In recent decades,single-cell(SC)technologies and applications have been a very hot topics in the field of biology and medicine.In fact,early studies involving a single cell were on the malignant process of acute nonl...In recent decades,single-cell(SC)technologies and applications have been a very hot topics in the field of biology and medicine.In fact,early studies involving a single cell were on the malignant process of acute nonlymphocytic leukemia and inherited or sporadic genetic disease in the 1980s.And since the RhD gene in a single cell from maternal plasma was detected by fluorescence-based polymerase chain reaction(PCR)in the 1990s,“SC biopsies”[1]were widely used for early diagnosis in clinical practice in the 2000s.Previous studies showed that SC RNA sequencing(scRNA-seq)was used to evaluate patterns of allelic gene expression at the allele-specific mRNA level,and SC genome analysis can be used for genetic diagnosis.展开更多
A protocol of simple rapid microdissection of single-chromosome, amplification and cloning of its DNA from Lilium regale Wilson is described. Single-chromosome, microdissected by micromanipulator, was put into a 0 5 m...A protocol of simple rapid microdissection of single-chromosome, amplification and cloning of its DNA from Lilium regale Wilson is described. Single-chromosome, microdissected by micromanipulator, was put into a 0 5 mL Eppendorf tube and digested with Sau3A, and then the Sau3A linker adaptors were ligated to the ends of DNA fragments. After 2 rounds of PCR amplification with one chain of linker adaptor as primer, the PCR products thus obtained have a length of 300-2 500 base pairs (bp) with predominant fragments at about 1 000 bp. Southern blot analysis confirmed that the PCR products originated from the genome of Lilium regale Wilson. By cloning the amplification products from the second round of PCR, single-chromosome DNA library was constructed, in which about as many as 100 000 recombinant clones were produced. A total number of 84 clones were analysed, and it was revealed that the inserts ranged in size from 300 to 1 800 bp, with an average of 780 bp. Compared with the methods described in other literature, this protocol, eliminating the need for enzymatic digestion and ligating micromanipulation of chromosomal DNA in nanoliter volumes, permits the efficient amplification of single chromosome (not tens of chromosomes as reported before) and the fragments (780 bp in average) cloned in this study are longer than those reported before (650 bp in average).展开更多
The single chromosome of Cunninghamia lanceolata was successfully dissected by "glass needles" under the micromanipulator in this research. The dissected Cunninghamia lanceolata chromosomes were dige...The single chromosome of Cunninghamia lanceolata was successfully dissected by "glass needles" under the micromanipulator in this research. The dissected Cunninghamia lanceolata chromosomes were digested by proteinase K in Eppendorf respectively. After degenerate olionucleotideprimedPCR amplification (DOPPCR), DNA fragments were acquired. The suitable method of making chromosome specimens for the microdissection and PCR amplification has been discussed.展开更多
对柚单染色体LA-PCR-AFLP技术体系中外源DNA污染问题中不同试验用水、加接头连接前后步骤对水污染的敏感度以及LA-PCR形成的非特异性亮带现象进行对比研究。结果表明:8种试验用水中BT水(Bioteke,RNase-free and DNase-free Water)的防...对柚单染色体LA-PCR-AFLP技术体系中外源DNA污染问题中不同试验用水、加接头连接前后步骤对水污染的敏感度以及LA-PCR形成的非特异性亮带现象进行对比研究。结果表明:8种试验用水中BT水(Bioteke,RNase-free and DNase-free Water)的防控效果最佳;水的质量对LA-PCR过程中接头连接之前的步骤影响最大,对加接头之后的步骤也有一定的影响;非特异性亮带为引物自连形成的多聚体,与外源污染无关。本试验通过对外源污染的来源和关键步骤实施严格防控,经过dot blot验证,使外源污染能够得到有效控制。这为建立高质量的柚单染色体文库奠定了基础。展开更多
文摘In recent decades,single-cell(SC)technologies and applications have been a very hot topics in the field of biology and medicine.In fact,early studies involving a single cell were on the malignant process of acute nonlymphocytic leukemia and inherited or sporadic genetic disease in the 1980s.And since the RhD gene in a single cell from maternal plasma was detected by fluorescence-based polymerase chain reaction(PCR)in the 1990s,“SC biopsies”[1]were widely used for early diagnosis in clinical practice in the 2000s.Previous studies showed that SC RNA sequencing(scRNA-seq)was used to evaluate patterns of allelic gene expression at the allele-specific mRNA level,and SC genome analysis can be used for genetic diagnosis.
文摘A protocol of simple rapid microdissection of single-chromosome, amplification and cloning of its DNA from Lilium regale Wilson is described. Single-chromosome, microdissected by micromanipulator, was put into a 0 5 mL Eppendorf tube and digested with Sau3A, and then the Sau3A linker adaptors were ligated to the ends of DNA fragments. After 2 rounds of PCR amplification with one chain of linker adaptor as primer, the PCR products thus obtained have a length of 300-2 500 base pairs (bp) with predominant fragments at about 1 000 bp. Southern blot analysis confirmed that the PCR products originated from the genome of Lilium regale Wilson. By cloning the amplification products from the second round of PCR, single-chromosome DNA library was constructed, in which about as many as 100 000 recombinant clones were produced. A total number of 84 clones were analysed, and it was revealed that the inserts ranged in size from 300 to 1 800 bp, with an average of 780 bp. Compared with the methods described in other literature, this protocol, eliminating the need for enzymatic digestion and ligating micromanipulation of chromosomal DNA in nanoliter volumes, permits the efficient amplification of single chromosome (not tens of chromosomes as reported before) and the fragments (780 bp in average) cloned in this study are longer than those reported before (650 bp in average).
文摘The single chromosome of Cunninghamia lanceolata was successfully dissected by "glass needles" under the micromanipulator in this research. The dissected Cunninghamia lanceolata chromosomes were digested by proteinase K in Eppendorf respectively. After degenerate olionucleotideprimedPCR amplification (DOPPCR), DNA fragments were acquired. The suitable method of making chromosome specimens for the microdissection and PCR amplification has been discussed.
文摘对柚单染色体LA-PCR-AFLP技术体系中外源DNA污染问题中不同试验用水、加接头连接前后步骤对水污染的敏感度以及LA-PCR形成的非特异性亮带现象进行对比研究。结果表明:8种试验用水中BT水(Bioteke,RNase-free and DNase-free Water)的防控效果最佳;水的质量对LA-PCR过程中接头连接之前的步骤影响最大,对加接头之后的步骤也有一定的影响;非特异性亮带为引物自连形成的多聚体,与外源污染无关。本试验通过对外源污染的来源和关键步骤实施严格防控,经过dot blot验证,使外源污染能够得到有效控制。这为建立高质量的柚单染色体文库奠定了基础。
