A simple method to create a chromosome-specific DNA library of rice, including microdissection, amplification,characterization and cloning, is described. Rice chromosome 4 from a metaphase cell has been isolated and a...A simple method to create a chromosome-specific DNA library of rice, including microdissection, amplification,characterization and cloning, is described. Rice chromosome 4 from a metaphase cell has been isolated and amplified by the Linker Adapter PCR (LA-PCR). The PCR products were labeled as probes with DIG-11-dUTP using the random priming method.Southern blot analysis with rice genomic DNA and specific RFLP markers demonstrated that the PCR products were derived from rice chromosome 4. A large library comprising over 100,000recombinant plasmid microclones from rice chromosome 4was constructed. Colony hybridization showed that 58% of the clones contained single or low-copy sequences and 42%contained repetitive sequences. The size of inserts generated by PCR ranged from 140bp to 500bp.This method will facilitate cloning of the specific chromosome DNA markers and important genes of rice.展开更多
上世纪90年代发展起来的LA-PCR （Long and accurate polymerase chain reaction）技术，以其扩增片段长，保真性（Fidelity）高的独特优势，被广泛地用于基因组测序、遗传多态性分析、病毒的遗传变异分析、病毒拯救、病理诊断等多个方...上世纪90年代发展起来的LA-PCR （Long and accurate polymerase chain reaction）技术，以其扩增片段长，保真性（Fidelity）高的独特优势，被广泛地用于基因组测序、遗传多态性分析、病毒的遗传变异分析、病毒拯救、病理诊断等多个方面。本文将对LA-PCR技术的基本原理和其独特的优势作一综述。展开更多
应用显微切割技术获得赤麂的Y2单条染色体,经LA-PCR(linker-adaptor PCR)扩增后用D IG标记制备探针,然后用Southern b lot杂交法对所制备的探针进行验证并对毛冠鹿的中期核型进行原位杂交.结果表明:用该法制备的涂染探针是成功的,原位...应用显微切割技术获得赤麂的Y2单条染色体,经LA-PCR(linker-adaptor PCR)扩增后用D IG标记制备探针,然后用Southern b lot杂交法对所制备的探针进行验证并对毛冠鹿的中期核型进行原位杂交.结果表明:用该法制备的涂染探针是成功的,原位杂交也获得了阳性结果,可以初步验证毛冠鹿中的Y染色体.展开更多
The univalent from the meiosis-metaphase spreads of F1 (Z2× wheat variety Wan7107) wasidentified to be Agropyrum intermedium 2Ai-2 chromosome by GISH. The 2Ai-2 chromosomes weremicroisolated and collected. After ...The univalent from the meiosis-metaphase spreads of F1 (Z2× wheat variety Wan7107) wasidentified to be Agropyrum intermedium 2Ai-2 chromosome by GISH. The 2Ai-2 chromosomes weremicroisolated and collected. After two rounds of PCR amplification, the PCR products wereranged from 150-3 000 bp,with predominant fragments at about 200-2 000 bp. Using Ag.intermedium genomic DNA as a probe, Southern blotting analysis confirmed the products originatedfrom Ag. intermedium genome. The products were purified, ligated to pUC18 and then transformedinto competence E.coli DH5αto produce a 2Ai-2 chromosome DNA library. The microcloningexperiments produced approximately 5 ×105 clones, the size range of the cloned inserts was 200-1 500 bp, with an average of 580 bp. Using Ag.intermedium genomic DNA as a probe, dot blottingresults showed that 56% clones are unique/low copy sequences, 44% are repetitive sequences inthe library. Four Ag. intermedium clones were screened from the library by RFLP, and threeclones(Mag065, Mag088, Mag139)belong to low/single sequences, one clone(Mag104)was repetitivesequence, and GISH results indicated that Mag104 was Ag.intermedium species-specific repetitiveDNA sequence.展开更多
文摘A simple method to create a chromosome-specific DNA library of rice, including microdissection, amplification,characterization and cloning, is described. Rice chromosome 4 from a metaphase cell has been isolated and amplified by the Linker Adapter PCR (LA-PCR). The PCR products were labeled as probes with DIG-11-dUTP using the random priming method.Southern blot analysis with rice genomic DNA and specific RFLP markers demonstrated that the PCR products were derived from rice chromosome 4. A large library comprising over 100,000recombinant plasmid microclones from rice chromosome 4was constructed. Colony hybridization showed that 58% of the clones contained single or low-copy sequences and 42%contained repetitive sequences. The size of inserts generated by PCR ranged from 140bp to 500bp.This method will facilitate cloning of the specific chromosome DNA markers and important genes of rice.
文摘上世纪90年代发展起来的LA-PCR （Long and accurate polymerase chain reaction）技术，以其扩增片段长，保真性（Fidelity）高的独特优势，被广泛地用于基因组测序、遗传多态性分析、病毒的遗传变异分析、病毒拯救、病理诊断等多个方面。本文将对LA-PCR技术的基本原理和其独特的优势作一综述。
文摘应用显微切割技术获得赤麂的Y2单条染色体,经LA-PCR(linker-adaptor PCR)扩增后用D IG标记制备探针,然后用Southern b lot杂交法对所制备的探针进行验证并对毛冠鹿的中期核型进行原位杂交.结果表明:用该法制备的涂染探针是成功的,原位杂交也获得了阳性结果,可以初步验证毛冠鹿中的Y染色体.
基金supported by National High-Tech R&D(863)ProgramNational Natural Science Foundation of China(101-04-03-03-97).
文摘The univalent from the meiosis-metaphase spreads of F1 (Z2× wheat variety Wan7107) wasidentified to be Agropyrum intermedium 2Ai-2 chromosome by GISH. The 2Ai-2 chromosomes weremicroisolated and collected. After two rounds of PCR amplification, the PCR products wereranged from 150-3 000 bp,with predominant fragments at about 200-2 000 bp. Using Ag.intermedium genomic DNA as a probe, Southern blotting analysis confirmed the products originatedfrom Ag. intermedium genome. The products were purified, ligated to pUC18 and then transformedinto competence E.coli DH5αto produce a 2Ai-2 chromosome DNA library. The microcloningexperiments produced approximately 5 ×105 clones, the size range of the cloned inserts was 200-1 500 bp, with an average of 580 bp. Using Ag.intermedium genomic DNA as a probe, dot blottingresults showed that 56% clones are unique/low copy sequences, 44% are repetitive sequences inthe library. Four Ag. intermedium clones were screened from the library by RFLP, and threeclones(Mag065, Mag088, Mag139)belong to low/single sequences, one clone(Mag104)was repetitivesequence, and GISH results indicated that Mag104 was Ag.intermedium species-specific repetitiveDNA sequence.
