Analysis of full-length sequence and 18 point mutations of HLA-B in a leukemia patient and her family / 中国组织工程研究

BACKGROUND: The human leukocyte antigen (HLA) has undergone long-term evolution to form diverse polymorphisms. In recent years, due to the increase in the number of examinees and the rapid development of HLA typing technology, novel HLA alleles have been discovered constantly. OBJECTIVE: To analyze the full-length sequence and 18 point mutations of HLA-B gene in a leukemia patient and her family using the next-generation sequencing technology. METHODS: Polymerase chain reaction and sequence-specific oligonucleotide probes (PCR-SSOP) and polymerase chain reaction-sequence based typing (PCR-SBT) revealed abnormalities in the patient’s HLA-B. To identify the genotype, we sequenced the full length of the gene by next-generation sequencing technology and collected blood samples from the patient’s father, mother and two sisters for genetic analysis of HLA genes. RESULTS AND CONCLUSION: Both PCR-SSOP and PCR-SBT indicated that the HLA-B sample had no perfectly matched genotype. Further detection using the next-generation sequencing technology revealed that the novel allele had 18 base mutations in the exon, intron and 3’UTR compared to the most homologous allele B*15:09:01. Five exon base mutations were located in the exons 3 and 4, which were: 486G→C, 583T→C, 636T→C, 652A→G, 756C→T, resulting in changes in the five corresponding codons, including 171 tyrosine (Tyr) → histidine (His) and 194 isoleucine (Ile) → valine (Val). A pedigree survey found that the patient’s novel HLA B allele was inherited from her father. The novel allele sequence was submitted to the Genbank database (MG595995). A novel HLA-B allele was confirmed by the next-generation sequencing, which was officially named HLA-B*15:435 by the World Health Organization HLA Factor Nomenclature Committee in December 2017.

Rapid detection of pathogenic mycoplasmas in genitourinary tract using PCR-reverse dot blot hybridization / 中华皮肤科杂志

Objective To develop a PCR-mverse dot blot hybridization(RDB)assay to rapidly detect pathogenic mycoplasmas in genitourinary tract.Methods Universal primers were designed and applied to amplify the 16S rRNA gone of ureaplasma parvum(Up),ureaplasma urealyticum(Uu),Mycoplasma genitalium(Mg),Mycoplasma hominis(Mh)by using nestcd PCR.Specific nucleotide probes of Up,Uu,Mg and Mh Were constructed and immobilized onto nylon membranes.PCR products were denatured and hybridized、with specific oligonucleofide probes on nylon membrane.The sensitivity and specificity of the PCR-RDB assay were evaluated based.on the hybddizafion results.Also,PCR-RDB Was utilized to detect pathogenic mycoplasmas from 60 clinical samples.Results The four probes selectively hybridized with the PCR product of corresponding mycoplasmas,and no cross hybridization was observed.The detection limit of PCR-RDB Was one colony forming unit(CFU)of mycoplasma.Out of the 60 clinical samples、19were positive for mycoplasm,Mixed infections were found in three samples,including two coinfected with Up and Uu and one with Uu and Mg.Conclusion PCR-RDB is a rapid,specific and sensitive approach to the identification of pathogenic mycoplasmas in urogenital tract.

Rapid detection of chromosomal aneuploidies by multiplex ligation-dependent probe amplification / 中华检验医学杂志

Objective To test whether multiplex ligation-dependent probe amplification(MLPA)could be used for the prenatal detection of the most common aneuploidies of chromosomes 13,18,21,X,and Y.Methods 34 cases including 22 blood samples(12 with trisomy 21,1 with monosomy X,one male witll extra Y and 8 healthy persons),4 cord blood samples with Down syndrome and 8 amniotic fluid samples ( 1 with trisomy 21 and 7 normal fetuses)were recruited into this study.All samples were confirmed by karvotype analysis. DNA was extracted from blood and amniotic lysate was incubated with proteinase K.MLPA was used to determine the relative copy numbers.Results The resuhs were available within 48 h and were concordant with karyotype analysis in all but one case of amniotic fluid that was suggested to be triploid sample 69,XXY by MLPA or contaminated by maternal blood.This sample actually was found containing a number of red blood cells after centfifugation in test. In total,the concordance rate with clinical characteristics was 97.1%.The Ratio values of 13,18,21,X in normal samples were approaching 1.0 except chromosome Y having slightly higher variation in relative copy number.The difference of ratio means between the normal and trisomy 21 samples was statistically significant by one-way ANOVA(F=298.906.P=0.000).Conclusion Computer assisted MLPA with high sensitivity is a rapid,simple,automatic and reliable method for detection of common chromosomal aneuploidies.

DISEÑO DE OLIGONUCLEÓTIDOS PARA EL ESTUDIO DE GENES CELULOLÍTICOS Y SOLVENTOGÉNICOS EN CEPAS COLOMBIANAS DE Clostridiumsp. (CLOSTRIDIACEAE) / Oligonucleotide Probe Design for the Study of Cellulolytic and Solventogenic Genes in Colombian Clostridiumsp. strains (Clostridiaceae)

El objetivo de este estudio fue analizar las rutas metabólicas para la producción de solventes y degradación de celulosa en cepas colombianas promisorias del género Clostridium. Para ello se diseñaron sondas de hibridación que sirvieran para posteriores estudios de mejoramiento genético de las cepas. Se construyó la base de datos denominada MULTICLOST en Microsoft Access® con las secuencias de 485 genes involucrados en las rutas metabólicas arriba mencionadas, provenientes de 45 especies bacterianas y 10 especies fúngicas. Los genes fueron agrupados de acuerdo al tipo de enzima y a los dominios catalíticos o de unión a sustrato en el caso de las celulasas. Cada grupo se sometió a alineamiento múltiple en ClustalW 1.83 y con base en los resultados se crearon subgrupos de similitud mayor al 50%. Se localizaron secuencias conservadas de longitud mayor a 19 nucleótidos en GeneDoc 2.6.002 y sus valores termodinámicos fueron estimados con GeneRunner v3.05, mientras que la sensibilidad y especificidad fue verificada por búsquedas en GenBank usando BLASTN 2.2.8. En total se obtuvieron 94 secuencias conservadas con las siguientes características: longitud promedio de 24 nucleótidos, Tm promedio de 65,8 ºC y contenido de (G+C) entre 14,3 y 60,0%. Se determinó que ninguna de las sondas diseñadas forma estructuras secundarias estables con Tm superior a 36,1 ºC. De acuerdo a sus características y valores termodinámicos, todas las sondas podrían ser utilizadas en la construcción de un microarreglo o en reacciones de PCR para la identificación de regiones relevantes en el mejoramiento del proceso por ingeniería metabólica.

The goal of the present study was to analyze the metabolic pathways involved in solvent production and cellulose consumption by promising Colombian native strains of the genus Clostridium. Therefore a set of oligonucleotide probes was designed, with the aim of analyzing potential targets for genetic improvement of the Colombian strains. The database named MULTICLOST was created in Microsoft Access® using the sequences from 485 genes involved in solventogenesis, 1,3propanodiol production and cellulolysis from 45 bacterial and 10 fungal species. The genes were grouped according to their respective enzyme function and to the catalytic domain or the substrate binding domain in the case of cellulases. ClustalW 1.83 was used for multiple alignment of every group. Subgroups of sequences with more than 50% identity among themselves were created. Conserved sequences longer than 19 nucleotides were identified using GeneDoc 2.6.002 and their thermodynamic values were calculated with GeneRunner v3.05, while their sensitivity and specificity were verified by searching in GenBank with BLASTN 2.2.8. Ninetyfour conserved sequences were obtained with an average 24nucleotide length, 65.8ºC average Tm and a (G+C) content between 14.3% and 60.0%. None of these probes forms stable secondary structures at temperatures higher than 36.1ºC. According to the former results, all of the probes could be used in an oligonucleotide microarray or in PCR reactions for the identification of metabolic targets for improvement of the industrial process.

Effect of NF-?B Decoys on development and maturation of dendritic cells and in itiation of Tcells immune response in vitro / 中华器官移植杂志

ObjectiveTo investigate the effect of NF-?B on the differentiation and maturation of mu rine bone marrow-derived dendritic cells (DC) in vitro.MethodsThe activity of NF-?B in dendritic cells was blocked by oligodeoxyribonucleot ide Decoys (ODN Decoys) containing two special binding sites for NF-?B. Morpho logical changes of DS were observed. The expression of assistant molecules on th e cell surface was detected by using flow cytometer, and the stimulating activit y of allogeneic T lymphocyte proliferative response was determined in culture mu rine DC. The effect of NF-?B on the maturation and immunobiological activity o f murine DC was studied.ResultsNF-?B ODN Decoys were efficiently incorporated by DC, markedly suppressed the maturation of DC, the expression of assistant costimulatory molecules (CD80, CD8 6 and CD40) on the surface of DC and the secretion of IL-12, blocked the develo pment of DC and this blocked function was not reversed by lipopolysaccharide (LP S). In mixed lymphocyte reactions, DC treated with NF-?B ODN Decoys could indu ce allogeneic T cells hyphoresponsiveness, and this was associated with the inhi bition of Th1-type cytokines (IL-2 and IFN-?) production.ConclusionsNF-? B is a key gene to the development and maturation of DC. Specific interference w ith NF-?B in DC using ODN Decoys approaches could offer a novel strategy for i nducing and generating tolerogeneic immature DC and provide a promising means to induce transplant immune tolerance.

SCREENING FOR PHENYLKETONURIA GENE MUTATION BY DNA AMPLIFICATION WITH THE POLYMERASE CHAIN REACTION / 西安交通大学学报(医学版)

Exon 3 termination mutation of phenylalanine hydroxylase (PAH) gene, the only identified one causing classical phenylketonuria (PKU) in Chinese, was detected in fourteen PKU children from Xi'an. The genomic DNA from these patients was amplified by polymerase chain reaction(PCR) and dot hybridied with specific oligonucleotide probes. This mutation is not present in any of these affected children, which indicates that phenylketonuria in Chinese may be caused by other mutations in phenylalanine hydroxylase locus. PCR amplification combining with oligonucleotide dot hybridization is technically feasible for prenatal diagnosis and carrier screening for PKU.