ABSTRACT
Objective: To develop a detection method for single nucleotide polymorphisms (SNPs) of bronchial asthma (BA) susceptibility genes (IL-13, IL-33, and GSDMA) based on fluorescence PCR melting curves.Methods: Peripheral blood samples from 33 patients with BA were collected. DNA was extracted, and positive plasmids were constructed. Probes and primers for fluorescence polymerase chain reaction (PCR) were designed according to IL-13, IL-33, and GSDMA sequences, and the SNPs were separately detected by gene sequencing and fluorescence PCR melting curve.Results: The system was successfully divided into 3 SNPs, including IL-13, IL-33, and GSDMA, and a comparison of sequencing methods showed that the results were completely consistent. The lowest detection limit was 1 ng/reaction, the sensitivity and specificity were 100%, and this method had high repeatability (CV = 2.8%).Conclusion: The fluorescence PCR melting curve method is suitable for the rapid and accurate classification of SNPs. The method is economical, simple, and efficient, and is suitable for the screening of the susceptible gene SNPs in a large-scale population of patients with BA.
Subject(s)
Asthma/diagnosis , Genetic Testing/methods , Genotyping Techniques/methods , Mass Screening/methods , Polymerase Chain Reaction/methods , Adolescent , Adult , Asthma/blood , Asthma/genetics , Feasibility Studies , Female , Fluorescence , Genetic Predisposition to Disease , Genotyping Techniques/economics , Humans , Interleukin-13/genetics , Interleukin-33/genetics , Male , Mass Screening/economics , Neoplasm Proteins/genetics , Polymerase Chain Reaction/economics , Polymorphism, Single Nucleotide , Sensitivity and Specificity , Young AdultABSTRACT
<p><b>OBJECTIVE</b>To identify and clone the gene encoding human M96 gene and study its expression spectrum in several blood cell lines.</p><p><b>METHODS</b>According to the sequence of human EST which was highly homologous to the mouse M96 gene, primers used for library screening were synthesized, then the human adult testis and fetal brain cDNA library were screened. The gene was analyzed by making use of BLAST and CLUSTAL W, and its expression spectrum was studied by multiple-cell lines Northern blot analysis. The expression change of M961 in cell differentiation was observed by use of K562 cell line induced by hemin.</p><p><b>RESULTS</b>Two cDNA clones encoding human M96 gene were isolated, identified and named as M961, and M962. They were found to be isoforms of each other. Northern, blot showed that M961 gene was expressed highly in CEM, Hel, Dami and K562 cell lines. However, during K562 cell line differentiation, process the expression of M961 elevated only slightly.</p><p><b>CONCLUSIONS</b>M961 gene was expressed highly in pluripotent cell lines with erythrocytic and megakaryocytic potentials.</p>
