A method for the preparation of normalized cDNA libraries enriched with full-length sequences

We developed a new method for the preparation of normalized cDNA libraries enriched with full-length sequences. It is based on the properties of the recently characterized duplex-specific nuclease from the hepatopancreas of the Kamchatka crab. The duplex-specific nuclease is thermostable, it effectively cleaves double-stranded DNA and is inactive toward single-stranded DNA (Shagin et al., Genome Res., 2002, vol. 12, pp. 1935-1942). Our method enables the normalization of cDNA samples enriched with full-length sequences without use of laborious and ineffective stages of physical separation. The efficiency of the method was demonstrated in model experiments using cDNA samples from several human tissues.     

A genetic strategy for differential screening of meiotic germ-cell cDNA libraries

The goals of this work were to create germ-cell-stage-specific cDNA libraries from mouse spermatogenic cells and to employ a novel two-step genetic screen to identify gene sequences present during the critical meiotic stage of spermatogenesis. Highly enriched germ-cell fractions were prepared from adult and juvenile mouse testes, and purity of these fractions was extensively analyzed by light and electron microscopy. Standard techniques were used to prepare cDNA libraries from populations of mixed leptotene and zygotene (L/Z) spermatocytes, pachytene (P) spermatocytes, and round spermatids. These libraries were analyzed with respect to representation of sequences from ubiquitously expressed genes, and from genes expressed at specific germ-cell stages as well as from genes expressed in testicular somatic cells. For the first step of the screening procedure, testicular cDNA was prepared from mutant mice carrying the T(X;11)38H chromosomal translocation that causes spermatogenic arrest at early meiotic prophase. This mixed cDNA probe was used to screen the libraries from L/Z and P spermatocytes to detect sequences that failed to hybridize. The clones identified were characterized for ability to hybridize to various germ-cell-specific cDNAs to verify that they represented sequences present in normal spermatogenic meiotic cells. These clones were then subjected to a second screening with another mutant probe; this time the cDNA probe was from testes of sterile mice bearing the T(X;16)16H chromosomal translocation that causes spermatogenic arrest at late meiotic prophase. This screen identified 27 clones that were not represented in testicular cDNA from T38-bearing mice or from T16-bearing mice. These clones may represent sequences essential for normal completion of the genetic events of meiosis during spermatogenesis. Likewise, the secondary screen identified 19 clones that were not represented in testicular cDNA from T38-bearing mice but were represented in testicular cDNA of T16-bearing mice. These clones are thus gene sequences present in spermatogenic cells during the time from early meiotic prophase to mid-to-late prophase. This strategy represents the first use of genetic aberrations in differential screening to identify genes expressed at specific times during mammalian spermatogenesis. © 1996 Wiley-Liss, Inc.     

Generation of full-length cDNA libraries enriched for differentially expressed genes for functional genomics

Here, we describe the application of a RecA-based cloning technology to generate full-length cDNA libraries enriched for genes that are differentially expressed between tumor and normal tissue samples. First, we show that the RecA-based method can be used to enrich cDNA libraries for several target genes in a single reaction. Then, we demonstrate that this method can be extended to enrich a cDNA library for many full-length cDNA clones using fragments derived from a subtracted cDNA population. The results of these studies show that this RecA-mediated cloning technology can be used to convert subtracted cDNAs or a mixture of several cDNA fragments corresponding to differentially expressed genes into a full-length library in a single reaction. This procedure yields a population of expression-ready clones that can be used for further high-throughput functional screening.     

Comparison of sequences of cDNA clones obtained from oligo-capping cDNA libraries with those from unigene.

We compared in detail the characteristics of the sequences of the cDNA clones obtained by the oligo-capping method (oligo-capping clones) with that of the sequences in the UniGene database. To compare the completeness of the sequences, three new variables, "fullness-proportion of clones" (the ratio of complete clones to total clones in a library), "fullness-proportion of genes" (the ratio of complete genes to total genes in a library), and "fullness-proportion of database" (the ratio of complete genes to total genes in a database sampled from a library), were defined. The fullness-proportion of clones of oligo-capping clones was 57.3%, 2.2 times larger than that of UniGene (25.9%). The fullness-proportion of genes of oligo-capping clones was 41.8%, 2.4 times larger than that of UniGene (17.8%). When gene length was restricted to > or = 1.5 kb, the fullness-proportion of genes of oligo-capping clones was four times larger than that of UniGene. The fullness-proportion of database of oligo-capping clones was approximately the same as that of UniGene. By simulating the clone redundancy, this coincidence was found to be due to the large redundancy of the UniGene database. Consequently, the cDNA sequence database of oligo-capping clones enabled high throughput selection of full-length cDNA clones.     

破色期番茄果实均一化cDNA沉默文库的构建和功能基因筛选模型的初步建立

番茄果实的成熟是由多基因精细调控的一个过程．利用破色期番茄果实,根据复性动力学原理在mRNA水平进行均一化操作使高丰度和低丰度的mRNA丰度接近．然后把均一化之后mRNA反转录得到cDNA,再与基因沉默载体pTRV重组,最后把构建好的载体通过电转化的方法转入到GV3101农杆菌中,从而建立起破色期番茄果实均一化cDNA沉默文库．通过番茄果实中病毒诱导基因沉默技术,对cDNA沉默文库进行初步筛选,从而确定功能基因筛选模型．在模型建立阶段,以番茄红素合成途径相关的PDS基因作为内标基因,在100个混合农杆菌样中,成功筛选到了PDS基因．     

A large scale analysis of cDNA in Arabidopsis thaliana: generation of 12,028 non-redundant expressed sequence tags from normalized and size-selected cDNA libraries.

For comprehensive analysis of genes expressed in the model dicotyledonous plant, Arabidopsis thaliana, expressed sequence tags (ESTs) were accumulated. Normalized and size-selected cDNA libraries were constructed from aboveground organs, flower buds, roots, green siliques and liquid-cultured seedlings, respectively, and a total of 14,026 5'-end ESTs and 39,207 3'-end ESTs were obtained. The 3'-end ESTs could be clustered into 12,028 non-redundant groups. Similarity search of the non-redundant ESTs against the public non-redundant protein database indicated that 4816 groups show similarity to genes of known function, 1864 to hypothetical genes, and the remaining 5348 are novel sequences. Gene coverage by the non-redundant ESTs was analyzed using the annotated genomic sequences of approximately 10 Mb on chromosomes 3 and 5. A total of 923 regions were hit by at least one EST, among which only 499 regions were hit by the ESTs deposited in the public database. The result indicates that the EST source generated in this project complements the EST data in the public database and facilitates new gene discovery.     

中华蜜蜂工蜂cDNA文库的构建及ESTs测序分析

【目的】为了解中华蜜蜂Apis cerana cerana工蜂的抗逆性,构建了中华蜜蜂工蜂的cDNA文库,并对文库质量进行分析。【方法】本研究利用SMART技术构建了中华蜜蜂工蜂的全长cDNA文库。【结果】文库库容为3.6×106 cfu/m L,文库重组率为97%,插入片段长度多数分布在1 000 bp左右。挑取cDNA克隆进行EST测序,共进行了306个成功反应,软件拼接共得到234个单基因簇(Unigene),其中包括207个单拷贝(Singletons)序列及27个重叠群(Contigs)。使用Blastx将这些序列同Gen Bank等数据库进行查询、比对和注释,结果显示141条序列有相关同源性,其他序列没有明显的同源性,这也为我们发现新功能基因提供了可靠依据。【结论】此文库的构建在中华蜜蜂功能基因的分离、克隆、筛选以及基因功能研究等方面具有重要作用。     

A hybridization and enzyme blocking method to enrich minor cDNA sequences

A method is presented which allows for the enrichment of low frequency cDNA sequences. The crucial step in the procedure is the hybridization of a pool of cDNA to homologous or heterologous RNA to a Rot value which will leave minor sequences in a single strand cDNA form while the major sequences form cDNA:RNA hybrids. This allows subsequent enzymatic differentiation between major and minor sequences resulting ultimately in the degradation of the major sequences. The procedure is general and simple as it requires no column chromatography step. The method is designed to integrate into a widely used cDNA cloning protocol and results either in double-stranded cDNA which can be used for molecular cloning or as a source of probes for hybridization.     

A simple and robust method for preparation of cDNA nylon microarrays.

DNA array technology has made remarkable progress in recent years and has become an indispensable tool in molecular biology research. However, preparing high-quality custom-made DNA arrays at a reasonable cost is still an important concern because we cannot abandon the use of DNA array systems designed for specific purposes. To address these problems, we here report the use of rolling circle amplification products of cDNA plasmids dissolved in 80% formamide as DNA probes immobilized on a nylon membrane. First, because formamide is practically non-volatile under ambient conditions and nucleic acids are easily dissolved in it, the use of formamide as a DNA solvent ensures that the DNA concentration of the solution will not change during arraying, which often takes several hours to a day depending on the number of DNA spots and arrays to produce. Secondly, the use of rolling circle amplification technology greatly reduced the labor needed to prepare the spotted DNA. The results in this study demonstrate that the introduction of these two modifications in preparation of nylon DNA array greatly improved its quality.     

Characterization of size-fractionated cDNA libraries generated by the in vitro recombination-assisted method.

We here modified a previously reported method for the construction of cDNA libraries by employing an in vitro recombination reaction to make it more suitable for comprehensive cDNA analysis. For the evaluation of the modified method, sets of size-selected cDNA libraries of four different mouse tissues and human brain were constructed and characterized. Clustering analysis of the 3' end sequence data of the mouse cDNA libraries indicated that each of the size-fractionated libraries was complex enough for comprehensive cDNA analysis and that the occurrence rates of unidentified cDNAs varied considerably depending on their size and on the tissue source. In addition, the end sequence data of human brain cDNAs thus generated showed that this method decreased the occurrence rates of chimeric clones by more than fivefold compared to conventional ligation-assisted methods when the cDNAs were larger than 5 kb. To further evaluate this method, we entirely sequenced 13 human unidentified cDNAs, named KIAA1990-KIAA2002, and characterized them in terms of the predicted protein sequences and their expression profiles. Taking all these results together, we here conclude that this new method for the construction of size-fractionated cDNA libraries makes it possible to analyze cDNAs efficiently and comprehensively.     

Comparative analysis of early embryonic sunflower cDNA libraries

To gain information concerning cell functions and activities during sunflower embryogenesis, an expressed sequence tag (EST) approach was used to analyse gene expression in the early stages of sunflower embryos development. Confocal microscopy observations of whole-mounted embryos allowed us to identify precisely the major steps of the zygotic embryonic development. A time-course analysis was then employed to collect the embryonic material. Three cDNA libraries were constructed from microdissected embryos, and three other cDNA libraries were created using a classical day after pollination schedule. A total of 7106 ESTs were produced and assembled. The total number of putative different genes represents about 43.1 (3064 tentative contigs and singlets) of the analysed sequences. The unigenes that showed similarity to proteins with known or predicted functions (50.3) were classified into 15 different functional categories. The functional profiles were found to be quite similar for all studied embryo stages but statistical analysis revealed that successive and coordinate sets of genes are expressed at each embryonic stage. The analysis allowed us to identify abundant and differentially expressed genes at the early stages of embryos development as well as some putatively interesting genes, showing strong similarities with genes playing key roles in plant and animal embryogenesis. The data presented in this study not only provide a first global overview of the genes expression profile during sunflower embryogenesis but also represent an original and valuable tool for developmental genomics studies on exalbuminous dicots.     

循环包装回收技术在筛选肝癌细胞相关耐药基因中的应用

肝癌先天性多表达多药耐药基因,严重影响肝癌的化疗效果,筛选肝癌细胞中的耐药基因,研究其耐药机制有助于提高肝癌化疗效果,提高肝癌的治愈率。首先构建肝癌细胞逆转录病毒的cDNA文库,感染成纤维细胞,使得逆转录病毒基因整合进细胞,加药筛选,存活细胞中的基因再次包装成病毒,用于下一轮筛选。采用循环包装回收(Cyclical packaging rescue,CPR)技术进行肝癌细胞耐药基因的筛选即是通过病毒包装将基因从细胞中钓取出来,相比于常规筛选方法,仅通过一轮筛选可能会出现很多假阳性基因,采用CPR技术则是通过多轮筛选,很大程度减少了假阳性细胞的出现。通过该方法经过四轮筛选获得核糖体蛋白S11(RPS11)、核糖体蛋白L6(RPL6)、核糖体蛋白L11(RPL11)、核糖体蛋白L24(RPL24)等几种基因,经初步检测,增加了细胞的耐药性。     

A powerful method for the preparation of cDNA libraries: isolation of cDNA encoding a 100-kDal nucleolar protein

An easy and quick method to synthesize large cDNA molecules and to clone them with very high efficiency in the expression vector lambda gt11 is described. The technique employs RNase H and Escherichia coli DNA ligase treatment during second-strand synthesis, followed by repair of the ds cDNA extremities by S1 nuclease and PolIk (Klenow fragment) treatment. This treatment allows efficient addition of suitable linkers and results in a 100-fold increase in the yield of cloned cDNA, when compared with other published techniques. Using 75 ng of poly(A)+ RNA from CHO cells, we have prepared a library of 1.1 X 10(7) clones. This library was screened with polyclonal antibodies raised against a 100-kDal nucleolar protein of CHO cells. Five recombinants were isolated with inserts of 500-2500 bp. The average size of cDNA obtained by this method is considerable: the 2500-bp cDNA represents 90% of the mRNA coding for the 100-kDal protein.     

Immunological screening of standard cDNA libraries in pBR322 vectors: detection of human fibrinogen and prothrombin cDNA clones

The in situ immunological detection of antigens encoded by cDNA inserted into the PstI site of pBR322 plasmids was optimized. It was found that sensitivity of the detection was dramatically increased by in situ amplification of the recombinant plasmids on chloramphenicol-containing medium followed by a brief incubation without chloramphenicol during which protein synthesis resumes. In addition, several modifications of the previously described methods which permit total suppression of background and false positives are described. These techniques allowed easy detection of cDNA clones for human B beta- and gamma-fibrinogen and -prothrombin using a human liver double-stranded cDNA recombinant plasmid library in pBR322 vectors.     

cDNA芯片差异表达基因检测的非转换方法

cDNA微阵列数据中包含许多变异因素,用于检测差异表达基因和其它统计分析前,必须将这些“噪音”剔除。对数比法（背景校正、对数比转换和数据标准化）已经被广泛应用于cDNA微阵列数据分析中,然而这种方法却存在着一些亟待解决的缺陷。对此,该文提出一种非转换方法,它可免去对数比的转化过程,直接在背景校正后进行数据标准化,可以有效剔除实验“噪音”。研究结果表明：在检测差异表达基因的效率方面,非转换方法比常规的对数比法具有更好的稳健性和更高的检测功效,基因检出率和准确性大大提高。     

A Non-transformation Method for Identifying Differentially Expressed Genes from cDNA Microarrays

cDNA microarray data are subject to many sources of variation that have to be removed before statistical tests can be applied for identifying genes that are expressed differentially. Background correction, log-ratio transformation, and normalization, referred as the log-ratio approach, have been widely used for this purpose. However, there are some problems associated with this procedure. In this study, we proposed an alternative approach that obviates the log-ratio transformation step and goes directly to normalization after background correction. The method can estimate the “noise” effect by utilizing the information more effectively. Simulation studies were carried out to compare the feasibility and efficiency of this approach for detecting the specifically and differentially expressed genes under various conditions with the log-ratio approach. The results showed that our approach worked well and was more robust and powerful than the log-ratio approach.