Enhanced concatemer cloning-a modification to the SAGE (Serial Analysis of Gene Expression) technique.
نویسنده
چکیده
The Serial Analysis of Gene Expression (SAGE) method, described in 1995 by Velculescu et al ., represents a powerful means to compare gene expression between two mRNA populations. An improvement to SAGE that removes contaminating linker molecules, which compromise the efficiency of the method, has been developed. This modification utilises biotinylated PCR primers, which generate biotinylated linkers at an early stage in the SAGE protocol, thus allowing removal of the unwanted linkers by binding to streptavidin-coated magnetic beads at a later stage. The application of this modification resulted in the rapid generation of high ditag yields and clones with large average insert sizes.
منابع مشابه
Substantially enhanced cloning efficiency of SAGE (Serial Analysis of Gene Expression) by adding a heating step to the original protocol.
The efficiency of the original SAGE (Serial Analysis of Gene Expression) protocol was limited by a small average size of cloned concatemers. We describe a modification of the technique that overcomes this problem. Ligation of ditags yields concatemers of various sizes. Small concatemers may aggregate and migrate with large ones during gel electrophoresis. A heating step introduced before gel el...
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Serial analysis of gene expression (SAGE) is a widely used technique for large-scale transcriptome analysis in mammalian systems. Recently, a modified version called LongSAGE (S. Saha, A.B. Sparks, C. Rago, V. Akmaev, C.J. Wang, B. Vogelstein, K.W. Kinzler [2002] Nat Biotechnol 20: 508–512) was reported by increasing tag length up to 21 bp. Although the procedures for these two methods are simi...
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BENCHMARKS The serial analysis of gene expression (SAGE) provides high-throughput information on researched samples. SAGE generates 9–13 bp tags from messenger RNA (mRNA) of the sample, and all tags are ligated to form concatemers for cloning and sequencing (1). LongSAGE uses similar procedures but a different restriction enzyme to obtain longer tags (19–21 bp). Both SAGE and LongSAGE tags are ...
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The serial analysis of gene expression (SAGE) has become an important technique for high-throughput transcriptome analysis. Since its first description in 1995 (1), SAGE has provided both quantitative and qualitative information concerning global gene expression in a variety of biological systems. Thereby, it has deepened our knowledge concerning developmental processes as well as pathogenic me...
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ورودعنوان ژورنال:
- Nucleic acids research
دوره 26 14 شماره
صفحات -
تاریخ انتشار 1998