Improved protocol for SAGE tag-to-gene allocation.
Serial analysis of gene expression (SAGE) is a powerful method for large-scale analysis of gene expression patterns. SAGE yields digital information on transcript abundance by the use of short sequence fragments (tags). Because SAGE does not require a priori knowledge of the expressed genes in the starting material, SAGE can be used for gene discovery. Unfortunately, correct tag-to-gene discovery. Unfortunately, correct tag-to-gene allocation after SAGE remains problematic when the short sequence of the tag corresponds to more than one gene in the reference database or when novel yet uncloned genes were detected. To overcome this problem, we developed an improved protocol for the proper identification of tag-corresponding genes. It relies on the isolation of 3'-terminal cDNA restriction fragments by the use of paramagnetic streptavidin beads and the ligation of linkers before the amplification step. Our protocol benefits from additional information encoded in each SAGE tag: its location 3'-terminal to the last NlaIII restriction site in the cDNA. In contrast to previously described protocols, stringent PCR conditions can be applied because of the length of the specific primers, which are composed of linker- and tag-specific sequences. Additionally, we demonstrate that our protocol yields quantitative information that can be used for further expression analysis of specific SAGE tags.
Kannbley
Ute
U
Humboldt University Berlin, Germany
Kapinya
Krisztian
K
Dirnagl
Ulrich
U
Trendelenburg
George
G
D000818:Animals; D001483:Base Sequence; D001709:Biotechnology; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D020224:Expressed Sequence Tags; D020869:Gene Expression Profiling / Q000379:methods* / Q000706:statistics & numerical data; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D016133:Polymerase Chain Reaction / Q000379:methods / Q000706:statistics & numerical data; D012680:Sensitivity and Specificity
D017931:DNA Primers; D018076:DNA, Complementary