滚环扩增(rolling circle amplification,RCA)是新近发展起来的一种恒温核酸扩增方法。这种方法不仅可以直接扩增DNA和RNA,还可以实现对靶核酸的信号放大,灵敏度达到一个拷贝的核酸分子,因此在核酸检测中具有很大的应用价值和潜力。本...滚环扩增(rolling circle amplification,RCA)是新近发展起来的一种恒温核酸扩增方法。这种方法不仅可以直接扩增DNA和RNA,还可以实现对靶核酸的信号放大,灵敏度达到一个拷贝的核酸分子,因此在核酸检测中具有很大的应用价值和潜力。本文结合了滚环扩增技术在医药领域中的最新研究进展,介绍了滚环扩增的原理及其在医药领域中的应用。展开更多
Blood samples have traditionally been used as the main source of DNA for genetic analysis. How-ever, this source can be difficult in terms of collection, transportation, and long-term storage. In this study, we invest...Blood samples have traditionally been used as the main source of DNA for genetic analysis. How-ever, this source can be difficult in terms of collection, transportation, and long-term storage. In this study, we investigated whether human nail clippings could be used as a source of DNA for SNP genotyping, null-allele detection, and whole-genome amplification. From extracted nail DNA, we achieved amplicons up to a length of ~400 bp and >96% concordance for SNP genotyping and 100% concordance for null-allele detection compared to DNA derived from matched blood sam-ples. For whole-genome amplification, OmniPlex performed better than Multiple Displacement Amplification with a success rate of 89.3% and 76.8% for SNP genotyping and null-allele detection, respectively. Concordance was ~98% for both methods. When combined with OmniPlex whole-genome amplification, human nail clippings could potentially be used as an alternative to whole blood as a less invasive and more convenient source of DNA for genotyping studies.展开更多
Using double-stranded RNA(dsRNA)technology and sequence-independent amplification(SIA),the molecular identification on infected Rehmannia glutinosa in the field with mosaic symptoms was performed and the whole-genome ...Using double-stranded RNA(dsRNA)technology and sequence-independent amplification(SIA),the molecular identification on infected Rehmannia glutinosa in the field with mosaic symptoms was performed and the whole-genome of the Rehmannia mosaic virus(ReMV)Shanxi isolate(ReMV-SX)was sequenced.Sequencing analysis showed that the virus that infected Rehmannia glutinosa was Rehmannia mosaic virus(ReMV).The full-length of the obtained ReMV-SX sequence(GenBank accession no.JX575184)was 6395 nt,containing four open reading frames(ORFs).The sequence homology analysis of the complete nucleotide sequence showed that ReMV-SX was 93.8%-97.0%homologous to ReMV in Tobamovirus subgroup Ⅰ,while only 49.8%-58.9%homologous to the isolates in subgroups Ⅱ and Ⅲ of the same genus.Phylogenetic analysis showed that ReMV-SX and ReMV-Henan formed a separate branch and had the closest genetic relationship.The results laid the foundation for ongoing researches in the taxonomic status and evolution of ReMV and for further investigating the pathogenic mechanism of ReMV infecting Rehmannia glutinosa.展开更多
Single-cell bisulfite sequencing (scBS-seq) was developed to assess DNA methylation heterogeneity in human and mouse. However, the reads are under-represented in regions with high DNA methylation, because these region...Single-cell bisulfite sequencing (scBS-seq) was developed to assess DNA methylation heterogeneity in human and mouse. However, the reads are under-represented in regions with high DNA methylation, because these regions are usually fragmented into long segments and are seldom sequenced on the lllumina plat. form. To reduce the read distribution bias and maximize the use of these long segments, we developed bisulfite-converted randomly integrated fragments sequencing (BRIF-seq), a method with high rates of read mapping and genome coverage. Single microspore of maize, which has a highly methylated and repetitive genome, was used to perform BRIF.seq. High coverage of the haploid genome was obtained to evaluate the methylation states of CG, CHG, and CHH (H = A, C, or T). Compared with scBS-seq, BRIF-seq produced reads that were distributed more evenly across the genome, including regions with high DNA methylation. Surprisingly, the methylation rates among the four microspores within one tetrad were similar, but differed significantly among tetrads, suggesting that non-simultaneous methylation reprogramming could occur among tetrads. Similar levels of heterogeneity, which often occur in lowcopy regions, were detected in different genetic backgrounds. These results suggest that BRIF-seq can be applied for single-cell methylome analysis of any species with diverse genetic backgrounds.展开更多
文摘滚环扩增(rolling circle amplification,RCA)是新近发展起来的一种恒温核酸扩增方法。这种方法不仅可以直接扩增DNA和RNA,还可以实现对靶核酸的信号放大,灵敏度达到一个拷贝的核酸分子,因此在核酸检测中具有很大的应用价值和潜力。本文结合了滚环扩增技术在医药领域中的最新研究进展,介绍了滚环扩增的原理及其在医药领域中的应用。
文摘Blood samples have traditionally been used as the main source of DNA for genetic analysis. How-ever, this source can be difficult in terms of collection, transportation, and long-term storage. In this study, we investigated whether human nail clippings could be used as a source of DNA for SNP genotyping, null-allele detection, and whole-genome amplification. From extracted nail DNA, we achieved amplicons up to a length of ~400 bp and >96% concordance for SNP genotyping and 100% concordance for null-allele detection compared to DNA derived from matched blood sam-ples. For whole-genome amplification, OmniPlex performed better than Multiple Displacement Amplification with a success rate of 89.3% and 76.8% for SNP genotyping and null-allele detection, respectively. Concordance was ~98% for both methods. When combined with OmniPlex whole-genome amplification, human nail clippings could potentially be used as an alternative to whole blood as a less invasive and more convenient source of DNA for genotyping studies.
基金Supported by the National Natural Science Foundation of China(31772130)China Agriculture Research System(CARS-21)。
文摘Using double-stranded RNA(dsRNA)technology and sequence-independent amplification(SIA),the molecular identification on infected Rehmannia glutinosa in the field with mosaic symptoms was performed and the whole-genome of the Rehmannia mosaic virus(ReMV)Shanxi isolate(ReMV-SX)was sequenced.Sequencing analysis showed that the virus that infected Rehmannia glutinosa was Rehmannia mosaic virus(ReMV).The full-length of the obtained ReMV-SX sequence(GenBank accession no.JX575184)was 6395 nt,containing four open reading frames(ORFs).The sequence homology analysis of the complete nucleotide sequence showed that ReMV-SX was 93.8%-97.0%homologous to ReMV in Tobamovirus subgroup Ⅰ,while only 49.8%-58.9%homologous to the isolates in subgroups Ⅱ and Ⅲ of the same genus.Phylogenetic analysis showed that ReMV-SX and ReMV-Henan formed a separate branch and had the closest genetic relationship.The results laid the foundation for ongoing researches in the taxonomic status and evolution of ReMV and for further investigating the pathogenic mechanism of ReMV infecting Rehmannia glutinosa.
基金the National Natural Science Foundation of China (31730064,31801125)National Key Research and Development Program of China (2016YFD0101003)+3 种基金National Postdoctoral Program for Innovative Talents (BX201700091)China Postdoctoral Science Foundation (2017M620325)the Hubei Provincial Natural Science Foundation of China (2015CFA008)and the Huazhong Agricultural University Scientific & Tech no logical Self-in novation Foundation.
文摘Single-cell bisulfite sequencing (scBS-seq) was developed to assess DNA methylation heterogeneity in human and mouse. However, the reads are under-represented in regions with high DNA methylation, because these regions are usually fragmented into long segments and are seldom sequenced on the lllumina plat. form. To reduce the read distribution bias and maximize the use of these long segments, we developed bisulfite-converted randomly integrated fragments sequencing (BRIF-seq), a method with high rates of read mapping and genome coverage. Single microspore of maize, which has a highly methylated and repetitive genome, was used to perform BRIF.seq. High coverage of the haploid genome was obtained to evaluate the methylation states of CG, CHG, and CHH (H = A, C, or T). Compared with scBS-seq, BRIF-seq produced reads that were distributed more evenly across the genome, including regions with high DNA methylation. Surprisingly, the methylation rates among the four microspores within one tetrad were similar, but differed significantly among tetrads, suggesting that non-simultaneous methylation reprogramming could occur among tetrads. Similar levels of heterogeneity, which often occur in lowcopy regions, were detected in different genetic backgrounds. These results suggest that BRIF-seq can be applied for single-cell methylome analysis of any species with diverse genetic backgrounds.
