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SAGE Libraries Construction and Sequencing
SAGE Libraries Construction and Sequencing
Place of Origin:
China
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Shanghai Huaguan Biochip Co., Ltd.
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Updated:
4/24/2011
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  • Product Detail
  • Company Profile
    SAGE, ie Serial Analysis of Gene Expression, is a patented technology owned and licensed by Genzyme Corporation. SAGE technology enables you to analyze every transcript in a cell without the need for any prior knowledge of the transcript and is unaffected by any PCR or cloning bias. The construction of SAGE libraries and sequencing provide a way to quantitatively analyze the patterns of gene expression.

    Concept ----SAGE is based on three principles:

    A short sequence tag (9~14 bp) obtained from a defined region within each transcript contains sufficient information to uniquely identify a transcript.

    Sequence tags can be linked together to form long DNA molecules (concatemers) that can be cloned and sequenced. Sequencing of the concatemer clones results in the identification of individual tags.

    The expression level of the transcript is quantified by the number of times a particular tag is observed.

    Description

    1. Synthesize double stranded cDNA with the templates of your sample mRNA.

    2. Digest the double stranded cDNA with a sequence specific restriction enzyme (an anchoring enzyme) that cleaves most transcripts at least once, eg. Nla III.

    3. Divide the cDNA into two fractions and ligate with two adapters (A and B, ~40 bp each). The adapters contain cohesive 4 bp overhangs complementary to the Nla III digested cDNA, a Type IIS restriction enzyme (tagging enzyme) recognition site at the 3′ end, and priming sites for PCR amplification.

    4. Cleave with Type IIS restriction enzyme (tagging enzyme), eg. BsmF I. The tagging enzyme binds to the recognition sequence in the adapter and cleaves the cDNA 10-14 bp downstream from the recognition site releasing a ~50 bp tag with a 4 bp overhang at the 5′ end. The tag consists of ~40 bp of adapter sequence and 10-14 bp of unique sequence from a single transcript.

    5. Perform a Klenow reaction to fill-in the 5′ overhangs created by BsmF I digestion and ligate the two fractions of tags to form ~100 bp ditags.

    6. Amplify the ~100 bp ditags using primers specific for the primer binding sites in the adapters to produce sufficient ditags for subsequent generation of concatemers. Since the tags are of short, uniform (~100 bp) size there is no PCR bias.

    7. Cleave the ~100 bp ditags with the anchoring enzyme to release a 26 bp ditag. These ditags are comprised entirely of sequences derived from transcript cDNAs and each ditag is punctuated by Nla III recognition sequence.

    8. Ligate the 26 bp ditags to form concatemers.

    9. Clone the concatemers into the vector to obtain a SAGE library. Sequence selected clones. Each transcript is identified by its unique 10-14 bp sequence tag and is quantified by the number of times the tag occurs within a given population of clones.

    Applications

    Obtain a comprehensive gene expression profile for a specific tissue or cell type

    Identify novel genes

    Characterize transcriptomes

    Study gene expression patterns in normal, developmental, or disease states.
    bio-equip.cn
    Shanghai Biochip Co., Ltd.（SBC） was founded in Aug. 2001. The existing shareholders of the company include some well-known universities, research institutes, hospitals and enterprises in China. In Feb. 2003, the National Development and Reform Commission authorized SBC to build up and operate the National Engineering Center for biochip at Shanghai project. The company is located in the Zhangjiang High-tech Park Biotech&Pharmaceutical Industrial Base in Pudong Shanghai with a total area of 40,000 square meters, a construction area of over 20,000 square meters, and a current employee number of more than 300 people.

    In accordance with company's development strategy and the construction requirements raised by National Development and Reform Commission for the National Engineering Center, SBC is committed to applying and promoting biotechnology development and products for life science research、health-care、 drug discovery and other fields. Through more than 10 years of construction and development, the technology innovation service system is established which is based on the core platforms of the biological sample bank, tissue chips, gene expression chips and the next-generation sequencing; The industrialized system which takes molecular medical diagnosis technology and product development as the core competitiveness has been set up; And three main business areas which include the services for research and drugs R&D, molecular medicine inspection services, and production and sales of molecular diagnostic product.

    SBC obeys the value idea of “Scientific innovation, Business orientation, Career development”, follows the construction requirements for the National Engineering Center, and strives to make the company an internationally-advanced national bio-chip industrialization base which integrates the function of technology innovation, achievements transformation, comprehensive service and personal training as a whole. What’s more, the company’s strategic goal is to be an innovative benchmark enterprise among the industry with an level internationally-advanced and has the capability of independent research and development. It would make a contribution to the goal of making Shanghai an influential technology innovation center and also contribute to the domestic field of biomedical science and technology innovation and industrialization in our country.
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