1) LIGATE BLUNT ENDS
2) Amplify by pcr using primers a and b
Cleave with anchoring enzyme
Ligate di-tags end to end il
Clone and sequence FIGURE 25.20 SAGE—The Procedure
The first step in making long concatemers of expressed sequences involves isolating the total cellular mRNA and making the corresponding cDNA. The total mRNA is bound via its poly(A) tail to an oligo(dT) primer linked to biotin. It is then converted to cDNA using reverse transcriptase.
The cDNAs are then truncated to short, tagged sequences. First, the cDNAs are cleaved with a restriction enzyme known as the anchoring enzyme. This generates a pool of shortened cDNA averaging 256 bp long, with some longer and others shorter. These are isolated using streptavidin, which binds to the biotin tag on the poly(A) tail end of the cDNA. This mixture is divided into two samples and each is ligated to a different linker. This linker has two features: (a) its overhang matches the overhang generated previously by the anchoring enzyme, and (b) it has a recognition site for a type II restriction enzyme (known as the tagging enzyme). Each sample is cut with the tagging enzyme. This enzyme recognizes the sequence in the linker, but actually makes a blunt end cut downstream in the cDNA sequence. This generates two pools of small cDNA sequence tags with different linkers.
Finally, the sequence tags are joined into one long sequence. First, fragments are linked by blunt-end ligation. Then PCR primers complementary to the linkers are used to amplify only those ligated molecules that have linker A and linker B flanking two different sequence tags. The PCR products are digested with the anchoring enzyme to remove the linkers and generate sticky ends. These are ligated and the resulting fragment is cloned and sequenced.
When th is approach is put into practice, the RNA is converted to DNA for sequencing and only a short segment from each RNA is sequenced.
ing enzyme") with a 4 bp recognition site and which generates sticky ends. This gives fragments of average length 256 bp, which are then bound to streptavidin-coated magnetic beads. This method generates a library of 3' ends, many just containing the 3' UTR region. The 3' UTR sequence is more divergent, therefore, mRNAs from highly homologous families can be more readily distinguished using this procedure.
The collected fragments are divided into two samples. The two sets of fragments are ligated to two different artificial linkers that contain recognition sites for a type II restriction enzyme (the "tagging enzyme"). Type II enzymes cut a fixed number of bases away from their recognition sites. A favorite choice is FokI which cuts 13 bp downstream and so leaves 9 bp of the original mRNA sequence (the "tag") plus the 4 bp anchoring enzyme site attached to the linker. The fragments are then blunt-end ligated head to tail, to give structures containing two mRNA-derived tags flanked by linker A and linker B. This structure is used as the target for PCR using two primers, a forward primer that binds to linker A and a backward primer that binds to linker B. (Other structures are formed during the ligation, but only those with the desired structure will be amplified by using this pair of PCR primers.) The PCR products are cleaved with the anchoring enzyme to give tag-dimers with sticky ends and these are ligated to give the DNA concatemer. Finally, the DNA concatemer is cloned and sequenced. The tags are identified and counted to indicate the relative levels of the original mRNA molecules.
type II restriction enzyme Type or restriction enzyme that cuts a fixed number of bases away from its recognition site
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