Haynaldia villosa is a wild relative of wheat and a valuable gene resource for wheat improvement.Owing to the limited number of probes available for fluorescence in situ hybridization(FISH),the resolution at which the...Haynaldia villosa is a wild relative of wheat and a valuable gene resource for wheat improvement.Owing to the limited number of probes available for fluorescence in situ hybridization(FISH),the resolution at which the karyotype of H.villosa can be characterized is poor,hampering accurate characterization of small segmental alien introgressions.We designed ten oligonucleotide probes using tandem repeats in DNA sequences derived from the short arm of H.villosa chromosome 6 V(6 VS).FISH with seven of them resulted in clear signals on H.villosa chromosomes.Using these,we constructed FISH karyotypes for H.villosa using oligo-6 VS-1 and oligo-6 VS-35 oligonucleotides and characterized the distribution of the two probes in five different H.villosa accessions.The new FISH probes can efficiently characterize H.villosa introgressions into wheat.展开更多
Systematic analysis of factors determining efficiency in discrimination of a point substitution (SNP) within specific DNA sequences was carried out in the context of hybridization approach. There are two types of sele...Systematic analysis of factors determining efficiency in discrimination of a point substitution (SNP) within specific DNA sequences was carried out in the context of hybridization approach. There are two types of selectivity that are critical for the rational design of highly specific oligonucleotides probes. The first type is the real selectivity of hybridization (fa) that is the ratio of association degrees of targets with an oligonucleotide probe upon the perfect and imperfect complex formation. This type of selectivity reflects the level of discrimination between matched and mismatched signals, which is determined both by experimental conditions and the thermodynamics of oligonucleotide hybridization. The second parameter characterizing the efficiency of SNP discrimination is the limit selectivity of hybridization, which determines the utmost value of fa at a given temperature. This value can be calculated as the ratio of corresponding equilibrium association constants of perfect and imperfect complex formation determined purely by thermodynamics. We have shown that the fa function is the most reliable characteristic describing the hybridization selectivity. For the analytical system designed to reveal any type of perturbation in DNA (e.g. SNP or modification), there is usually a temperature at which fa has its maximum value. The dependency of the fa maximum on different experimental parameters as well as the structural characteristics of a probe are described in details. The results allowed us to postulate points of principle to rationally design the most selective probes on the basis of oli- gonucleotides or their derivatives.展开更多
单分子荧光原位杂交(single-molecule fluorescence in situ hybridization,smFISH)技术是一种通过用偶联荧光基团的寡核苷酸探针,对固定细胞或组织中单个mRNA分子进行成像的方法。smFISH可对RNA进行定位、定量,以此对目标转录本进行实...单分子荧光原位杂交(single-molecule fluorescence in situ hybridization,smFISH)技术是一种通过用偶联荧光基团的寡核苷酸探针,对固定细胞或组织中单个mRNA分子进行成像的方法。smFISH可对RNA进行定位、定量,以此对目标转录本进行实时研究。sm FISH适用于细胞、组织切片等多种类型生物样本。近年来,多种基于基础smFISH的改进技术被发明,进一步促进了该技术的实际应用。smFISH良好的RNA单分子可视化能力,使得其在发育生物学、神经生物学及肿瘤生物学等基础生物学科中得到了广泛的应用。本文综述了smFISH技术基本原理、smFISH技术的局限性、smFISH衍生技术方法、smFISH在不同生物学科中的应用进展,并对smFISH技术的发展前景做出展望。展开更多
荧光原位杂交技术(fluorescence in situ hybridization,FISH)是植物分子细胞遗传学研究最为重要的手段之一。近些年,基于参考基因组设计的低拷贝寡聚核苷酸探针在FISH中应用得越来越广泛。然而,由于植物基因组中分布大量的重复序列,这...荧光原位杂交技术(fluorescence in situ hybridization,FISH)是植物分子细胞遗传学研究最为重要的手段之一。近些年,基于参考基因组设计的低拷贝寡聚核苷酸探针在FISH中应用得越来越广泛。然而,由于植物基因组中分布大量的重复序列,这使得oligo-FISH的分辨率存在一定局限性。利用包含多个荧光基团的荧光PCR引物,扩增出甘蔗染色体特异oligo探针,并进一步优化甘蔗的荧光原位杂交体系,提高了甘蔗oligo探针识别近缘物种染色体的效率。通过开发多荧光标记的甘蔗oligo探针以及甘蔗荧光杂交体系的优化,有效拓宽荧光信号的最小分辨率,提高信噪比(signal-to-noise ratio,SNR),并成功基于甘蔗oligo探针对高粱1-10号染色体分型。多荧光标记引物增强oligo探针信号的新方法及FISH体系的优化为今后在其他物种中提高oligo-FISH鉴定染色体及捕捉微弱的荧光信号提供了参考。展开更多
利用rpoB基因芯片技术快速进行分枝杆菌菌种鉴定。以分枝杆菌rpoB基因编码序列为靶基因,用基因芯片技术检测21种分枝杆菌标准株;8种其它细菌标准株;126株临床分离株。分枝杆菌与其它细菌标准株经PCR扩增后,分枝杆菌标准株均扩增出360 bp...利用rpoB基因芯片技术快速进行分枝杆菌菌种鉴定。以分枝杆菌rpoB基因编码序列为靶基因,用基因芯片技术检测21种分枝杆菌标准株;8种其它细菌标准株;126株临床分离株。分枝杆菌与其它细菌标准株经PCR扩增后,分枝杆菌标准株均扩增出360 bp DNA片段,在其它细菌中,除甲型溶血性链球菌和假白喉棒状杆菌出现同样片段外,其它细菌均未见扩增。21种寡核苷酸探针除海分枝杆菌与偶然分枝杆菌的探针有交叉杂交外,其余均为特异性杂交。对126株临床分离株进行鉴定,89株为结核分枝杆菌,占70.6%(89/126),非结核分枝杆菌(NTM)占9.2%(9/98)。应用rpoB基因芯片技术鉴定分枝杆菌菌种,是一种快速、准确的方法,具有较高的临床应用价值。展开更多
基金supported by the National Key Research and Development Program of China(2016YFD0102001)the National Natural Science Foundation of China(31571653,31771782,31201204,31501305)+3 种基金International Cooperation and Exchange Programme of the National Natural Science Foundation of China(31661143005)Introducing the Technique to Exploring the Genetic Germplasm Based on the Chromosome Sorting and Sequencing(2015-Z41)the Special Fund of Jiangsu Province for the Transformation of Scientific and Technological Achievements(BA2017138)supported by European Regional Development Fund Project“Plants as a Tool for Sustainable Global Development”(CZ.02.1.01/0.0/0.0/16_019/0000827)。
文摘Haynaldia villosa is a wild relative of wheat and a valuable gene resource for wheat improvement.Owing to the limited number of probes available for fluorescence in situ hybridization(FISH),the resolution at which the karyotype of H.villosa can be characterized is poor,hampering accurate characterization of small segmental alien introgressions.We designed ten oligonucleotide probes using tandem repeats in DNA sequences derived from the short arm of H.villosa chromosome 6 V(6 VS).FISH with seven of them resulted in clear signals on H.villosa chromosomes.Using these,we constructed FISH karyotypes for H.villosa using oligo-6 VS-1 and oligo-6 VS-35 oligonucleotides and characterized the distribution of the two probes in five different H.villosa accessions.The new FISH probes can efficiently characterize H.villosa introgressions into wheat.
文摘Systematic analysis of factors determining efficiency in discrimination of a point substitution (SNP) within specific DNA sequences was carried out in the context of hybridization approach. There are two types of selectivity that are critical for the rational design of highly specific oligonucleotides probes. The first type is the real selectivity of hybridization (fa) that is the ratio of association degrees of targets with an oligonucleotide probe upon the perfect and imperfect complex formation. This type of selectivity reflects the level of discrimination between matched and mismatched signals, which is determined both by experimental conditions and the thermodynamics of oligonucleotide hybridization. The second parameter characterizing the efficiency of SNP discrimination is the limit selectivity of hybridization, which determines the utmost value of fa at a given temperature. This value can be calculated as the ratio of corresponding equilibrium association constants of perfect and imperfect complex formation determined purely by thermodynamics. We have shown that the fa function is the most reliable characteristic describing the hybridization selectivity. For the analytical system designed to reveal any type of perturbation in DNA (e.g. SNP or modification), there is usually a temperature at which fa has its maximum value. The dependency of the fa maximum on different experimental parameters as well as the structural characteristics of a probe are described in details. The results allowed us to postulate points of principle to rationally design the most selective probes on the basis of oli- gonucleotides or their derivatives.
文摘荧光原位杂交技术(fluorescence in situ hybridization,FISH)是植物分子细胞遗传学研究最为重要的手段之一。近些年,基于参考基因组设计的低拷贝寡聚核苷酸探针在FISH中应用得越来越广泛。然而,由于植物基因组中分布大量的重复序列,这使得oligo-FISH的分辨率存在一定局限性。利用包含多个荧光基团的荧光PCR引物,扩增出甘蔗染色体特异oligo探针,并进一步优化甘蔗的荧光原位杂交体系,提高了甘蔗oligo探针识别近缘物种染色体的效率。通过开发多荧光标记的甘蔗oligo探针以及甘蔗荧光杂交体系的优化,有效拓宽荧光信号的最小分辨率,提高信噪比(signal-to-noise ratio,SNR),并成功基于甘蔗oligo探针对高粱1-10号染色体分型。多荧光标记引物增强oligo探针信号的新方法及FISH体系的优化为今后在其他物种中提高oligo-FISH鉴定染色体及捕捉微弱的荧光信号提供了参考。
文摘利用rpoB基因芯片技术快速进行分枝杆菌菌种鉴定。以分枝杆菌rpoB基因编码序列为靶基因,用基因芯片技术检测21种分枝杆菌标准株;8种其它细菌标准株;126株临床分离株。分枝杆菌与其它细菌标准株经PCR扩增后,分枝杆菌标准株均扩增出360 bp DNA片段,在其它细菌中,除甲型溶血性链球菌和假白喉棒状杆菌出现同样片段外,其它细菌均未见扩增。21种寡核苷酸探针除海分枝杆菌与偶然分枝杆菌的探针有交叉杂交外,其余均为特异性杂交。对126株临床分离株进行鉴定,89株为结核分枝杆菌,占70.6%(89/126),非结核分枝杆菌(NTM)占9.2%(9/98)。应用rpoB基因芯片技术鉴定分枝杆菌菌种,是一种快速、准确的方法,具有较高的临床应用价值。
