Detection of RHD zygosity in China: using Syber Green Ⅰ real-time polymerase chain reaction based on high-resolution melting curve analysis

Due to relatively higher mutation frequencies in Chinese individuals with the RHD-negative phenotype25% for 1227 G>A RHD elution and 5% for RHD1-RHCE(2-9)-RHD10, Rhesus box analysis is rarely used in China. Here, quantitative real-time polymerase chain reaction (qPCR) with a high-resolution melting curve mode and a matrix mix containing Syber Green Ⅰ were used to sequence specific primers of 1227 G>A and RHD exons 1, 5, and 10 in two families, consisting of two parents and two children per family (n = 8). The samples with RHD gene dele- tion homozygous/heterozygous, 1227 G>A heterozygous with RHD gene deletion and normal RHD, normal RHD homozygous/heterozygous, and RHD1-RHCE(2-9)-RHD10 homozygous/heterozygous status were all included. All samples were screened using RHD exon genotyping, Sanger sequencing, and Rhesus box analysis. DNA sample quality was maintained at 68~72 ng/μL, and OD_260/280 at 1.7~1.9. The Tm ratio of RHD exon 1 (87 ℃ ) to internal control (77℃ ) was 2.49~2.67 and 2.09~2.35 in subjects with RHD exon 1 homozygous and heterozygous, respectively; the Tm ratio of RHD exon 10 (81℃ ) to internal control (77℃ ) was 5.01~6.11 and 3.34~4.31 in subjects with RHD exon 10 homozygous and heterozygous, respectively; the Tm ratio of RHD exon 5 (83℃ ) to internal control (77℃ ) was 3.98~4.75, 3.02~3.45, and 0.03 in subjects with RHD exon 5 homozygous, heterozygous, and deletion homozygous, respectively; the Tm ratio of 1227A (87℃ ) to internal control (77℃ ) was 1.11, 0.51, and <0.03 in subjects with 1227A heterozygous, 1227A homozygous (exon 9 deletion), and wild type, respectively. The results suggest that using the primers of Tm ratio in comparison with an internal control is an effective way to detect RHD gene deletion or RHD-RHCE hybrid variant allele carrier. The method can also be used to calculate the mother-newborn RHD phenotype proportion and assist pedigree analysis.     

Molecular basis of β-thalassemia in Thailand: analysis of β-thalassemia mutations using the polymerase chain reaction

Summary -Thalassemia mutations in 71 chromosomes of Thai patients from the northeast, the middle and the south of the country were investigated using dot blot hybridization of PCR (polymerase chain reaction)-amplified DNA with allelespecific oligonucleotide probes. Eight different known molecular defects were detected, at different frequencies. There was an amber mutation in codon 17, a C-T transversion at position 654 of IVS-2, a frameshift mutation between codons 71 and 72, an A-G transition at nucleotide -28 within the TATA box (known as Chinese mutations), a G-T transversion at position 1 of IVS-1 (an Indian mutation), a 4bp deletion in codons 41/42 and a G-C transversion at position 5 of IVS-1 (described as both Chinese and Indian mutations) and a Thai original mutation, an ochre mutation in codon 35. Analysis of the three unknown alleles by DNA sequencing of the cloned DNA fragment amplified by PCR revealed an A-G substitution at the second position of the codon for amino acid 19 (AAC-AGC). The analytic approach used in the present study and the characteristic distribution of mutations in each region of Thailand will prove useful for setting up a prenatal diagnosis program.     

A DNA marker assay based on high-resolution melting curve analysis for distinguishing species of the Festuca–Lolium complex

The grass breeding industry is interested in a fast and cheap method of identifying contamination in seeds of Italian and perennial ryegrass (Lolium perenne L. and L. multiflorum Lam., respectively). This study shows that high-resolution melting curve analysis in combination with an unlabelled probe assay is an effective method of detecting single nucleotide polymorphisms (SNPs) in diverse Italian and perennial ryegrass backgrounds. This method proved efficient in differentiating ryegrass species and reducing the effect of additional DNA sequence polymorphisms close to the target SNP on the melting curve profiles. For the identification of contamination in Italian and perennial ryegrass seed production, high-resolution melting curve analysis shows great potential, as it is a single closed-tube PCR reaction with an easy workflow, providing results in <2 h after DNA extraction.     

Use of extremely short F?rster resonance energy transfer probes in real-time polymerase chain reaction

Described in the article is a new approach for the sequence-specific detection of nucleic acids in real-time polymerase chain reaction (PCR) using fluorescently labeled oligonucleotide probes. The method is based on the production of PCR amplicons, which fold into dumbbell-like secondary structures carrying a specially designed ‘probe-luring’ sequence at their 5′ ends. Hybridization of this sequence to a complementary ‘anchoring’ tail introduced at the 3′ end of a fluorescent probe enables the probe to bind to its target during PCR, and the subsequent probe cleavage results in the florescence signal. As it has been shown in the study, this amplicon-endorsed and guided formation of the probe-target duplex allows the use of extremely short oligonucleotide probes, up to tetranucleotides in length. In particular, the short length of the fluorescent probes makes possible the development of a ‘universal’ probe inventory that is relatively small in size but represents all possible sequence variations. The unparalleled cost-effectiveness of the inventory approach is discussed. Despite the short length of the probes, this new method, named Angler real-time PCR, remains highly sequence specific, and the results of the study indicate that it can be effectively used for quantitative PCR and the detection of polymorphic variations.     

Preimplantation genetic diagnosis of β-thalassemia using real-time polymerase chain reaction with fluorescence resonance energy transfer hybridization probes

Preimplantation genetic diagnosis (PGD) is employed increasingly to allow transfer of embryos to the uterus in assisted reproduction procedures. There are three stages of biopsy: polar bodies, one or two blastomeres from the cleavage-stage embryos, and trophectoderm cells (∼5 cells) from the blastocyst-stage embryos. Validation of polymerase chain reaction (PCR)-based assays are challenging because only limited genetic material can be obtained for PGD. In the current study, we modified a valid single-cell PCR protocol for PGD using real-time PCR assay with fluorescence resonance energy transfer (FRET) hybridization probes followed by melting curve analysis. We optimized and clinically applied the protocol, permitting molecular genetic analysis to amplify a specific region on the beta-globin (HBB) gene for a couple, carriers of two mutations: c.-78A>G and c.52A>T. Among a total of eight embryos obtained after ovarian stimulation, a single blastomere per embryo at the six- to eight-cell stage was biopsied. This PGD method showed that four embryos were unaffected, two embryos were selected for transfer, and one pregnancy was achieved. Finally, a healthy male baby was delivered at 38 weeks’ gestation. The results obtained using the new method, FRET hybridization probes, were compared with findings using an existing method, primer extension minisequencing.     

Haplotyping using a combination of polymerase chain reaction–single-strand conformational polymorphism analysis and haplotype-specific PCR amplification

A single nucleotide polymorphism (SNP) may have an impact on phenotype, but it may also be influenced by multiple SNPs within a gene; hence, the haplotype or phase of multiple SNPs needs to be known. Various methods for haplotyping SNPs have been proposed, but a simple and cost-effective method is currently unavailable. Here we describe a haplotyping approach using two simple techniques: polymerase chain reaction–single-strand conformational polymorphism (PCR–SSCP) and haplotype-specific PCR. In this approach, individual regions of a gene are analyzed by PCR–SSCP to identify variation that defines sub-haplotypes, and then extended haplotypes are assembled from the sub-haplotypes either directly or with the additional use of haplotype-specific PCR amplification. We demonstrate the utility of this approach by haplotyping ovine FABP4 across two variable regions that contain seven SNPs and one indel. The simplicity of this approach makes it suitable for large-scale studies and/or diagnostic screening.     

A quantitative assay to detect α-thalassemia deletions and triplications using multiplex nested real-time quantitative polymerase chain reaction

Increasing evidence indicates that copy number variants (CNVs) have great relevance to common human diseases. In α-thalassemia, clinical phenotypes are related to genotypes, specifically copy number changes in the human α-globin gene cluster. Assays are available for high-throughput screening of unknown CNVs genome-wide and also for targeted CNV genotyping at loci associated with genetic disorders. Here we describe a universal quantitative approach based on nested real-time quantitative polymerase chain reaction for accurate determination of copy numbers at multiple particular gene loci. We used the α-globin gene as a model system, obtaining the reproducibility and sensitivity to analyze different gene copies and testing 95 DNA samples with 16 different known genotypes. Our results showed that this approach has high sensitivity and low standard deviations for correctly genotyping DNA samples containing different copy numbers of the α1 and α2 globin genes. Our method is rapid, simple, and reliable, and it could be used to simultaneously screen for α-thalassemia deletions or triplications. Moreover, it has potential as a versatile technology for the rapid genotyping of known CNVs in a targeted region.     

The influence of drinking-water pollution with heavy metal on the expression of IL-4 and IFN-γ in mice by real-time polymerase chain reaction

In recent years, water pollution has been converted to a challenging discussion in health area of human being. Heavy elements are one of the most important water pollutants and their negative adverse effects on body systems have been confirmed. In this study, investigation of effects of two heavy elements including lead (Pb) and copper (Cu) on expression of interlukin-4 (IL-4) and interferon-gamma (IFN-γ) as humoral and cellular immunity biomarkers, respectively, was aimed and PCR, real-time PCR and electrophoresis techniques were used. In this study, BALB/c mice were studied that had free access to drinking water which contained Cu or Pb salts. After 2 weeks, spleens of mice were removed, RNA extracted, and cDNA was prepared for RT-PCR. Then the expression of IL-4 and IFN-γ genes were assessed by real-time PCR. The expression of IFN-γ was up-regulated in both treated groups and the expression of IL-4 was only up-regulated in the group treated with Cu and down-regulated in the group treated with Pb. This study shows that the presence of heavy elements as drinking-water pollutants results in a disproportion of natural cytokines balances, and thus may result in a negative effect on immune system.     

Detection of Leishmania braziliensis in human paraffin-embedded tissues from Tucumán, Argentina by polymerase chain reaction

American cutaneous leishmaniasis (ACL) is an endemic disease in Northern Argentina. We applied the polymerase chain reaction (PCR) followed by a hybridization labelled probe to 21 paraffin embedded human skin biopsies, already analyzed histologically, from leishmaniasis endemic areas in the province of Tucumán, Argentina. We used primers previously designed to detect a Leishmania-specific 120-base-pair fragment of kinetoplast DNA minicircle, other two primer pairs that amplify kDNA minicircles belonging to the L. braziliensis and L. mexicana complexes respectively, and specific oligonucleotide primers to detect L. (V.) braziliensis which amplify the sequence of the ribosomal protein L-14 of this species. The PCR-hybridization showed a sensitivity of 90.5% when compared to the histopathology test which was 61.9%. Five of the total samples analyzed were positive for the L. braziliensis complex whilst none was positive for the L. mexicana complex. The specific primers for L. (V.) braziliensis detected the parasite in four samples. These results are consistent with those reported for close endemic areas and demonstrate that the causative agent of human leishmaniasis in the analyzed cases was L. (V.) braziliensis. PCR should be used as a diagnostic tool for tegumentary leishmaniasis, especially in the mucosal form, and as a valuable technique for the identification of the Leishmania species that causes the disease in certain areas.     

Detection by the polymerase chain reaction of two polymorphisms in exon 14 of the human inter-α-trypsin inhibitor heavy chain H1 gene

Two polymorphisms were detected within exon 14 of the inter-alpha-trypsin inhibitor heavy chain H1 (ITIH1) gene. The polymorphisms are detected by digesting the same 202-bp polymerase reaction product with the PstI and HphI restriction endonucleases. These gene polymorphisms lead to the change of two amino acids in the mature protein. The polymorphisms can be used for the analysis of 3p21 deletion in human carcinomas as well as to develop a better understanding of the protein polymorphism already described.     

Detection of Brazilian spotted fever infection by polymerase chain reaction in a patient from the state of São Paulo

Brazilian spotted fever (BSF) cases have been increasing in the state of S?o Paulo but no genomic information about local rickettsia isolated from humans has been well documented. We recovered spotted-fever group rickettsiae from a sample of patient blood cultured in Vero cells using the shell vial technique. Rickettsial DNA fragments (gltA, ompA, and, ompB genes) were detected, and analysis of the ompB gene base sequences showed identity with the Rickettsia rickettsii ompB sequence available in the GenBank.     

Chromosomal assignment and linkage analysis of the human glutathione S-transferase μ gene (GSTM1) using intron specific polymerase chain reaction

Oligonucleotide primers specific for intron 5 sequences were used to amplify a unique 718 bp fragment in the human GST gene. Using DNA from a panel of somatic cell hybrids it was possible to confirm the assignment of the GST1 locus to chromosome 1p and to refine localisation to 1p13 using Southern blot analysis of DNA from three-generation CEPH families and a GST specific DNA probe.     

Mitochondrial DNA mutations in Japanese detected by polymerase chain reaction—restriction fragment—single strand conformation polymorphism analysis

PCR—restriction fragment—SSCP (PCR—RF—SSCP) analysis of mitochondrial DNA by HhaI/HincII in Japanese revealed 46 polymorphic patterns. The determinations of nucleotide sequence of these 46 patterns revealed 56 mutations compared with the Cambridge Sequence.     

Easy and rapid method of zygosity determination in transgenic mice by SYBR<Superscript>®</Superscript> Green real-time quantitative PCR with a simple data analysis

Establishment and maintenance of transgenic mouse strains require being able to distinguish homozygous from heterozygous animals. To date, the developed real-time quantitative PCR techniques are often complicated, time-consuming and expensive. Here, we propose a very easy and rapid method with a simple data analysis to determine zygosity in transgenic mice. We show that the real-time quantitative PCR using SYBR Green fluorescent dye can be applied to discriminate two-fold differences in copy numbers of the transgene. Our procedure has to fit only three simple requirements: (1) to design primers capable of detecting one Ct difference for two-fold differences in DNA amounts (2) to measure genomic DNA concentrations accurately and (3) to have a reference animal of known zygosity in each run. Then, if the Ct values for the control gene are similar in all samples, we are able to compare directly the Ct values for the transgene in every sample, and so, to deduce the zygosity status of each mouse relative to the reference animal. This method is really simple and reliable, and it may be valuable as a rapid screening tool for zygosity status in transgenic animals.     

The influence of exogenous peptide on β2–microglobulin exchange in the HLA complex: analysis in real-time

 We used an optical biosensor to determine the relative binding affinity of peptides to purified HLA class I molecules. In this assay we monitor β₂–microglobulin (β₂m) exchange within the HLA-A2 molecule, whereby native β₂m in the complex is replaced by β₂m immobilized at the surface of the biosensor. Quantitative kinetic measurements permit us to obtain association rate (k_ass), dissociation rate (k_diss) and affinity constants (K_A) for the β₂m exchange reaction, alone, (control) and in the presence of exogenous peptide. We tested a panel of six peptides which had been designed and synthesized with an HLA-A2 binding motif, and had also been tested by the T2-cell binding assay, along with control peptides. The biosensor results demonstrate that exogenous peptide influences the dynamics of β₂m exchange in a sequence-specific manner. Five of six peptides increased the association rate, decreased the dissociation rate, and significantly increased the affinity (K_A=1.55–1.88×10⁹ M^–1) of HLA-A2 for immobilized β₂m compared with the control (K_A =1.14±0.04×10⁹ M^–1), demonstrating stabilization of the complex. One peptide was unable to stabilize the complex, as also shown in the T2 binding assay. However, analysis of peptide sequences demonstrated that the HLA-A2 secondary motif as well as primary motif residues are required for HLA-A2 stabilization. Further experiments demonstrated that β₂m exchange alone cannot stabilize the HLA class I complex at the cell surface until a peptide of sufficient binding affinity is bound. Hence kinetics equal to or below the control values in our biosensor assay probably represent an unstable complex in vivo. Unlike other methods described for the analysis of peptide stabilization, this approach is significantly faster, provides full kinetic analysis, and is simpler, since it requires no labeling of peptides. Furthermore, this may have important implications in the assessment of peptide vaccines. Received: 9 October 1997 / Revised: 20 January 1998