High resolution melting (HRM), based on melting curve analysis, requires not only saturating dyes that fluoresce in the presence of double-stranded DNA, but also higher resolution detection equipment. The melting curve is a novel method for sequence matching, genotyping and mutation scanning. The technology is simple, accurate, rapid, closed-tube, low-cost, and high-throughput, which make it gain more and more applications. This review article presents the basic principles, key factors and both the advantage and limitations of HRM. The potential application is discussed in the study of molecular identity of traditional Chinese medicine.
DNA Mutational Analysis ; methods ; Drugs, Chinese Herbal ; classification ; Genotyping Techniques ; methods ; Medicine, Chinese Traditional ; Nucleic Acid Denaturation

OBJECTIVETo study the effect of Tiantai No. 1 [symbol in text] on gene expression profile in hippocampus of Alzheimer's disease (AD) rat, molecular genetic target points of the effect of this drug were defined, its molecular genetic pharmacodynamic mechanism of anti-AD was further explored at molecular gene level, and a scientific basis was provided for its clinical availability and promotion.

METHODSThirty male Sprague-Dawley rats were divided into three groups with 10 rats per group: sham-operation group, model group and Tiantai No. 1 group. Sterile surgical procedure was applied, the model group with bilateral hippocampal injection of Aβ1-40 was established, and normal saline was used instead of Aβ1-40 in the sham-operation group. One week after the models was made, rats were administered by gastric lavage once every day for three consecutive weeks. The rats of the sham-operation group and the model group were daily fed with purified water by lavage; the rats of the Tiantai No.1 group treated group were administered with Tiantai No.1 by lavage. Total RNAs of hippocampus tissues were extracted with Trizol, the changes of hippocampus gene expression profiles in the above three groups were analyzed by using Affymetrix rat whole genome expression profile microarray.

RESULTSMicroarray analysis showed that, compared with the sham-operation group, the hippocampus of the model group had 50 up-regulated genes with significant difference (fold change >2), and 21 down-regulated genes with significant difference (fold change <0.5); compared with the hippocampus of the model group, the hippocampus of the Tiantai No. 1 group was found to have 5 up-regulated genes with significant difference (fold change >2) and 20 down-regulated genes with significant difference (fold change <0.5). The functions of differentially expressed genes of the groups were involved in nervous system's development, neuronic differentiation and function-regulation, cellular growth and differentiation and apoptosis, synaptic occurrence and plasticity, inflammation and immune response, ion channels/transporters, cellular signal transduction, cellular material/energy metabolism and so on.

CONCLUSIONTiantai No. 1 can regulate hippocampal function, and further regulate the brain function of animals in multiple gene target points by a number of ways.

Alzheimer Disease ; genetics ; pathology ; Animals ; Body Weight ; drug effects ; Computational Biology ; methods ; Drugs, Chinese Herbal ; pharmacology ; Electrophoresis, Agar Gel ; Gene Expression Profiling ; Gene Expression Regulation ; drug effects ; Hippocampus ; drug effects ; metabolism ; pathology ; Male ; Nucleic Acid Denaturation ; Organ Size ; drug effects ; RNA ; isolation & purification ; metabolism ; Rats, Sprague-Dawley

OBJECTIVETo explore the pharmacological anti-inflammatory mechanism of Chinese formula Qingwen Baidu Decoction (清瘟败毒饮, QBD) from the view of holistic biology.

METHODSThe rats were randomly divided into a normal conrol group, a lipopolysaccharide (LPS) group, the low- and high-dose QBD groups, and a dexamethasone (DXM) group. NR8383 cells were treated with culture fluid containing 6% serum from rats of each group respectively. Inflammatory mediators were detected by reverse transcription polymerase chain reaction (RT-PCR), Western blotting hybridization, enzyme linked immunosorbent assay (ELISA), polymerase chain reaction (PCR) gene array and antibody array.

RESULTSIt is showed that the levels of interleukin (IL)-1α, IL-4 and IL-12 were enhanced in the low-dose QBD group; levels of IL-1α, IL-12 and IL-18 were augmented in the high-dose QBD group, compared with the LPS group after ELISA detection. Western blot showed that IL-1β and tumor necrosis factor (TNF)-α expression of the control group were lower than other groups. IL-1β level of the low-dose and high-dose QBD groups detected by RT-PCR was higher in early stage but lower after 24 h than that of the control group (P<0.01). Expression of 84 main inflammatory cytokines and receptors was detected by rat inflammatory cytokines and receptors PCR array. Up-regulation genes were 22 in both the LPS group and the low-dose QBD group, among which 16 up-regulating genes were the same. In these 16 genes, the up-regulating amplitude of 9 genes in the low-dose QBD group was less than that in the LPS group, 4 were similar to and 3 were more. Twenty-nine main cytokines were inspected by rat cytokine antibody array. Intergroup gray value differences were found in 7 expressed cytokines. The levels of these 7 cytokines in the low-dose QBD group were all lower than those in the the LPS group.

CONCLUSIONSQBD has anti-inflammatory effect on sepsis by changing the level of inflammatory mediators.

Animals ; Anti-Inflammatory Agents ; pharmacology ; therapeutic use ; Blotting, Western ; Cell Line ; Cytokines ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Electrophoresis, Agar Gel ; Inflammation Mediators ; metabolism ; Lung ; drug effects ; metabolism ; pathology ; Male ; Nucleic Acid Denaturation ; Rats, Sprague-Dawley ; Reverse Transcriptase Polymerase Chain Reaction ; Sepsis ; drug therapy ; pathology

OBJECTIVES: The present study was designed to systematically characterize the denaturation and the renaturation of double stranded DNA (dsDNA), which is suitable for DNA hybridization. METHODS: A series of physical and chemical denaturation methods were implemented on well-defined 86-bp dsDNA fragment. The degree of each denaturation was measured and the most suitable denaturation method was determined. DNA renaturation tendency was also investigated for the suggested denaturation method. RESULTS: Heating, beads mill, and sonication bath did not show any denaturation for 30 minutes. However probe sonication fully denatured DNA in 5 minutes. 1 mol/L sodium hydroxide (alkaline treatment) and 60% dimethyl sulfoxide (DMSO) treatment fully denatured DNA in 2-5 minutes. CONCLUSIONS: Among all the physical methods applied, the direct probe sonication was the most effective way to denature the DNA fragments. Among chemical methods, 60% DMSO was the most adequate denaturation method since it does not cause full renaturation during DNA hybridization.
Baths ; Dimethyl Sulfoxide ; DNA* ; Heating ; Hot Temperature ; Nucleic Acid Denaturation ; Sodium Hydroxide ; Sonication

OBJECTIVE: To develop the single-tube one-step methylation variable position (MVP) analysis technology-single-tube post-digestion PCR-melting curve analysis (PDP-MCA). METHODS: Based on differentially methylated region (DMR) reported previously as the model, a set of primers with different melting temperatures of products in the two sides of MVP were designed. By using the FastDigest methylation-sensitive restriction enzyme (MSRE), DNA digestion, multiplex amplification, MCA detection and MCA profiles were performed in a single reaction tube. Same samples (peripheral venous blood, semen, and vaginal fluid, 5 samples each type) were tested by single-tube one step MVP and traditional MSRE-PCR MCA technology. To verify the feasibility of this method, the results were compared with that of the traditional technology. The MCA/HRM profiles of different samples were analyzed and compared. RESULTS: When the melting temperature of the fragments had a differential of 2 degrees C, the MCA melting peaks separated well, and MCA detection after multiplex amplification was successful. The single-tube PDP-MCA assay was developed, which integrated multiple reactions (digestion, amplification and detection) into one tube. By this method, the sample-specific profiles and data were analyzed in 2 h, which is similar to that of the traditional method. The rapid classifications of the samples were also realized. CONCLUSION Multiplex MVPs can be analyzed in a single closed-tube. The single-tube PDP-MCA technology is a simple, fast, and automatable method. It can be used for detection of DNA methylation variations.
DNA/isolation & purification* ; DNA Methylation/genetics* ; DNA Primers/genetics* ; Humans ; Multiplex Polymerase Chain Reaction/standards* ; Nucleic Acid Denaturation

OBJECTIVETo assess the feasibility of high-resolution melting (HRM) analysis for screening patients with neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD).

METHODSBased on previous studies on SLC25A13 gene in Chinese patients with NICCD, four hotspot mutations (851del4, 1638ins23, IVS6+5G>A and IVS16ins3kb) were selected. Results of the HRM analysis was validated using 50 negative controls and 20 patients with NICCD whose genotypes were confirmed previously by direct sequencing. With the established protocol, 171 suspected patients were enrolled. Samples with abnormal melting curves were further validated by DNA sequencing.

RESULTSHRM analysis can accurately determine the genotypes of all negative controls and patients. The sensitivity and specificity of the technique reached 100% (70/70). The melting curves of samples with the same genotype were highly reproducible. In 171 suspected patients, seven NICCD patients were detected by HRM. Identified mutations have included one case of 851del4 homozygote, one case of IVS6+5G>A heterozygote, 3 cases of 851del4 heterozygotes, one case of [IVS6+5G>A]+[ 851del4] and one case of [1638ins23+IVS16ins3kb]+[1638ins23]. All mutations were subsequently confirmed by DNA sequencing.

CONCLUSIONHRM analysis is a convenient, high-throughput and rapid technique for the screening of NICCD patients.

Anion Transport Proteins ; genetics ; Base Sequence ; Calcium-Binding Proteins ; deficiency ; China ; Citrullinemia ; diagnosis ; genetics ; metabolism ; DNA ; chemistry ; genetics ; Genetic Predisposition to Disease ; Genotype ; Humans ; Mitochondrial Proteins ; genetics ; Molecular Sequence Data ; Mutation ; Nucleic Acid Denaturation ; Organic Anion Transporters ; deficiency ; Sensitivity and Specificity

OBJECTIVETo establish a simple, rapid, inexpensive and sensitive method for detecting hot region for mutations in exon 7 of PAH gene.

METHODSHigh-resolution melting (HRM) technology was used to detect a c.728G>A mutation in exon 7 in 88 patients with classical type phenylketonuria. Suspected mutations were validated by direct DNA sequencing.

RESULTSThe results detected by HRM are in good agreement with the results obtained by direct sequencing.

CONCLUSIONHRM analysis is a simple, rapid, inexpensive and sensitive method for detecting hot mutational region in exon 7 of PAH gene.

Base Sequence ; Child ; Child, Preschool ; DNA Mutational Analysis ; methods ; Exons ; Female ; Humans ; Infant ; Infant, Newborn ; Male ; Mutation ; Nucleic Acid Amplification Techniques ; methods ; Nucleic Acid Denaturation ; Phenylalanine Hydroxylase ; genetics ; Phenylketonurias ; diagnosis ; genetics ; Transition Temperature

BACKGROUND: We developed and evaluated the utility of a multiplex real-time PCR assay that uses melting curve analysis and allows simultaneous identification of vancomycin-resistant genotypes and clinically relevant enterococci. METHODS: The specificity of the assay was tested using 4 reference strains of vancomycin-resistant enterococci (VRE) and 2 reference strains of vancomycin-susceptible enterococci. Ninety-three clinical isolates of enterococci with different glycopeptide-resistant phenotypes were genotyped and identified using a multiplex real-time PCR assay and melting curve analysis. RESULTS: Representative melting curves were obtained for Enterococcus faecium, Enterococcus faecalis, vanA-containing E. faecium, vanB-containing E. faecalis, Enterococcus gallinarum, and Enterococcus casseliflavus. Phenotypic and genotypic analysis of the isolates revealed same results for 82 enterococcal isolates, while in 4 isolates, the glycopeptide-resistant phenotypes were inconsistent with the glycopeptide-resistant genotypes and in the 4 other isolates, species could not be accurately identified. Three isolates with mixed strains, which were detected by the PCR assay, could not be correctly identified using phenotypic methods. CONCLUSIONS: VRE genotyping and identification of clinically relevant enterococci were rapidly and correctly performed using multiplex real-time PCR assay and melting curve analysis.
Bacterial Proteins/genetics ; Carbon-Oxygen Ligases/genetics ; DNA, Bacterial/genetics ; Enterococcus/genetics/*isolation &purification ; Enterococcus faecalis/genetics/isolation &purification ; Enterococcus faecium/genetics/isolation &purification ; Genotype ; Nucleic Acid Denaturation ; Peptide Synthases/genetics ; Phenotype ; *Polymerase Chain Reaction ; Vancomycin Resistance/*genetics

OBJECTIVETo establish a quantitative technique for assaying gene methylation in hepatocellular carcinoma (HCC) and evaluate its feasibility for clinical application.

METHODSFollowing bisulfite modification and PCR amplification, the fragments of CDKN2A and ACTB were cloned into plasmids to generate calibration curves using SYBR Green quantitative PCR, and then these two genes were quantitatively analyzed in 41 cases of HCC specimen.

RESULTSThe amplification curve, dissociation curve, calibration curve and electrophoresis analysis showed that SYBR Green fluorescent quantitative PCR could assay 10(2)-10(8) copies/microL of recombinant plasmids with high specificity, high sensitivity and a wide detection range. The tests on 41 cases of HCC specimens further confirmed its feasibility for quantitative analysis of methylation.

CONCLUSIONSYBR Green fluorescent PCR is an easy, fast and high-throughout quantitative tool, and it can be used for methylation analysis in basic research or clinical assay.

Actins ; genetics ; Biopsy ; Calibration ; Carcinoma, Hepatocellular ; genetics ; pathology ; DNA Methylation ; Feasibility Studies ; Female ; Fluorescence ; Genes, p16 ; Humans ; Luminescent Measurements ; Male ; Middle Aged ; Nucleic Acid Denaturation ; Polymerase Chain Reaction ; methods ; Sensitivity and Specificity ; Transition Temperature

OBJECTIVETo detect the most prevalent mutations, R778L and P992L of ATP8B gene, in Chinese Wilson disease(WD) patients by high resolution melting (HRM) analysis after polymerase chain reaction (PCR).

METHODSGenomic DNA was extracted from peripheral blood samples obtained from 30 cases of WD by the standard phenol/chloroform method. DNA fragments encompassing ATP7B exons 8 and 13 were produced by PCR amplification. The amplicons containing the R778L or P992L mutations were then generated by nested PCR. The nested PCR products were subjected to HRM analysis using the HR-1 instrument. Mutations detected in HRM analysis were verified by restriction analysis using restriction enzyme (MspI or AluI or AfaI) or DNA sequencing.

RESULTSHRM analysis of the fragments encompassing ATP7B exon 8 showed four curve patterns. Subsequent restriction analysis and DNA sequencing proved that the four different curves represent four different genotypes: the wild type, the R778L/R778L homozygote, the R778L heterozygote, and the R778L/752.33delG compound heterozygote. Three HRM curve patterns were observed for the fragments encompassing ATP7B exon 13, representing the wild type, the P992L heterozygote, and the P992L/S975Y compound heterozygote. In our studied samples, allele frequencies of the R778L, P992L and S975Y mutations were 25%, 15% and 1.67%, respectively.

CONCLUSIONHRM analysis is a simple, accurate and sensitive approach for rapid detection of the ATP7B mutations and could be used as an optimized method for genetic testing in WD.

Adenosine Triphosphatases ; genetics ; Base Sequence ; Cation Transport Proteins ; genetics ; Copper-transporting ATPases ; DNA ; genetics ; metabolism ; DNA Mutational Analysis ; methods ; DNA Restriction Enzymes ; metabolism ; Exons ; genetics ; Freezing ; Gene Frequency ; Genotype ; Hepatolenticular Degeneration ; genetics ; Humans ; Nucleic Acid Denaturation ; Polymerase Chain Reaction ; Time Factors