共查询到20条相似文献,搜索用时 31 毫秒
1.
2.
3.
Oligonucleotide microarray analysis of aminoallyl-labeled cDNA targets from linear RNA amplification 总被引:2,自引:0,他引:2
Kaposi-Novak P Lee JS Mikaelyan A Patel V Thorgeirsson SS 《BioTechniques》2004,37(4):580, 582-586, 588
4.
5.
6.
Bacterial populations in samples of bioleached copper ore as revealed by analysis of DNA obtained before and after cultivation. 总被引:8,自引:4,他引:4
下载免费PDF全文
![点击此处可从《Applied microbiology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
The composition of bacterial populations in copper bioleaching systems was investigated by analysis of DNA obtained either directly from ores or leaching solutions or after laboratory cultures. This analysis consisted of the characterization of the spacer regions between the 16 and 23S genes in the bacterial rRNA genetic loci after PCR amplification. The sizes of the spacer regions, amplified from DNAs obtained from samples, were compared with the sizes of those obtained from cultures of the main bacterial species isolated from bioleaching systems. This allowed a preliminary assessment of the bacterial species present in the samples. Identification of the bacteria was achieved by partial sequencing of the 16S rRNA genes adjacent to the spacer regions. The spacer regions observed in DNA from columns leached at different iron concentrations indicated the presence of a mixture of different bacteria. The spacer region corresponding to Thiobacillus ferrooxidans was the main product observed at high ferrous iron concentration. At low ferrous iron concentration, spacer regions of different lengths, corresponding to Thiobacillus thiooxidans and "Leptospirillum ferrooxidans" were observed. However, T. ferrooxidans appeared to predominate after culture of these samples in medium containing ferrous iron as energy source. Although some of these strains contained singular spacer regions, they belonged within previously described groups of T. ferrooxidans according to the nucleotide sequence of the neighbor 16S rRNA. These results illustrate the bacterial diversity in bioleaching systems and the selective pressure generated by different growth conditions. 相似文献
7.
8.
Microarray-based analysis of microbial community RNAs by whole-community RNA amplification 总被引:1,自引:0,他引:1
Gao H Yang ZK Gentry TJ Wu L Schadt CW Zhou J 《Applied and environmental microbiology》2007,73(2):563-571
A new approach, termed whole-community RNA amplification (WCRA), was developed to provide sufficient amounts of mRNAs from environmental samples for microarray analysis. This method employs fusion primers (six to nine random nucleotides with an attached T7 promoter) for the first-strand synthesis. The shortest primer (T7N6S) gave the best results in terms of the yield and representativeness of amplification. About 1,200- to 1,800-fold amplification was obtained with amounts of the RNA templates ranging from 10 to 100 ng, and very representative detection was obtained with 50 to 100 ng total RNA. Evaluation with a Shewanella oneidensis Deltafur strain revealed that the amplification method which we developed could preserve the original abundance relationships of mRNAs. In addition, to determine whether representative detection of RNAs can be achieved with mixed community samples, amplification biases were evaluated with a mixture containing equal quantities of RNAs (100 ng each) from four bacterial species, and representative amplification was also obtained. Finally, the method which we developed was applied to the active microbial populations in a denitrifying fluidized bed reactor used for denitrification of contaminated groundwater and ethanol-stimulated groundwater samples for uranium reduction. The genes expressed were consistent with the expected functions of the bioreactor and groundwater system, suggesting that this approach is useful for analyzing the functional activities of microbial communities. This is one of the first demonstrations that microarray-based technology can be used to successfully detect the activities of microbial communities from real environmental samples in a high-throughput fashion. 相似文献
9.
Quantitation of mRNA content in samples of total cellular RNA is required for the analysis of Northern blot hybridization
to estimate the relative level of specific gene expression. Commonly used methods based on UV absorbance and dye staining
measure only total RNA, and mRNA normalization by probing for mRNA levels of housekeeping genes, such as β-actin and glyceraldehyde-3-phosphate
dehydrogenase, assumes a constant level of their expression, which, in fact, may vary as a function of cell proliferation
and differentiation. We describe here a nonradioactive, slot-blotting method for quantifying eukaryotic mRNA levels using
a biotinylated oligo(dT) probe, which hybridizes directly to the 3′-polyadenylated sequence of eukaryotic mRNAs. The method
provides a more accurate estimation of mRNA content in total RNA samples and should be applicable for quantitative Northern
analysis. 相似文献
10.
11.
Microarray-Based Analysis of Microbial Community RNAs by Whole-Community RNA Amplification 总被引:6,自引:1,他引:5
下载免费PDF全文
![点击此处可从《Applied microbiology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Haichun Gao Zamin K. Yang Terry J. Gentry Liyou Wu Christopher W. Schadt Jizhong Zhou 《Applied microbiology》2007,73(2):563-571
A new approach, termed whole-community RNA amplification (WCRA), was developed to provide sufficient amounts of mRNAs from environmental samples for microarray analysis. This method employs fusion primers (six to nine random nucleotides with an attached T7 promoter) for the first-strand synthesis. The shortest primer (T7N6S) gave the best results in terms of the yield and representativeness of amplification. About 1,200- to 1,800-fold amplification was obtained with amounts of the RNA templates ranging from 10 to 100 ng, and very representative detection was obtained with 50 to 100 ng total RNA. Evaluation with a Shewanella oneidensis Δfur strain revealed that the amplification method which we developed could preserve the original abundance relationships of mRNAs. In addition, to determine whether representative detection of RNAs can be achieved with mixed community samples, amplification biases were evaluated with a mixture containing equal quantities of RNAs (100 ng each) from four bacterial species, and representative amplification was also obtained. Finally, the method which we developed was applied to the active microbial populations in a denitrifying fluidized bed reactor used for denitrification of contaminated groundwater and ethanol-stimulated groundwater samples for uranium reduction. The genes expressed were consistent with the expected functions of the bioreactor and groundwater system, suggesting that this approach is useful for analyzing the functional activities of microbial communities. This is one of the first demonstrations that microarray-based technology can be used to successfully detect the activities of microbial communities from real environmental samples in a high-throughput fashion. 相似文献
12.
13.
T7启动子在哺乳类动物细胞中启动外源基因表达的研究 总被引:2,自引:0,他引:2
人低密度脂蛋白(LDL)受体基因cDNA和氯霉素已酞转移酶基因(CAT)及PolyA信号序列被克隆进pGEM4载体的T7噬茵体启动子下游,构建成质粒pT7LDLR和pT7CAT.两个重组质粒转化CHO细胞.PCR和CAT酶实验显示:两个基因被T7噬菌体启动子所启动.结果证实真核生物RNA聚合酶能够识别T7启动子,转录外源基因.常用的含有T7启动子的质粒可同时作为原核生物和真核生物的表达载体. 相似文献
14.
15.
16.
17.
Optimized T7 amplification system for microarray analysis. 总被引:8,自引:0,他引:8
C Pabón Z Modrusan M V Ruvolo I M Coleman S Daniel H Yue L J Arnold 《BioTechniques》2001,31(4):874-879
18.
Directional random oligonucleotide primed (DROP) global amplification of cDNA: its application to subtractive cDNA cloning.
下载免费PDF全文
![点击此处可从《Nucleic acids research》网站下载免费的PDF全文](/ch/ext_images/free.gif)
We describe a method of global PCR amplification of cDNA such that the strand sense is maintained. The products of this process are random primed fragments ranging in size from 100 to 500 bp which facilitates uniform PCR amplification of total cDNA. Directional incorporation of a T7 RNA polymerase initiator/promoter sequence allows efficient synthesis of total sense RNA from this material and the use of a biotinylated primer permits the separation of single-stranded cDNA. Isolation of these products from different cell types provides a renewable source of target single-stranded cDNA and driver RNA from limited cell numbers and we demonstrate their use for subtractive hybridisation cloning of differentially expressed cDNAs. 相似文献
19.
Two methods of constructing linear functional expression elements (LFEE) using hybrid DNA and RNA primers in DNA amplification for rapid gene expression are described. In both methods, it is not necessary to have additional transformation or bacterial propagation. The promoter, open reading frame (ORF) and terminator are amplified using Pfu or Taq DNA polymerase. Three elements containing DNA or RNA overhang are covalently ligated by T4 DNA ligase. The recombinant molecule is amplified with element-specific primers. The LFEE can be generated by both methods in a few hours and can be expressed in mammalian cells. 相似文献
20.
Linear RNA amplification using T7 RNA polymerase is useful in genome-wide analysis of gene expression using DNA microarrays, but exponential amplification using polymerase chain reaction (PCR) is still required for cDNA library preparation from single-cell quantities of RNA. We have designed a small RNA molecule called chum-RNA that has enabled us to prepare a single-cell cDNA library after four rounds of T7-based linear amplification, without using PCR amplification. Chum-RNA drove cDNA synthesis from only 0.49 femtograms of mRNA (730 mRNA molecules) as a substrate, a quantity that corresponds to a minor population of mRNA molecules in a single mammalian cell. Analysis of the independent cDNA clone of this library (6.6 × 105 cfu) suggests that 30-fold RNA amplification occurred in each round of the amplification process. The size distribution and representation of mRNAs in the resulting one-cell cDNA library retained its similarity to that of the million-cell cDNA library. The use of chum-RNA might also facilitate reactions involving other DNA/RNA modifying enzymes whose Michaelis constant (Km) values are around 1 mM, allowing them to be activated in the presence of only small quantities of substrate. 相似文献