SAGE Libraries Construction and Sequencing
——Brief Technique Introduction
( According to Invitrogen Corporation )
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.
----SAGE is based on three principles:
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.