A simple,rapid, efficient and inexpensive strategy for sequencing clones from cDNA libraries

In this report, we describe a simple, rapid, efficient and inexpensive strategy for sequencing inserted DNAs from clones of cDNA or gDNA libraries. This strategy uses PCR products directly amplified from transformed bacterial colonies, with universal primers within the vector. The method can be applied for sequencing cDNA or gDNA libraries with up to 4 ∼ 5 kb insert sizes, without overnight liquid culture or plasmid DNA preparation steps. We successfully used this method to analyze clones from full-length, enriched cDNA libraries. Although simple, following this strategy will significantly help researchers to avoid unnecessary steps in the analysis of a cDNA library.     

Random sequencing of cDNA libraries reveals a variety of expressed genes in cultured cells of rice (Oryza sativa L.)

Large scale sequencing of randomly selected cDNA clones was carried out to investigate the feasibility of this method for isolating plant genes. cDNA libraries were made using mRNA prepared from suspension-cultured cells of rice (Oryza sativa L.). Partial nucleotide sequences of 830 individual cDNA clones have been determined and compared with the GenBank database. Approximately 8% of the cDNA clones could be identified as particular genes. This method provides the opportunity to isolate large numbers of plant genes.     

Systematic sequencing of cDNA clones using the transposon Tn5

In parallel with the production of genomic sequence data, attention is being focused on the generation of comprehensive cDNA-sequence resources. Such efforts are increasingly emphasizing the production of high-accuracy sequence corresponding to the entire insert of cDNA clones, especially those presumed to reflect the full-length mRNA. The complete sequencing of cDNA clones on a large scale presents unique challenges because of the generally small, yet heterogeneous, sizes of the cloned inserts. We have developed a strategy for high-throughput sequencing of cDNA clones using the transposon Tn5. This approach has been tailored for implementation within an existing large-scale ‘shotgun-style’ sequencing program, although it could be readily adapted for use in virtually any sequencing environment. In addition, we have developed a modified version of our strategy that can be applied to cDNA clones with large cloning vectors, thereby overcoming a potential limitation of transposon-based approaches. Here we describe the details of our cDNA-sequencing pipeline, including a summary of the experience in sequencing more than 4200 cDNA clones to produce more than 8 million base pairs of high-accuracy cDNA sequence. These data provide both convincing evidence that the insertion of Tn5 into cDNA clones is sufficiently random for its effective use in large-scale cDNA sequencing as well as interesting insight about the sequence context preferred for insertion by Tn5.     

A novel system for large-scale sequencing of cDNA by PCR amplification.

We have developed a method for constructing a library containing the 3' end fragment of cDNA for large-scale sequencing of cDNA clones. The average size of the insert was 270 bp. Cell lysates that carry plasmids having the cDNA insert were subjected to PCR amplification of the cDNA moiety and the products were subjected to sequencing analysis using an autosequencer. With this protocol, sample preparation became a non-limiting step, that allowed us to sequence as many samples as the autosequencer could handle.     

Generation of expressed sequence tags of random root cDNA clones of Brassica napus by single-run partial sequencing.

Two hundred thirty-seven expressed sequence tags (ESTs) of Brassica napus were generated by single-run partial sequencing of 197 random root cDNA clones. A computer search of these root ESTs revealed that 21 ESTs show significant similarity to the protein-coding sequences in the existing data bases, including five stress- or defense-related genes and four clones related to the genes from other kingdoms. Northern blot analysis of the 10 data base-matched cDNA clones revealed that many of the clones are expressed most abundantly in root but less abundantly in other organs. However, two clones were highly root specific. The results show that generation of the root ESTs by partial sequencing of random cDNA clones along with the expression analysis is an efficient approach to isolate genes that are functional in plant root in a large scale. We also discuss the results of the examination of cDNA libraries and sequencing methods suitable for this approach.     

An efficient strategy for large-scale high-throughput transposon-mediated sequencing of cDNA clones

We describe an efficient high-throughput method for accurate DNA sequencing of entire cDNA clones. Developed as part of our involvement in the Mammalian Gene Collection full-length cDNA sequencing initiative, the method has been used and refined in our laboratory since September 2000. Amenable to large scale projects, we have used the method to generate >7 Mb of accurate sequence from 3695 candidate full-length cDNAs. Sequencing is accomplished through the insertion of Mu transposon into cDNAs, followed by sequencing reactions primed with Mu-specific sequencing primers. Transposon insertion reactions are not performed with individual cDNAs but rather on pools of up to 96 clones. This pooling strategy reduces the number of transposon insertion sequencing libraries that would otherwise be required, reducing the costs and enhancing the efficiency of the transposon library construction procedure. Sequences generated using transposon-specific sequencing primers are assembled to yield the full-length cDNA sequence, with sequence editing and other sequence finishing activities performed as required to resolve sequence ambiguities. Although analysis of the many thousands (22 785) of sequenced Mu transposon insertion events revealed a weak sequence preference for Mu insertion, we observed insertion of the Mu transposon into 1015 of the possible 1024 5mer candidate insertion sites.     

用PCR直接筛选和鉴定虎纹捕鸟蛛毒素Ⅰ的重组阳性克隆

以合成的两段插入序列为上、下游引物用PCR法直接筛选插入有虎纹捕鸟蛛毒素Ⅰ（HWTX－Ⅰ）cDNA的重组阳性克隆。并用PCR法快速鉴定重组体中插入片段的正、反连接方向,扩增用引物是以位于克隆位点上游的一段载体序列上游引物,以插入序列为下游引物。对100个单克隆进行了上述两次PCR筛选鉴定,选取2个有靶片段插入并且为正向连接的重组子进行测序,其结果证实了插入片段及其方向的正确性。     

Construction of a Normalized cDNA Library for the Trypanosoma cruzi Genome Project

Sequencing of the Trypanosoma cruzi genome is underway. Expressed sequence tags, obtained from cDNA libraries, facilitate mapping and gene discovery. The efficiency of large-scale generation of such tags is increased when using normalized cDNA libraries, where the frequency of individual clones is brought within a narrow range. Repetitive sequencing of abundant clones is therefore minimized. We constructed a normalized cDNA library from epimastigotes of clone CL Brener, and the efficiency of normalization of representative clones was assessed and shown to be adequate. The normalized cDNA library has been distributed to several groups and large-scale sequencing is currently in progress.     

Rapid verification of lambda-cDNA clones using mixed-base oligonucleotide as screening probe and sequencing primer

A rapid procedure has been developed for the isolation and verification of cDNA clones isolated from a cDNA library based on lambda vectors. Using information of the partial amino acid sequence of a protein, synthetic mixed-base oligonucleotides are first employed as a screening probe using the plaque hybridization procedure. The cDNA inserts of the clones obtained are then directly amplified by polymerase chain reaction (PCR) using primers flanking the cloning site of the vector. Besides being used for cloning into a plasmid vector, the amplified DNA's are also subjected to nucleotide sequence analysis using the same mixed-base oligonucleotides as sequencing primers. This approach allows sequencing through the region of the known amino acid sequence for direct verification of the authenticity of the clones obtained. This procedure has successfully been used for cloning and partial characterization of the gene coding for a platelet aggregation inhibitor.     

Construction of cDNA libraries for highly efficient DNA sequencing from the 3' end of expressed genes

The majority of expressed sequence tag (EST) sequences available today have been derived from the 5' ends of cDNA clones. Obtaining high-quality DNA sequences from the 3' ends of oligo(dT)-primed cDNA on a large scale has been difficult because of slippage of the DNA polymerase enzyme used in direct PCR and cycle sequencing. With the completion of whole genome sequencing for more and more organisms, mRNA 3'-UTR sequences can be particularly useful for clustering large numbers of ESTs for the effective discrimination of individual genes and gene families. We have identified a flaw in the widely used oligo(dT) primers for cDNA synthesis, and here we describe an improved priming approach to effectively synthesize cDNA devoid of homopolymeric nucleotide stretches from mRNA poly(A) tails to enable highly efficient and reliable DNA sequence determination from 3' mRNA ends. Using this method, we produced a rat lung cDNA library and successfully sequenced the 3' ends of 98% of all attempted clones.     

Use of high coverage reference libraries of Drosophila melanogaster for relational data analysis. A step towards mapping and sequencing of the genome

Three differently made, primary Drosophila cosmid libraries of 16-fold genome coverage have been generated. Also, a jumping library has been created by a new method that takes advantage of methylation differences between genomic DNA and vector. Thirdly, two cDNA libraries have been picked. All these libraries have been arrayed on high-density in situ filters, each containing 9216 clones. As a reference system, such filters are distributed and identified clones are provided. Single-copy probes have identified on average 1.4 cosmids per genome equivalent. Together with cytogenetically mapped yeast artificial chromosomes, the libraries are also being used for physically mapping the genome, mainly by oligonucleotide fingerprinting and pool hybridizations. cDNA clones are further examined by a partial sequencing analysis by oligomer hybridization.     

Isolation and characterization of expressible cDNA clones for mouse Thy-1: a model system for cDNA expression of cell surface proteins

By using the mouse Thy-1 gene as a model, we have developed a procedure to distinguish functional vs nonfunctional cDNA of lymphocyte surface antigens by transfecting COS-7 monkey cells and testing for expression of cell surface products encoded by the cDNA inserts. By cross-hybridization with a mouse Thy-1 probe, we isolated cDNA clones from a pcD-expression library prepared from mRNA of C5 cells. Two functional clones were distinguished from the remainder by detection of Thy-1.2 on the surface of 0.5% of COS-7 cells transiently transfected by the DEAE-Dextran method. Inclusion of chloroquine in the transfection procedure greatly facilitated the detection of functional cDNA by raising the percentage of expressing cells to 30%. Nucleotide sequencing of one functional cDNA, about 1700 bp long, confirmed that the gene encodes a protein whose sequence agrees with the published Thy-1.2 protein sequence with the additional 31 amino acids attached at the COOH-terminus. A 75 bp 5' untranslated region preceding the coding region contains 50 bp not found in the genomic clones. Comparison indicates that one or more introns are present in the 5' untranslated region, but are not found in the mature mRNA. The first exon may be separated by at least 1 kb intron from the initiation codon. Because the expressible clones are approximately the size of the mRNA seen on Northern blots, we believe that these clones are nearly full-length cDNA. Dilution experiments indicate that this strategy should also be useful for identifying functional cDNA clones for cell surface proteins solely on the basis of their expression in mammalian cells.     

Cloning full-length, cap-trapper-selected cDNAs by using the single-strand linker ligation method.

We have developed the single-strand linker ligation method (SSLLM), which uses DNA ligase to add a dsDNA linker to single-stranded (ss) full-length cDNA. The linkers have random 6-bp (dN6 or dGN5) 3' overhangs that can ligate to any cDNA sequence, thereby facilitating the production of cDNA libraries with titers exceeding 1 x 10(6) independent clones. We confirmed that the 5' ends of cDNA inserts cloned by using SSLLM are full-length and include the 5' untranslated regions. The great advantage of our method is that the elimination of the GC tail simplifies the sequencing and protein translation of the full-length clones. Further, our method tags ss cDNAs more efficiently than does the traditional RNA ligase reaction.