A Draft Sequence of the Rice Genome ( Oryza sativa L. ssp. indica )
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Tài liệu tham khảo
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For the plasmid shotgun libraries a DNA isolation protocol was modified from Sambrook and Russell (29). Fresh leaves at the seeding stage were ground in liquid nitrogen before complete lysis (30). Purified high–molecular weight genomic DNA was sonicated and sized on agarose gels selecting for fragments of size 1.5 to 3.0 kb. QIAEX Gel Extraction Kit (QIAGEN) was used to purify DNA from the gel slices. Genomic fragments were ligated to Sma I–linearized pUC18 plasmids and transformed into DH10B-competent cells by electroporation.
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Single colonies were grown in 96-deep-well plates and plasmid DNA was prepared by alkaline lysis (32). Quality of DNA and insert sizes were examined by agarose gel electrophoresis. Purified plasmid DNA (200 ng; Amersham Pharmacia Biotech Beijing) was used for the sequencing reactions. DNA sequencing was done with MegaBACE 1000 capillary sequencers (Amersham Pharmacia Biotech Beijing). Machine parameters were adjusted for high output (10 to 11 runs a day on average).
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For each protein query we created an array with one element for each amino acid position. Blast_hits() recorded the number of times that each position was covered by a TblastN hit. Each hit had associated with it a score for the percentage of identically matched amino acids. AA_identity() recorded the maximum and minimum score at each position across all TblastN hits. “Extent of hit ” quoted as a percentage of the protein length is the number of nonzero elements in Blast_hits(). “AA identity” and “hits per gene” are the median values of AA_identity() and Blast_hits() computed over positions with one or more hits. We used the median instead of the mean to minimize the likelihood of counting a highly duplicated domain when the entire protein is not duplicated.
Source for BAC-end sequences: .
We are indebted to faculty and staff at the Beijing Genomics Institute whose names were not listed but who also contributed to the team effort (www.genomics.org.cn). We are indebted to our scientific advisors M. V. Olson L. Bolund R. Waterston E. Lander and M.-C. King for their long-term support. We are grateful to R. Wu and C. Herlache for editorial assistance on the manuscript. We thank Amersham Pharmacia Biotech (China) Ltd. SUN Microsystems (China) Inc. and Dawning Computer Corp. for their support and service. This project was jointly sponsored by the Chinese Academy of Science the Commission for Economy Planning the Ministry of Science and Technology the Zhejiang Provincial Government the Hangzhou Municipal Government the Beijing Municipal Government and the National Natural Science Foundation of China. The analysis was supported in part by the National Institute of Environmental Health Sciences (grant 1 RO1 ES09909).