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S.Prasanth Kumar, Bioinformatician Gene Expression Studies Serial Analysis of Gene Expression (SAGE) Technology S.Prasanth Kumar Dept. of Bioinformatics Applied Botany Centre (ABC) Gujarat University, Ahmedabad, INDIA www.facebook.com/Prasanth Sivakumar FOLLOW ME ON ACCESS MY RESOURCES IN SLIDESHARE prasanthperceptron CONTACT ME [email_address]

Why SAGE ? Each organism can supply required amount of products at an appropriate time that confer functions proper to the organism e.g. cell cycle proteins To generally understand the cellular functions under the certain conditions at a certain time, it can be attained by measuring the species and respective numbers of mRNAs at a point of time Species= mRNAs of different genes Each cell contains more than 10 000 species , copies of each species ranging from less than one to more than 10 000, and, as a total, up to half a million mRNA transcript copies. It was therefore practically impossible to determine them

Large-scale Random cDNA sequencing by EST project mRNA Species 1 ……………. mRNA Species n Plasmid Insertion cDNA clones RE Assemble EST1…n Hence, sequencing = n x n times cDNA Assemble EST1…n Assemble EST1…n of all seq. projects All steps

SAGE Scheme SAGE method allows for a quantitative and simultaneous analysis of a large number of transcripts in any particular cells or tissues mRNA species 1 mRNA species 2 mRNA species 3 9–10 bp tag AAAAA AAAAA AAAAA clone Extract tags ,concatenate in plasmid

SAGE Scheme Isolate insertion seq from plasmid sequencing TAGCGG.. ATGCGGC.. TATTTTAGC… mRNA tag of species 1 mRNA tag of species 2 mRNA tag of species 3 Use BLAST service Human genome ATCGCC TAGCGG TACGCCG ATGCGGC ATAAAATCGTATTTTAGC Annotated Gene 1 Annotated Gene 12 Annotated Gene 34 Result : gene 1, 12, 34 are expressed during certain time say mitosis

SAGE procedure AAAAA mRNA mRNa-cDNA hybrid TTTTT Oligo(dT)-primer AAAAA Remove RNA by RNase H TTTTT ds cDNA synthesis TTTTT AAAAA

SAGE procedure AAAAA TTTTT Cleave with anchoring enzyme (AE) e.g. NlaIII TTTTT AAAAA 5’ GTAC Bind to streptavidin beads TTTTT 5’ GTAC Divide in half TTTTT 5’ GTAC AAAAA AAAAA TTTTT AAAAA 5’ GTAC

SAGE procedure GTAC AAAAA TTTTT CATG GGGA CCCT A GTAC CATG GGGA CCCT B AAAAA TTTTT Linkers A Linkers B Cleave Tagging Enzyme (TE) e.g. Bsm FI. Linkers have RE site for BsmFI TE RE site TE RE site GTAC CATG GGGA CCCT A NNNNN NNNNNNNNNNNNN Staggered end CATG GGGA CCCT B NNNNN NNNNNNNNNNNNN GTAC T4 DNA polymerase GTAC CATG GGGA CCCT A NNNNNN NNNNNNN NNNNNNNNNNNNN CATG GGGA CCCT B NNNNN NNNNNNNN NNNNNNNNNNNNN GTAC Blunt end

SAGE procedure GTAC CATG GGGA CCCT A NNNNNNNNNNNNN NNNNNNNNNNNNN CATG GGGA CCCT B NNNNNNNNNNNNN NNNNNNNNNNNNN GTAC 5’ 5’ Ligate tail-to-tail orientation GTAC CATG GGGA CCCT A NNNNNNNNNNNNN NNNNNNNNNNNNN CATG CCCT GGGA B NNNNNNNNNNNNN NNNNNNNNNNNNN Amplify by primers A and B GTAC CATG GGGA CCCT A NNNNNNNNNNNNN NNNNNNNNNNNNN NNNNNNNNNNNNN NNNNNNNNNNNNN primer A primer B GTAC CATG CCCT GGGA B GTAC

SAGE procedure After 1 round of amplification GTAC CATG GGGA CCCT A NNNNNNNNNNNNN NNNNNNNNNNNNN NNNNNNNNNNNNN NNNNNNNNNNNNN GTAC CATG GGGA CCCT A NNNNNNNNNNNNN NNNNNNNNNNNNN NNNNNNNNNNNNN NNNNNNNNNNNNN Cleave with AE AE RE site AE RE site NNNNNNNNNNNNN NNNNNNNNNNNNN NNNNNNNNNNNNN NNNNNNNNNNNNN GTAC CATG CATG GGGA CCCT A CATG CCCT GGGA B CATG CCCT GGGA B GTAC GTAC GTAC CCCT GGGA B GTAC NNNNNNNNNNNNN NNNNNNNNNNNNN NNNNNNNNNNNNN NNNNNNNNNNNNN GTAC CATG GTAC Isolate ditags

SAGE procedure NNNNNNNNNNNNN NNNNNNNNNNNNN NNNNNNNNNNNNN NNNNNNNNNNNNN GTAC CATG NNNNNNNNNNNNN NNNNNNNNNNNNN NNNNNNNNNNNNN NNNNNNNNNNNNN GTAC concatenate NNNNNNNNNNNNN NNNNNNNNNNNNN NNNNNNNNNNNNN NNNNNNNNNNNNN GTAC CATG NNNNNNNNNNNNN NNNNNNNNNNNNN NNNNNNNNNNNNN NNNNNNNNNNNNN GTAC Insert into plasmid & clone CATG CATG You can concatenate n number of species Remember, 1 mRNA species gives 2 ds cDNA joined by Palindromic Sequences

SAGE procedure NNNNNNNNNNNNN NNNNNNNNNNNNN NNNNNNNNNNNNN NNNNNNNNNNNNN GTAC CATG NNNNNNNNNNNNN NNNNNNNNNNNNN NNNNNNNNNNNNN NNNNNNNNNNNNN GTAC CATG 1 mRNA species mRNA species no. 1 mRNA species no. 2 mRNA species no. 3 mRNA species no. n plasmid

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