Unlabeled Probe Melting Research Articles

High throughput identification of tuna (Thunnus spp.) larvae in the Gulf of Mexico using unlabeled-probe high-resolution melting analysis (UP-HRMA).

The genus Thunnus (family Scombridae) comprises eight species of tunas of which all but one are targeted by industrialized fisheries. While intact individuals of these species can be distinguished by morphological characteristics, researchers and managers often rely on dressed, frozen, juvenile, or larval fish samples which often necessitates molecular species identification. Here we investigate short amplicon (SA) and unlabeled probe (UP) high-resolution melting analysis (HRMA) as a low-cost, high-throughput molecular genotyping assay capable of distinguishing between albacore tuna (T. alalunga), blackfin tuna (T. atlanticus), bigeye tuna (T. obesus), Atlantic bluefin tuna (T. thynnus), and yellowfin tuna (T. albacares) in the Gulf of Mexico. While SA-HRMA of variable regions in the NADH dehydrogenase subunit 4 (ND4) and subunit 5 (ND5), and subunit 6 (ND6) of the mitochondrial DNA (mtDNA) genome did yield some species-specific diagnostic melting curves (ex. ND4 assay can reliably distinguish Atlantic bluefin tuna) genotype masking produced excessive variation in melting curves for reliable multi-species identification. To minimize the genotyping masking of SA-HRMA a 26 base pair long UP containing four SNPs was developed within a 133 bp segment of ND4. The UP-HRMA is able to reliably distinguish Gulf of Mexico species T. thynnus, T. obesus, T. albacares, and T. atlanticus by UP melting temperature at 67°C, 62°C, 59°C, and 57°C respectively. The developed UP-HRMA assay is a lower-cost, higher-throughput, alternative to previously published molecular assays for tuna identification that can be easily automated for large data sets including ichthyological larval surveys, fisheries specimens lacking distinguishing morphological characteristics, or detection of fraudulent trading of tuna species.

Association of JAG1 gene polymorphism with systemic blood pressure in patients with obstructive sleep apnea: a prospective cohort study

AimTo assess the effects of single nucleotide polymorphisms (SNPs) on blood pressure control in patients with obstructive sleep apnea (OSA).MethodsThis prospective observational cohort study, conducted between 2004 and 2014, examined the associations of SNPs of JAG1, GUCY1A3-GUCY1B3, SH2B3, and NPR3-C5orf23 genes with systolic and diastolic blood pressure (SBP, DBP) in 1179 adults evaluated for OSA with overnight polysomnography. Genotyping was performed by unlabeled probe melting analysis.ResultsThe patients were predominantly male (69.6%, mean age 52 ± 11 years, apnea-hypopnea index 34 ± 31 episodes/h). Only JAG1 genotype was associated with SBP and DBP: compared with AA homozygotes, G allele carriers (pooled GG and AG genotype) had significantly higher morning SBP (132 ± 19 vs 129 ± 18 mm Hg; P = 0.009) and morning and evening DBP (85 ± 11 vs 83 ± 10 mm Hg, P = 0.004; 86 ± 10 vs 84 ± 10 mm Hg, P = 0.012, respectively); the differences remained significant after the correction for multiple SNPs testing. In multivariate analyses, oxygen desaturation index and JAG1 genotype independently predicted morning SBP (P = 0.001, P = 0.003, respectively) and DBP (P < 0.001, P = 0.005, respectively), and evening SBP (P = 0.019, P = 0.048, respectively) and DBP (P = 0.018, P = 0.018, respectively).ConclusionThis is the first replication study of the SNPs recently linked to arterial hypertension in general population by genome-wide association studies. Our findings suggest that JAG1 genotype is related to blood pressure control in OSA: G allele was associated with higher morning and evening SBP and DBP.

A portable microfluidic platform for rapid molecular diagnostic testing of patients with myeloproliferative neoplasms

The ability to simultaneously detect JAK2 V617F and MPL W515K/L mutations would substantially improve the early diagnosis of myeloproliferative neoplasms (MPNs) and decrease the risk of arterial thrombosis. The goal of this study is to achieve a point of care testing platform for simultaneous analysis of major genetic alterations in MPN. Here, we report a microfluidic platform including a glass capillary containing polypropylene matrix that extracts genomic DNA from a drop of whole blood, a microchip for simultaneous multi-gene mutation screening, and a handheld battery-powered heating device. The µmLchip system was successfully used for point-of-care identification of the JAK2 V617F and MPL W515K/L mutations. The µmLchip assays were then validated by mutation analysis with samples from 100 MPN patients who had previously been analyzed via unlabeled probe melting curve analysis or real-time PCR. The results from the µmLchip were in perfect agreement with those from the other methods, except for one discrepant result that was negative in the unlabeled probe melting curve analysis but positive in the µmLchip. After T-A cloning, sequences of cloned PCR products revealed JAK2 V617F mutation in the sample. The portable microfluidic platform may be very attractive in developing point-of-care diagnostics for MPL W515K/L and JAK2 V617F mutations.

Genotyping Sugarcane for the Brown Rust Resistance Locus Bru1 Using Unlabeled Probe Melting

Brown rust, caused by the fungus Puccinia melanocephala, is a major disease of sugarcane (Saccharum spp. hybrid) in Florida, Louisiana, and other sugarcane growing regions. The Bru1 locus has been used as a durable and effective source of resistance, and markers are available to select for the trait. The markers currently being used by the USDA Sugarcane Field Station in Canal Point, FL for Bru1 genotyping have two disadvantages. One marker (here Bru1B) is dominant, which means that a Bru1-negative individual cannot be distinguished from a failed PCR reaction. The second marker (here Bru1A) is codominant, but genotyping requires enzyme restriction and gel electrophoresis, adding time and cost to the analysis. A closed-tube, codominant assay for Bru1 would significantly decrease the time and cost currently required for Bru1 genotyping. By sequencing the Bru1A PCR product from Bru1-positive and Bru1-negative individuals, we indentified two SNPs (here Bru1A1 and Bru1A2) that alter the relevant restriction enzyme recognition sequence. An unlabeled probe assay was designed to target each of the SNPs. Unlabeled probe assays are DNA melting assays that rely on the dissociation of an oligonucleotide probe from its target DNA strand. The dissociation is detected by the decrease in fluorescence of double-stranded DNA binding dyes and is sensitive enough to detect single base pair changes. A comparison between the traditional genotyping method and unlabeled probe melting using 344 genotypes showed a 94.7 % success rate, with 100 % concordance between Bru1A and unlabeled probe genotyping. We also confirmed prior unpublished results that discovered a rare recombination between Bru1A and Bru1B. We have demonstrated that unlabeled probe melting can be used to detect the Bru1 locus, eliminates the risk of false negatives, and is faster and less expensive than the current method.

Toward point-of-care testing for JAK2 V617F mutation on a microchip

Molecular genetics now plays a crucial role in diagnosis, the identification of prognostic markers, and monitoring of hematological malignancies. Demonstration of acquired changes such as the JAK2 V617F mutation within myeloproliferative neoplasms (MPN) has quickly moved from a research setting to the diagnostic laboratory. Microfluidics-based assays can reduce the assay time and sample/reagent consumption and enhance the reaction efficiency; however, no current assay has integrated isothermal amplification for point-of-care MPN JAK2 V617F mutation testing with a microchip. In this report, an integrated microchip that performs the whole human blood genomic DNA extraction, loop-mediated isothermal nucleic acid amplification (LAMP) and visual detection for point-of-care genetic mutation testing is demonstrated. This method was validated on DNA from cell lines as well as on whole blood from patients with MPN. The results were compared with those obtained by unlabeled probe melting curve analysis. This chip enjoys a high accuracy, operability, and cost/time efficiency within 1h. All these benefits provide the chip with a potency toward a point-of-care genetic analysis. All samples identified as positive by unlabeled probe melting curve analysis (n=27) proved positive when tested by microchip assay. None of the 30 negative controls gave false positive results. In addition, a patient with polycythemia vera diagnosed as being JAK2 V617F-negative by unlabeled probe melting curve analysis was found to be positive by the microchip. This microchip would possibly be very attractive in developing a point-of-care platform for quick preliminary diagnosis of MPN or other severe illness in resource-limited settings.

Association of XRCC1 and XRCC3 gene haplotypes with the development of radiation-induced fibrosis in patients with nasopharyngeal carcinoma.

Radiation-induced fibrosis is one of the late complications of radiotherapy (RT) for nasopharyngeal carcinoma (NPC). The aim of this study was to investigate the association between X-ray repair cross-complementing protein 1 and 3 (XRCC1 and XRCC3, respectively) gene haplotypes and radiation-induced fibrosis in NPC patients. Genomic DNA was extracted from blood samples of 120 NPC patients previously treated with RT. In total, 12 tag single-nucleotide polymorphisms (SNPs) were selected from the XRCC1 and XRCC3 genes and were genotyped using restriction fragment length polymorphism analysis or unlabeled probe melting analysis. Single-marker and haplotype analyses were performed using multivariate logistic regression analysis. The functional variant rs861539 of XRCC3 may be associated with radiation-induced fibrosis [asymptotic P-value (Pasym)<0.05]. No significant association was observed between radiation-induced fibrosis and any of the tag SNPs of XRCC1 and XRCC3 in either single-marker or haplotype analysis after 10,000 permutations [empirical P-value (Pemp)>0.05]. Our preliminary results indicated that the rs861539 variant of XRCC3 may be associated with an increased risk of radiation-induced fibrosis; however, a large-scale study is required to confirm this result.

Development and Inter-Laboratory Validation of Unlabeled Probe Melting Curve Analysis for Detection of JAK2 V617F Mutation in Polycythemia Vera

BackgroundJAK2 V617F, a somatic point mutation that leads to constitutive JAK2 phosphorylation and kinase activation, has been incorporated into the WHO classification and diagnostic criteria of myeloid neoplasms. Although various approaches such as restriction fragment length polymorphism, amplification refractory mutation system and real-time PCR have been developed for its detection, a generic rapid closed-tube method, which can be utilized on routine genetic testing instruments with stability and cost-efficiency, has not been described.Methodology/Principal FindingsAsymmetric PCR for detection of JAK2 V617F with a 3′-blocked unlabeled probe, saturate dye and subsequent melting curve analysis was performed on a Rotor-Gene® Q real-time cycler to establish the methodology. We compared this method to the existing amplification refractory mutation systems and direct sequencing. Hereafter, the broad applicability of this unlabeled probe melting method was also validated on three diverse real-time systems (Roche LightCycler® 480, Applied Biosystems ABI® 7500 and Eppendorf Mastercycler® ep realplex) in two different laboratories. The unlabeled probe melting analysis could genotype JAK2 V617F mutation explicitly with a 3% mutation load detecting sensitivity. At level of 5% mutation load, the intra- and inter-assay CVs of probe-DNA heteroduplex (mutation/wild type) covered 3.14%/3.55% and 1.72%/1.29% respectively. The method could equally discriminate mutant from wild type samples on the other three real-time instruments.ConclusionsWith a high detecting sensitivity, unlabeled probe melting curve analysis is more applicable to disclose JAK2 V617F mutation than conventional methodologies. Verified with the favorable inter- and intra-assay reproducibility, unlabeled probe melting analysis provided a generic mutation detecting alternative for real-time instruments.

A comparison of restriction fragment length polymorphism, tetra primer amplification refractory mutation system PCR and unlabeled probe melting analysis for LTA + 252 C > T SNP genotyping

BackgroundFrom the wide range of methods currently available for genotyping, we wished to identify a quick, reliable and affordable approach for routine use in our laboratory for LTA+252 C>T SNP screening. MethodsWe set up and compared three genotyping methods for SNP detection: restriction fragment length polymorphism (RFLP), tetra primer amplification refractory mutation system PCR (TPAP) and unlabeled probe melting analysis (UPMA). The SNP model used was LTA+252 C>T, a cytokine gene polymorphism that has been associated with response to treatment in rheumatoid arthritis. The study was performed using 46 samples from healthy Caucasian volunteers. ResultsAllele and genotype distribution was similar to that previously described in the same population. All three genotyping methods showed good reproducibility and are suitable for a medium scale throughput molecular platform. UPMA was the most cost effective, reliable and safe method since it required the shortest technician time, could be performed in a single closed tube and involved automatic data analysis. ConclusionThis work is the first to compare these three genotyping techniques and provides evidence for UPMA being the method of choice for LTA+252 C>T SNP genotyping.

Unlabeled Probes for the Detection and Typing of Herpes Simplex Virus

Unlabeled probe detection with a double-stranded DNA (dsDNA) binding dye is one method to detect and confirm target amplification after PCR. Unlabeled probes and amplicon melting have been used to detect small deletions and single-nucleotide polymorphisms in assays where template is in abundance. Unlabeled probes have not been applied to low-level target detection, however. Herpes simplex virus (HSV) was chosen as a model to compare the unlabeled probe method to an in-house reference assay using dual-labeled, minor groove binding probes. A saturating dsDNA dye (LCGreen Plus) was used for real-time PCR. HSV-1, HSV-2, and an internal control were differentiated by PCR amplicon and unlabeled probe melting analysis after PCR. The unlabeled probe technique displayed 98% concordance with the reference assay for the detection of HSV from a variety of archived clinical samples (n = 182). HSV typing using unlabeled probes was 99% concordant (n = 104) to sequenced clinical samples and allowed for the detection of sequence polymorphisms in the amplicon and under the probe. Unlabeled probes and amplicon melting can be used to detect and genotype as few as 10 copies of target per reaction, restricted only by stochastic limitations. The use of unlabeled probes provides an attractive alternative to conventional fluorescence-labeled, probe-based assays for genotyping and detection of HSV and might be useful for other low-copy targets where typing is informative.

Characterization of Aberrant Melting Peaks in Unlabeled Probe Assays

An unlabeled probe assay relies on a double-stranded DNA-binding dye to detect and verify target based on amplicon and probe melting. During the development and application of unlabeled probe assays, aberrant melting peaks are sometimes observed that may interfere with assay interpretation. In this report, we investigated the origin of aberrant melting profiles observed in an unlabeled probe assay for exon 10 of the RET gene. It was determined that incomplete 3' blocking of the unlabeled probe allowed polymerase-mediated probe extension resulting in extension products that generated the aberrant melting profiles. This report further examined the blocking ability of the 3' modifications C3 spacer, amino-modified C6, phosphate, inverted dT, and single 3' nucleotide mismatches in unlabeled probe experiments. Although no 3' blocking modifications in these experiments were 100% effective, the amino-modified C6, inverted dT, and C3 spacer provided the best blocking efficiencies (1% or less unblocked), phosphate was not as effective of a block (up to 2% unblocked), and single nucleotide mismatches should be avoided as a 3' blocking modification.