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Predicting full-length transcripts 总被引:2,自引:0,他引:2
Brent MR 《Trends in biotechnology》2002,20(7):273-5; discussion 275
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Shinji Kondo Akira Shinagawa Tetsuya Saito Hidenori Kiyosawa Itaru Yamanaka Katsunori Aizawa Shiro Fukuda Ayako Hara Masayoshi Itoh Jun Kawai Kazuhiro Shibata Yoshihide Hayashizaki 《Mammalian genome》2001,12(9):673-677
Although the sequencing of the human genome is complete, identification of encoded genes and determination of their structures
remain a major challenge. In this report, we introduce a method that effectively uses full-length mouse cDNAs to complement
efforts in carrying out these difficult tasks. A total of 61,227 RIKEN mouse cDNAs (21,076 full-length and 40,151 EST sequences
containing certain redundancies) were aligned with the draft human sequences. We found 35,141 non-redundant genomic regions
that showed a significant alignment with the mouse cDNAs. We analyzed the structures and compositional properties of the regions
detected by the full-length cDNAs, including cross-species comparisons, and noted a systematic bias of GENSCAN against exons
of small size and/or low GC-content. Of the cDNAs locating the 35,141 genomic regions, 3,217 did not match any sequences of
the known human genes or ESTs. Among those 3,217 cDNAs, 1,141 did not show any significant similarity to any protein sequence
in the GenBank non-redundant protein database and thus are candidates for novel genes.
Received: 18 January 2001 / Accepted: 17 May 2001 相似文献
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Large-scale identification and characterization of alternative splicing variants of human gene transcripts using 56,419 completely sequenced and manually annotated full-length cDNAs 下载免费PDF全文
Takeda J Suzuki Y Nakao M Barrero RA Koyanagi KO Jin L Motono C Hata H Isogai T Nagai K Otsuki T Kuryshev V Shionyu M Yura K Go M Thierry-Mieg J Thierry-Mieg D Wiemann S Nomura N Sugano S Gojobori T Imanishi T 《Nucleic acids research》2006,34(14):3917-3928
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Current strategies for cDNA cloning are based on construction of cDNA libraries and colony screening. The process of obtaining a full-length cDNA clone can be highly time and labor intensive. Using the human actin gene as a model target cDNA, we have developed an RNA-capture method for rapid cloning of full-length cDNAs. The approach involves the capture of mRNA with expressed sequence tag (EST)-derived, biotin labeled antisense "capture" primers and streptavidin-coated magnetic beads. Full-length cDNA is then synthesized from purified EST-specific mRNA and cloned directly into plasmid vectors. The results of using beta-actin-based capture primers on cytoplasmic RNA were the isolation of both beta- and gamma-actin cDNA clones. Of the 16 actin-specific cDNA clones analyzed, 15 (93%) were full-length. This approach for cloning full-length cDNAs from available ESTs or partial cDNA sequences will facilitate a more rapid and efficient characterization of gene structure and function. 相似文献
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The ability to generate and obtain full-length (FL) cDNAs is of critical importance to the field of genomics. Most cDNAs in a traditional cDNA library lack the initiating 5' ATG, making it difficult to obtain a FL clone. We report here on an improved protocol for the preparation of FL enriched cDNA libraries. We demonstrate that if good quality RNA is used in the cDNA synthesis, high-quality, FL cDNA can be generated for messages upward of 7 kb. In addition, we demonstrate the utility of size fractionation as a means to produce libraries containing a high percentage of initiating 5' ATG containing clones with insert sizes greater than 4 kb. The method is simple, cost efficient, and can be performed in most laboratories equipped to perform molecular biology. Lastly, the novel methodologies used in the analysis of the cDNA and library should prove useful to others working to create high-quality cDNA libraries. 相似文献
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Hirozane-Kishikawa T Shiraki T Waki K Nakamura M Arakawa T Kawai J Fagiolini M Hensch TK Hayashizaki Y Carninci P 《BioTechniques》2003,35(3):510-6, 518
The normalization and subtraction of highly expressed cDNAs from relatively large tissues before cloning dramatically enhanced the gene discovery by sequencing for the mouse full-length cDNA encyclopedia, but these methods have not been suitable for limited RNA materials. To normalize and subtract full-length cDNA libraries derived from limited quantities of total RNA, here we report a method to subtract plasmid libraries excised from size-unbiased amplified lambda phage cDNA libraries that avoids heavily biasing steps such as PCR and plasmid library amplification. The proportion of full-length cDNAs and the gene discovery rate are high, and library diversity can be validated by in silico randomization. 相似文献
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Antisense transcripts at the EMX2 locus in human and mouse 总被引:2,自引:0,他引:2