A novel strategy for identifying differential gene expression: An improved method of differential display analysis.

We propose a novel alternative approach, an advanced method for recently developed strategies, for identifying differentially expressed genes. Firstly, double-stranded cDNAs were digested using Sau3AI and the 3'-end restriction fragments of the cDNA were ligated to a double-stranded adapter. Next, the restriction fragments were directly amplified using several combinations of adapter-specific primers and FITC-labeled oligo dT primers. The selected cDNA fragments were displayed on a polyacrylamide gel. Neither nested PCR nor purification of 3'-end fragments are necessary. We examined the validity of this approach by evaluating gene expression changes during granulocytic differentiation of HL-60 cells. This method can theoretically detect almost all gene expression changes more rapidly and through simpler manipulations than by any other approach.     

Generation of high-quantity and quality tag/ditag cDNAs for SAGE analysis

The serial analysis of gene expression (SAGE) technique is an important tool for genome-wide gene expression analysis. However, the requirement of a large amount of mRNA for the analysis and the difficulties in generating high-quality tag and ditag fragments for the construction of a SAGE library often interfere with the successful performance of the SAGE technique. We developed two procedures to solve these issues: (i) introducing low-cycle PCR amplification of the 3' cDNA before the BsmFI digestion of the 3' cDNAs and (ii) gel purifying the BsmFI-released tag fragments before ditag formation. These modifications provide a large quantity of initial 3' cDNAs and high-quality tags and ditags for the construction of SAGE libraries.     

Global single-cell cDNA amplification to provide a template for representative high-density oligonucleotide microarray analysis

We describe here a protocol for the representative amplification of global mRNAs from typical single mammalian cells to provide a template for high-density oligonucleotide microarray analysis. A single cell is lysed in a tube without purification and first-strand cDNAs are synthesized using a poly(dT)-tailed primer. Unreacted primer is specifically eliminated by exonuclease treatment and second strands are generated with a second poly(dT)-tailed primer after poly(dA) tailing of the first-strand cDNAs. The cDNAs are split into four tubes, which are independently directionally amplified by PCR, and then recombined. The amplified products (approximately 100 ng) show superior representation and reproducibility of original gene expression, especially for genes expressed in more than 20 copies per cell, compared with those obtained by a conventional PCR protocol, and can effectively be used for quantitative PCR and EST analyses. The cDNAs are then subjected to another PCR amplification with primers bearing the T7 promoter sequence. The resultant cDNA products are gel purified, amplified by one final cycle and used for isothermal linear amplification by T7 RNA polymerase to synthesize cRNAs for microarray hybridization. This protocol yields cDNA templates sufficient for more than 80 microarray hybridizations from a single cell, and can be completed in 5-6 days.     

PCR检定OSM cDNA转染细胞中基因组整合与转录

用PCR和RT-PCR方法对人OSM cDNA转染的小鼠黑色素瘤细胞进行基因组整合和mRNA转录的检定.基因组整合检定时,采用与调控序列和cDNA序列相对应的上、下游引物,以连续的转录单位进行扩增,能够更准确地反映整合与表达的关系;mRNA检定时,采用与cDNA序列和质粒克隆位点与加polyA信号之间序列相对应的上、下游引物,可以区分宿主细胞中内源性与外源性基因的转录.     

Rapid amplification of genomic DNA ends bynla III partial digestion and polynucleotide tailing

We report a simple and efficient method, which combines restriction endonuclease digestion and deoxynucleotide tailing, for cloning unknown genomic sequences adjacent to a known sequence. Total genomic DNA is partially digested with the frequent-cutting restriction enzymeNla III. A homo-oligomeric cytosine tail is added by terminal transferase. The tailed DNA fragments are used as the template for cloning flanking regions from all sequences of interest. A first round PCR amplification is performed with a gene-specific primer and the selective (modified polyguanine) anchor primer complementary to the cytosine tail and theNla III recognition site, with a universal amplification primer sequence at its 5′ end. This is followed by another PCR amplification with a nested gene-specific primer and the universal amplification primer. Finally, the amplified products are fractionated, cloned, and sequenced. Using this method, we cloned the upstream region of a salt-induced gene based upon a partial cDNA clone (RSC5-U) obtained from sunflower (Helianthus annuus L.).