Melting Curve Profiles Research Articles

A Novel Proximal −13914G>A Base Replacement in the Vicinity of the Common-13910T/C Lactase Gene Variation Results in an Atypical LightCycler Melting Curve in Testing with the MutaREAL Lactase Test

The common −13910T/C exchange located 13910-bp upstream of the lactase gene ( LCT ) is associated with the persistence/nonpersistence trait of adult-type hypolactasia, which leads to symptoms of lactose malabsorption, including bloating, flatulence, diarrhea, and abdominal pain. The typing of this variant is used as a genetic test for lactase (non)persistence. We used the MutaReal Lactase real-time PCR test (Immundiagnostik) with the Roche LightCycler capillary system to analyze genomic DNA and observed in 1 sample an aberrant melting curve profile attributable to a novel base replacement, −13914G>A, in the vicinity of the common −13910T/C lactase gene variation. Lactose intolerance results primarily from the physiological decline in activity of the LCT gene-encoded enzyme lactase-phorozin hydrolase, which breaks down milk sugar. Use of linkage disequilibrium and haplotype analysis of affected Finnish families revealed a defined DNA transition (−13910T/C) proximal to the LCT gene in intron 13 of the adjacent minichromosome maintenance type 6 gene ( MCM6 ), which is associated with verified lactase nonpersistence and reduced bone mineral density predisposing for bone fractures (1)(2). Persons homozygous …

Abnormal melt curve profile during prothrombin 20210G → A analysis due to the 20209C → T variant

The common factor II 20210G --> A mutation, located in the 3'-untranslated region, is an important risk factor for the development of thromboembolic disorders, especially in Caucasians. A number of methods are employed for clinical laboratory diagnosis of this mutation, some of which are capable of detecting adjacent 3'-end sequence variations. We present results from an African deep vein thrombosis patient tested for the 20210G --> A mutation by real-time polymerase chain reaction and melt-curve analysis using hybridization probes that incidentally detected an adjacent 3'-untranslated region variant. The patient sample was tested using the Factor II (Prothromobin) G20210A Kit (Roche Diagnostics, Indianapolis, Indiana, USA), in conjunction with the Roche LightCycler. A polymerase chain reaction fragment from the 3'-end of the F2 gene was subsequently sequenced for identification of the variant. Melt-curve analysis revealed a normal 20210*G peak and an unknown aberrant allelic peak. Following sequence analysis, the patient was determined to be heterozygous for 20209C --> T. The presence of the 20209C --> T variant in the current patient and in eight other reported individuals of African descent, most with thrombosis-associated complaints, suggests that this rare variant poses a potential increased risk for thromboembolic disease in this ethnic group.

A cost-effective melting temperature assay for the detection of single-nucleotide polymorphism in the MBL2 gene of HIV-1-infected children

We report a fast (less than 3 h) and cost-effective melting temperature assay method for the detection of single-nucleotide polymorphisms in the MBL2 gene. The protocol, which is based on the Corbett Rotor Gene real time PCR platform and SYBR Green I chemistry, yielded, in the cohorts studied, sensitive (100%) and specific (100%) PCR amplification without the use of costly fluorophore-labeled probes or post-PCR manipulation. At the end of the PCR, the dissociation protocol included a slow heating from 60 degrees to 95 degrees C in 0.2 degrees C steps, with an 8-s interval between steps. Melting curve profiles were obtained using the dissociation software of the Rotor Gene-3000 apparatus. Samples were analyzed in duplicate and in different PCR runs to test the reproducibility of this technique. No supplementary data handling is required to determine the MBL2 genotype. MBL2 genotyping performed on a cohort of 164 HIV-1-positive Brazilian children and 150 healthy controls, matched for age and sex and ethnic origin, yielded reproducible results confirmed by direct sequencing of the amplicon performed in blind. The three MBL2 variants (Arg52Cys, Gly54Asp, Gly57Glu) were grouped together and called allele 0, while the combination of three wild-type alleles was called allele A. The frequency of the A/A homozygotes was significantly higher among healthy controls (0.68) than in HIV-infected children (0.55; P = 0.0234) and the frequency of MBL2 0/0 homozygotes was higher among HIV-1-infected children than healthy controls (P = 0.0296). The 0 allele was significantly more frequent among the 164 HIV-1-infected children (0.29) than among the 150 healthy controls (0.18; P = 0.0032). Our data confirm the association between the presence of the mutated MBL2 allele (allele 0) and HIV-1 infection in perinatally exposed children. Our results are in agreement with the literature data which indicate that the presence of the allele 0 confers a relative risk of 1.37 for HIV-1 infection through vertical transmission.

PolydA and polyrA self-structured by a europium and amino acid complex

Different DNA selectivity was found for the newly synthesized europium– l-valine complex. Unexpected DNA and RNA selection results showed that europium– l-valine complex can cause single-stranded polydA and polyrA to self-structure. The sigmoidal melting curve profiles indicate the transition is cooperative, similar to the cooperative melting of a duplex DNA. This is different from another europium amino acid complex, europium– l-aspartic acid complex which can induce B-Z transition under the low salt condition. To our knowledge, there is no report to show that a metal–amino acid complex can cause the self-structuring of single-stranded DNA and RNA.

Involvement of the Mouse Prp19 Gene in Neuronal/Astroglial Cell Fate Decisions

The molecular mechanisms involved in neuronal/astroglial cell fate decisions during the development of the mammalian central nervous system are poorly understood. Here, we report that PRP19beta, a splice variant of mouse PRP19alpha corresponding to the yeast PRP19 protein, can function as a neuron-astroglial switch during the retinoic acid-primed neural differentiation of P19 cells. The beta-variant possesses an additional 19 amino acid residues in-frame in the N-terminal region of the alpha-variant. The forced expression of the alpha-variant RNA caused the down-regulation of oct-3/4 and nanog mRNA expression during the 12-48 h of the late-early stages of neural differentiation and was sufficient to convert P19 cells into neurons (but not glial cells) when the cells were cultured in aggregated form without retinoic acid. In contrast, the forced expression of the beta-variant RNA suppressed neuronal differentiation and conversely stimulated astroglial cell differentiation in retinoic acid-primed P19 cells. Based on yeast two-hybrid screening, cyclophilin A was identified as a specific binding partner of the beta-variant. Luciferase reporter assay mediated by the oct-3/4 promoter revealed that cyclophilin A could act as a transcriptional activator and that its activity was suppressed by the beta-variant, suggesting that cyclophilin A takes part in the induction of oct-3/4 gene expression, which might lead to neuroectodermal otx2 expression within 12 h of the immediate-early stages of retinoic acid-primed neural differentiation. These results show that the alpha-variant gene plays a pivotal role in neural differentiation and that the beta-variant participates in neuronal/astroglial cell fate decisions.

FRET hybridization probes for the rapid detection of disease resistance alleles in plants: detection of corky root resistance in lettuce

We describe the development of a non-electrophoresis PCR-based assay for allele discrimination at a disease resistance locus. The assay is based on the emission of light by fluorescence resonance energy transfer (FRET) upon annealing of two hybridization probes. The analysis of melting curve profiles of the probes and templates allowed the detection of single nucleotide polymorphisms. The assay was applied to the detection of alleles at the cor locus in lettuce (Lactuca sativa) that confers recessive resistance to corky root disease. Probes and primers for the assay were designed after the characterization of a single nucleotide polymorphism between alleles of PCR products amplified using a linked marker. That polymorphism was validated in a collection of lettuce varieties representing different genetic backgrounds. The FRET hybridization probes approach provided fast and accurate genotyping of breeding material directly in a one-tube reaction. The absence of electrophoresis makes this approach suitable for applications that require automation and high-throughput genotyping analyses such as marker-assisted selection programs.

Quantification of ribozyme target RNA using real-time PCR.

An important part of the ribozyme efficiency-screening process is to have a fast and accurate way to measure steady-state levels of the target RNA. Here, we describe the use of real-time polymerase chain reaction (PCR) for quantification of ribozyme target transcripts. In contrast to classical quantitative PCR, real-time PCR does not require extensive manipulation or generation of relatively complex reagents, thus reducing the risk of contamination. PCR products generated by Taq polymerase in the presence of SYBR Green dye I can be monitored each cycle by collecting fluorescence signals emitted only as the double-stranded DNA is formed. The temperature at which the fluorescent data used for quantification are collected is based on the melting-curve analysis of the amplified product. After constructing a standard curve by plotting the log of the standards' copy number vs their fractional cycle number, the copy number of the unknown samples is automatically determined by interpolation of this curve. However it is very important to validate the melting curve profile with standard gel electrophoresis, particularly while setting up the technique. Real-time PCR is fast and reproducible. Excluding the isolation of RNA and synthesis of cDNA, the results can be obtained in less than 1 h. The coefficient of variance is 15% in the range of 104-106 gene copies.

Homogeneous Amplification and Mutation Scanning of the p53 Gene Using Fluorescent Melting Curves

In malignancy, gene mutations frequently occur in tumor suppressor genes such as p53 and are sporadically located. We describe a homogeneous method for amplification and mutation scanning, and apply the method to the p53 gene. Using a series of overlapping fluorescein-labeled oligonucleotides complementary to a wild-type p53 sequence, we detected somatic mutations in colorectal cancers by aberrant probe:target melting temperatures (T(m)). The probes were designed so that fluorescence decreased on target annealing as a result of deoxyguanosine quenching. Probes were walked along the sequence to be scanned, using two to three probes per cuvette and placing overlapping probes in separate reactions. After amplification, the reaction was cooled to anneal probes and then slowly heated (0.1 degrees C/s) while fluorescence was continuously monitored. Somatic mutations in tumor tissue were detected by changes from a characteristic wild-type melting curve profile using leukocyte DNA. A complete scanning of the DNA binding domain (exons 5-8) of the p53 gene was completed in a single run ( approximately 30 min) starting from genomic leukocyte DNA. To show proof-of-principle, p53 exons 6-8 from 63 colon cancers were probe-scanned and showed 100% agreement with direct sequencing for detecting alterations from wild-type DNA. p53 mutation scanning by single-labeled hybridization probes is a homogeneous, rapid, and sensitive method with application in both research and clinical diagnostics.

Melting Curve SNP (McSNP) Genotyping: a Useful Approach for Diallelic Genotyping in Forensic Science

The increasing availability of Single Nucleotide Polymorphisms (SNPs) and Deletion/Insertion Polymorphisms (DIPs), as well as the outstanding progress in SNP genotyping technologies, will impact forensics profoundly. We have developed a new method for genotyping SNPs and DIPs, which is based on the determination of melting curve profiles of amplified DNA in solution. We have termed this method Melting curve SNP (McSNP) genotyping. Melting curve profiles are composites of the particular melting temperatures (Tm) of the individual fragments that comprise the DNA sample. Simple mixtures of DNA can be resolved in a very robust and efficient fashion, since the samples can be scored in the plates in which they were amplified with no or very few post-PCR manipulations. As such, McSNP is one of the least expensive genotyping methods available and can and should be useful in forensic science.

An unusual melting curve profile in LightCycler multiplex genotyping of the hemochromatosis H63D/C282Y gene mutations

Objectives: Real time polymerase chain reaction followed by melting curve analysis using hybridization probes has become an important tool in routine diagnosis of the HFE mutations, which are associated with hereditary hemochromatosis. Design and methods: We used the LightCycler technology for simultaneous detection of the H63D and C282Y mutations of the HFE gene in patients with a higher prevalence for hemochromatosis. Results: In our cohort we identified two siblings with a variant pattern of the HFE-LightCycler melting profiles preventing allelic discrimination. Conclusions: As a consequence, in these patients DNA sequencing or RFLP analysis is necessary to unequivocally assign the correct HFE genotype.

The use of real-time PCR and fluorogenic probes for rapid and accurate genotyping of newborn mice

Real-time PCR and fluorogenic probes were combined in a simple, rapid and sensitive method to genotype murine breeding stocks and their progeny for a point mutation. DNA from tail biopsies of newborn mice was mixed with amplification primers and fluorogenic hybridization probes in a PCR mixture. The primers were designed to amplify a region of the Fas-Ligand gene including the site for the gld natural point mutation. The fluorogenic hybridization probes overlaid this target sequence and were used to detect amplification of the PCR fragment as well as determine the presence of the point mutation using fluorescence resonance energy transfer (FRET). Both mutated and wild-type forms of the gene fragment were amplified as detected with real-time PCR. Melting curve profiles completed on each amplified sample revealed the genotype for each mouse. These genotypes were confirmed by sequencing the amplified fragments. These results suggest real-time spectrofluorometric PCR techniques incorporating FRET-based hybridization probes may be used for rapid, sensitive, inexpensive and reliable genotyping.

Conservation and chromosomal localization of DNA satellites in balenopterid whales.

DNA satellites were isolated from three balenopterid species, viz. the minke, sei, and fine whales. In each of them at least two DNA satellites were recognizable with buoyant densities in neutral CsCl of rho = 1.702/1.703 and rho = 1.710/1;711, respectively. cRNAs from each satellite group were used for filter and in situ hybridisations. Homo-and heterologous DNA-cRNA hybrids within each satellite group yielded virtually identical melting curve profiles showing conservation of at least a considerable part of the DNA satellite sequences. There was no evident sequence homology between the rho = 1.702/1.703 and the rho = 1.710/1;711 satellites by filter hybridisation.--The in situ hybridisation showed that in each species the rho = 1.702/1.703 satellite was located in centromeric-paracentromeric C-bands in a few pairs, whereas the rho = 1.710/1.711 satellite was located in terminal C-bands throughout the karyotypes.--The data on the whale DNA satellites indicate that the quantitative evolution of the sateliite DNA sequences preceded species divergence of the balenopterids and that the satellite sequences have remained relatively unaltered since the divergence took place. The function of satellite DNA is considered to imply the introduction of both chromosomal and genic polymorphisms and thus being of great importance in speciation, Based upon these concepts a model is postulated for the function of satellite DNA. According to this model at meiotic pairing euchromatinheterochromatin overlapping between homologous chromosomes is considered to be of a general occurrence. This overlapping is presumed to be accentuated by the size heteromorphism frequently observed between homologous heterochromatic segments (C-bands). In the region of such euchromatinheterochromatin overlapping, cross-over would be excluded. The overlapping is suggested to be rectified progresssively in the chromosome arms, leaving unaffected crossing-over distant to the euchromatin-heterochromatin junctions. The consequence of this will be that genes in the proximity of the junctions are collectively inherited and selected, whereas genes distant to the the heterochromatin will be independently assorted and selected.

Template activity of brominated DNA for DNA polymerase

Bromination of DNA affects only guanine and cytosine which are converted into 8bromoguanine (8-BrC) and S-bromocytosine (5.BrC) respectively [I] . If the Br:nucleotide ratio is below 1, the percentage of brominated bases is proportional to the amount of bromine added. The brominated DNA molecules are more flexible than native DNA [2] and electronmicroscope photographs of material brominated to a low degree reveal that they are composed of two species: clusters resembling the denatured DNA and extended chains simulating native molecules [3]. The presence of two distinct species is also revealed by the melting curve profile of partially brominated DNA and these two species can be separated by fractionation on a hydroxylapatite column. DNA isolated from antibiotic-resistant strains of Haemophilus influenzae looses some of its transforming activity after bromination. A similar inactivation is observed in brominated Bacillus subtilis DNA; however, in both cases, a significantly different degree of inactivation is found for various markers [4] . In the course of this study, the template activity of calf thymus DNA, brominated to different extents, was tested with two DNA polymerases: E. coli DNA polymerase, insensitive to the secondary structure of the primer; and regenerating rat liver replicative DNA polymerase requiring preferentially denatured DNA [51. The action of both polymerases is inhibited by the presence of 8BrC and S-BrC in the template DNA and Ear the mammalian enzyme, modifications of the secondary structure of DNA are not responsible for the inhibition. North-Holland Publishing Company Amsterdam 2. Methods

Effect of kinetin on the ecteola cellulose elution profile and other properties of RNA from the excised first seedling leaves of barley

The total RNA isolated by phenol-sodium lauryl sulfate procedure from fresh barley leaves and from excised barley leaves floated for 4 days in the dark on water or on 10 mg per liter kinetin solution was compared regarding its nucleotide composition, melting curve, and elution profile on Ecteola anion exchanger. Only quantitative differences were noted in the Ecteola elution profile of RNA from the three sources. The RNA from all the three sources was also similar in the nucleotide composition and the shape of the melting curve. Quantitative differences in the amount of components (designated from “a” to “m”) of RNA from three sources were noted. The most significant difference was a 50% decrease in the amount of the principal RNA component, “K,” in leaves floated on water. The component “K” was eluted from the Ecteola column with 0.1 N NaOH and was not retarded by chromatography on Sephadex G-200. This component, which is regarded as representative of the major component of high molecular weight RNA, was preserved almost 100% in leaves floated on kinetin solution. It is suggested that kinetin, by stimulating synthesis and suppressing destruction of heavy RNA, may preserve the polyribosomes of the excised leaf and thus maintain protein synthesis, which is essential for the survival of the excised leaf.