One-tube Assay Research Articles

Real-Time Polymerase Chain Reaction MicroRNA Detection Based on Enzymatic Stem-Loop Probes Ligation

MiRNAs (microRNAs) are a group of endogenous, small noncoding RNA with the length of 18-25 nucleotides, which have recently been demonstrated to play important roles in a wide range of biological processes. In this work, we developed a simple, sensitive, specific, and inexpensive assay through the combination of enzymatic probe ligation and real-time PCR amplification for the measurement of mature miRNAs. A couple of novel DNA probes with a stem-loop structure were implemented to reduce nonspecific ligation by at least 100-fold. The assay has several remarkable features including wide dynamic range, low total RNA input (0.02-0.2 ng), distinct anti-interference from precursor miRNAs (signal-to-noise ratio > 500), and single-base mismatch discrimination among miRNA sequences. In addition, a one-tube assay could be accomplished by designing a couple of universal probes, which makes it feasible to examine the expression of a whole family of miRNA (such as let-7) at one time. Finally, we validated the method for quantifying the expression of four mature miRNAs including miR-122, miR-1, miR-34a, and let-7a across 10 mouse tissues, where U6 snRNA could be simultaneously examined as an endogenous control. Thus, this method revealed a great potential for miRNA quantitation in ordinary laboratory studies and clinical diagnoses.

Simultaneous detection of DNA from 10 food allergens by ligation-dependent probe amplification

The simultaneous detection of DNA from different allergenic food ingredients by a ligation-dependent probe amplification (LPA) system is described. The approach allows detection of several targets in a one-tube assay. Synthetic oligonucleotides were designed to detect DNA from peanuts, cashews, pecans, pistachios, hazelnuts, sesame seeds, macadamia nuts, almonds, walnuts and brazil nuts. The specificity of the system was tested with DNA from more than 50 plant and animal species. The sensitivity of the method was suitable to detect allergenic ingredients in the low mg kg−1 range. The limit of detection (LOD) for single allergens in different food matrices was 5 mg kg−1. The novel analytical strategy represents a useful tool for the surveillance of established legislation on food allergens within the European Union.

Easy detection of 5,10-methylenetetrahydrofolate reductase 1298A/C genotype by mutagenically separated PCR assay

5,10-Methylenetetrahydrofolate reductase (MTHFR) plays a central role in folate metabolism. Previous studies have suggested an association between the MTHFR 1298A/C polymorphism and several diseases, such as cardiovascular and psychiatric diseases, neural tube defects, diabetes, and cancer. Currently, either PCR-restriction fragment length polymorphism (RFLP) technique or real-time PCR using Taqman assay are used to determine the MTHFR 1298 genotype. We developed a simple and efficient approach that employs mutagenically separated PCR to genotype MTHFR 1298A/C polymorphism. Two forward mutagenic allele-specific primers of different lengths for MTHFR 1298A/C were paired with the same reverse primer in a one-tube assay to genotype 20 genomic DNA samples. Electrophoresis on 2.5% agarose gel showed two allele-specific fragments, a 113-bp A allele-specific and a 93-bp C allele-specific PCR product. The results were confirmed by the conventional PCR-RFLP method. We conclude that mutagenically separated PCR could be used as an alternative simple, reliable, and cost-effective method for the genotyping of MTHFR 1298A/C polymorphism.

Development of a modular system for detection of genetically modified organisms in food based on ligation-dependent probe amplification

The application of a method based on ligation-dependent probe amplification to the simultaneous detection of different genetically modified organisms in food is described. The ligation reaction of target-specific probes with characteristic lengths and universal primer binding sites is followed by PCR amplification using fluorescein-labeled primers. The separation and the detection of DNA fragments are achieved by capillary electrophoresis via laser-induced fluorescence. The approach allows the simultaneous detection of several targets corresponding to different levels of specificity in a one-tube assay. Synthetic oligonucleotides were designed to detect (1) reference genes in the genome from maize, soya and rapeseed, (2) the CaMV 35S-promotor as screening element, (3) the construct-specific 35S-pat junction, and (4) the event-specific regions of the transgenic maize line MON 810 and of Roundup Ready soya. Specificity and sensitivity (GMO content 0.1%) of the approach were shown for all targets. This novel analytical strategy represents a flexible, modular system for the surveillance of GMO-derived products that can be readily complemented by further target sequences of interest.

Simultaneous analysis of multiple staphylococcal enterotoxin genes by an oligonucleotide microarray assay.

Staphylococcal enterotoxins (SEs) are a family of 17 major serological types of heat-stable enterotoxins that are one of the leading causes of gastroenteritis resulting from consumption of contaminated food. SEs are considered potential bioweapons. Many Staphylococcus aureus isolates contain multiple SEs. Because of the large number of SEs, serological typing and PCR typing are laborious and time-consuming. Furthermore, serological typing may not always be practical because of antigenic similarities among enterotoxins. We report on a microarray-based one-tube assay for the simultaneous detection and identification (genetic typing) of multiple enterotoxin (ent) genes. The proposed typing method is based on PCR amplification of the target region of the ent genes with degenerate primers, followed by characterization of the PCR products by microchip hybridization with oligonucleotide probes specific for each ent gene. We verified the performance of this method by using several other techniques, including PCR amplification with gene-specific primers, followed by gel electrophoresis or microarray hybridization, and sequencing of the enterotoxin genes. The assay was evaluated by analysis of previously characterized staphylococcal isolates containing 16 ent genes. The microarray assay revealed that some of these isolates contained additional previously undetected ent genes. The use of degenerate primers allows the simultaneous amplification and identification of as many as nine different ent genes in one S. aureus strain. The results of this study demonstrate the usefulness of the oligonucleotide microarray assay for the analysis of multitoxigenic strains, which are common among S. aureus strains, and for the analysis of microbial pathogens in general.

Two-color multiplex ligation-dependent probe amplification: Detecting genomic rearrangements in hereditary multiple exostoses

Genomic deletions and duplications play an important role in the etiology of human disease. Versatile tests are required to detect these rearrangements, both in research and diagnostic settings. Multiplex ligation-dependent probe amplification (MLPA) is such a technique, allowing the rapid and precise quantification of up to 40 sequences within a nucleic acid sample using a one-tube assay. Current MLPA probe design, however, involves time-consuming and costly steps for probe generation. To bypass these limitations we set out to use chemically synthesized oligonucleotide probes only. The inherent limitations of this approach are related to oligonucleotide length, and thus the number of probes that can be combined in one assay is also limited. This problem was tackled by designing a two-color assay, combining two sets of probes, each amplified by primers labeled with a different fluorophore. In this way we successfully combined 28 probes in a single reaction. The assay designed was used to screen for the presence of deletions and duplications in patients with hereditary multiple exostoses (HME). Screening 18 patients without detectable point mutations in the EXT1 and EXT2 genes revealed five cases with deletions of one or more exons: four in EXT1 and one in EXT2. Our results show that a two-color MLPA assay using only synthetic oligonucleotides provides an attractive alternative for probe design. The approach is especially suited for cases in which the number of patients to be tested is limited, making it financially unattractive to invest in cloning.

Expression profiling via novel multiplex assay allows rapid assessment of gene regulation in defined signalling pathways.

The current interest in expression of groups of functionally related genes creates a demand for novel experimental tools. We describe a multiplex ligation-dependent amplification procedure (RT-MLPA), which accurately quantifies up to 45 transcripts of interest in a one-tube assay. The output, a set of fluorescent DNA fragments, is analysed via capillary sequencer and spreadsheet software. The procedure is highly sensitive and reproducible over a 100-fold range of input RNA, with excellent compatibility with RT-PCR and microarrays. We targeted two comprehensive sets of human genes: 35 apoptosis regulators and 30 genes involved in inflammation. Both probe sets accurately assessed specific changes in gene expression in two relevant model systems. Stimulation of lymphocytes with various Toll-like receptor (TLR) ligands induced distinct inflammatory profiles. Furthermore, osteosarcoma cells treated with cytostatic drugs showed as primary response strong up-regulation of the apoptogenic p53-inducible PUMA transcript. Suppression by RNAi validated that indeed Puma expression was responsible for apoptosis induction. Thus, RT-MLPA enables relevant changes in transcription patterns to be quickly pinpointed and guide further experiments. This can be an advantage compared to hypothesis-free whole genome screens where large numbers of differentially expressed genes can obscure functional interpretation.

One-Step Detection of c-kit Point Mutations Using Peptide Nucleic Acid-Mediated Polymerase Chain Reaction Clamping and Hybridization Probes

The prognostic significance of somatic activating codon 816 c-kit mutations in pediatric urticaria pigmentosa has not yet been established in detail. Detection of such mutations in archival paraffin-embedded biopsies is usually hampered by an abundance of surrounding normal cells. Here we describe a method for the selective amplification and specific detection of c-kit mutation Asp816-->Val in complete tissue sections cut from up to 24-year-old paraffin blocks. Peptide nucleic acid-mediated polymerase chain reaction clamping of the wild-type allele was combined with on-line mutation detection using oligonucleotide hybridization probes. In DNA extracted from HMC-1 cells heterozygously carrying the c-kit mutation Asp816-->Val, the one-tube assay allowed specific detection of this mutation in a more than 1000-fold excess of normal background DNA within 1 hour and without the need for additional analytical steps. In a series of 38 cases with pediatric urticaria pigmentosa we detected c-kit codons 815 and 816 mutations in 16 cases. Mutation detection did not correlate with clinical outcome after a mean follow-up of 11.2 years. In conclusion, the procedure described may represent an ideal screening tool for all kinds of clinical applications, using point mutations as markers of, for example, early events in carcinogenesis, circulating metastatic tumor cells, and minimal residual disease.

The mutagenically separated polymerase chain reaction is a rapid and reliable method for genotyping of the tumour necrosis factor-α promoter polymorphism (−308 G/A)

Background: The tumour necrosis factor-α (TNFα) promoter polymorphism (−308 G/A) has been shown to be associated with the susceptibility to and/or the severity of diverse diseases such as infections, autoimmunity, and malignancies. We developed a genotyping technique based on the mutagenically separated polymerase chain reaction (MS-PCR) which may be useful in the clinical risk assessment. Methods: Different length allele-specific primers and an unspecific complementary strand primer were used in a one-tube assay. At least one PCR product was generated in a single reaction obviating the need for an internal control amplification. Introduction of additional base substitutions into the allele-specific primers led to a clear-cut separation between the alleles through the reduction of cross-reactions during amplification. The only post-PCR step required was the separation of allelic PCR products by size upon agarose gel electrophoresis. Results: The allele frequencies in 300 German healthy Caucasians were 0.84 for TNF1 (−308 G) and 0.16 for TNF2 (−308 A) in accordance with published data obtained with the conventional RFLP method. No significant deviation from Hardy–Weinberg equilibrium was observed. The specificity of MS-PCR was confirmed by sequence-based typing. Conclusions: MS-PCR is a rapid, reliable, and cost-effective technique for genotyping of the TNFα promoter polymorphism (−308 G/A).

High‐resolution typing for HLA‐DRB1*15 and ‐DRB1*16 by fluorescence‐marked sequence‐specific priming (TaqMan assay)

Sequence-specific primed polymerase chain reaction (PCR-SSP) is widely used in HLA laboratories. The TaqMan method, which is described here for high-resolution typing of HLA-DRB1*15 and -DRB1*16, does not require elaborate and time-consuming post-PCR detection steps. In this one-tube assay, conventional PCR-SSP and fluorescence detection of the amplicon with a doubly labeled fluorescent probe are combined: a fluorogenic hybridization probe (FHP) labeled with a spectral resolvable fluorescent reporter dye (FAM or TET) at its 5' terminus and a common quencher dye (TAMRA) at its 3' terminus is cleaved by the 5' nuclease activity of Taq DNA polymerase only if the target sequence is amplified. An increase of fluorescence intensity indicates a successful amplification. For high-resolution typing of HLA-DRB1*15 and -DRB1*16 alleles we designed two FHPs and 14 specific primer mixes (7 for DR15 and 7 for DR16). Amplification of the specific sequence was detected by a FAM-labeled FHP, whereas amplification of the internal control was detected by a TET-labeled FHP. We were able to type all heterozygous DRB1*15/DRB1*16 subtype combinations. For evaluation, 60 HLA-DRB1*15-positive and 40 HLA-DRB1*16-positive individuals were typed and the results were compared with conventional PCR-SSP DR15/16 subtyping. There were no discrepancies between the two methods. The TaqMan method is an alternative to conventional PCR-SSP typing which is suitable for routine use in HLA laboratories.