns for L. rhamnosus ATCC 7469. The profiles of all L. rhamnosus strains tested were similar and could be grouped; these strains shared four common fragments. Wild strains first identified with classic methods shared common patterns with the L. rhamnosus species and could be reclassified. One fragment of the profile was purified, cloned, used as a probe and found to be specific to the L. rhamnosus species. These results may help to localize this species within its ecological niche and to elucidate the progression of the carious process.

Rapid DNA extraction and sensitive alkaline blotting protocol: application for detection of gene rearrangement and amplification for clinical molecular diagnosis.

A simple and sensitive DNA extraction/Southern blotting method is described for the routine detection of gene rearrangement and amplification in DNA samples. Significant features of the procedure are: (1) sequential digestion of RNA and protein in the same DNA buffer; and (2) use of a modified alkaline transfer protocol which increases sensitivity and decreases the autoradiographic exposure period. The method lends itself to handling multiple samples containing minimal amounts of starting material, requires equipment commonly found in a clinical laboratory, and yields results within 36 h of specimen receipt. Accordingly, these procedures may find utility in the establishment of molecular diagnostics in a conventional clinical setting.

Isolation of a Salmonella-specific DNA hybridization probe.

A Salmonella-specific DNA fragment of the Salmonella typhimurium LT2 chromosome has been isolated. The fragment (2.3 kilobases (kb)) was used as a probe in a colony hybridization assay, where 185 strains of 93 different Salmonella serovars were correctly identified as belonging to Salmonella. The specificity of the probe was evaluated in colony hybridization assays on pure cultures of non-Salmonella bacteria and on specimens with an indigenous flora. Sixty-three strains of 34 non-Salmonella Gram-negative species did not hybridize to the fragment. By DNA hybridization to faecal samples from calves, pigs and chickens, and samples of animal feed, three samples out of 10 positive by traditional culture methods gave negative results by hybridization, 45 samples were negative in both methods, while one sample was positive only in the hybridization assay. From this sample, Salmonella livingstone was isolated by a replica plate hybridization technique. The probe therefore proved 100% specific for the genus Salmonella. The 2.3 kb fragment may form the basis of hybridization assays for specific detection of Salmonella in food, environmental and clinical specimens.

Use of RAPD-PCR to isolate a species specific DNA probe for Phytophthora cinnamomi.

The products of RAPD-PCR amplification of Phytophthora cinnamomi DNA were separated by electrophoresis in agarose. Parallel Southern blots of the gels were hybridized with nick translated DNA from different species of Phytophthora. Fragments that hybridized specifically to P. cinnamomi DNA were identified. These fragments were purified and cloned into pUC18. Their specificity for P. cinnamomi was confirmed.

Development of mycobacterial species-specific DNA probes by subtraction hybridization.

Subtraction hybridization was used to identify sequences of Mycobacterium bovis DNA which might be of diagnostic value. Genomic DNA from Mycobacterium avium, isolated commonly from cattle and whose tuberculin is used in the comparative intradermal tuberculin test, was subtracted from M. bovis genomic DNA. A novel sequence, of 131 bp, which appears to be Mycobacterium tuberculosis complex-specific was identified. The specificity of this sequence was stringently tested by a probe and polymerase chain reaction (PCR) assay. Nucleotide identity determination and sequence comparisons revealed that the 131-bp sequence is located directly upstream of a potential isocitrate dehydrogenase (IDH) coding gene and may be of diagnostic value, enabling differentiation of M. tuberculosis complex mycobacteria from other mycobacterial species.

Dual Alu polymerase chain reaction primers and conditions for isolation of human chromosome painting probes from hybrid cells.

A method for rapid and efficient production of chromosome- and chromosome-region specific probes for fluorescent in situ hybridization (FISH) detectable by simple fluorescent microscopy is described. The procedure is based on simultaneous use of two inter-Alu-polymerase chain reaction (PCR) primers for extraction of highly heterogeneous human DNA from interspecific somatic cell hybrids containing the chromosome regions of interest. Probes so produced do not hybridize to centromeric sequences and simultaneously band the target chromosomes, making them useful for unambiguous identification of chromosomal elements and breakpoints associated with cancer.