Download HOW SAGE WORKS (Reference http://www

HOW SAGE WORKS (Reference http://www.embl-heidelberg.de/info/sage)
Each type of RNA has a unique chemical composition that is a direct transcription of
information stored in a particular gene. The basic units that make up DNA and RNAs
are called nucleotides. The alphabet of nucleotides is very small (with only four
letters), but it suffices to spell out the unique, long words that make up the genetic
code. Cells and viruses contain molecular tools that can transform DNA into RNA.
Researchers use a method called "sequencing" to read the nucleotide spelling of a
molecule. It’s a complex procedure, and reading the entire sequence of every RNA in
a cell would probably take decades. But scientists don’t need to read long sequences.
They’ve discovered that just fourteen letters are enough to match an RNA to the
precise gene that produced it. This is much easier than decoding each whole
molecule – a single RNA might consist of thousands of nucleotides.
What SAGE does is to capture the RNAs, "rewrite" them into DNA, and cut a small,
fourteen–letter tag from each one. Since it would take a long time to load tens of
thousands of single tags into a sequencing machine, the method glues a lot of tags
together into long molecules called concatemers. The sequencer reads these
molecules, counts them and analyzes them, and computer programs give you a list
of the genes that they belong to.
Some of the steps of SAGE:
i.
Trap RNAs with beads
ii.
Convert the RNA into cDNA
iii. Make a cut in each cDNA so that there is a broken end sticking out
iv. Attach a "docking module" to this end; here a new enzyme can dock, reach
down the molecule, and cut off a short tag
v.
Combine two tags into a unit, a di-tag
vi. Make billions of copies of the di-tags (using a method called PCR)
vii. Remove the modules and glue the di-tags together into long concatamers
viii. Put the concatamers into bacteria and copy them millions of times
ix. Pick the best concatamers and sequence them
x.
Use software to identify how many different cDNAs there are, and count them;
xi. Match the sequence of each tag to the gene that produced the RNA.
Microscopic Bead and mRNA
Graphic: Jason Soffe
RNA binds to bait and is copied into DNA
Graphic: Jason Soffe
An enzyme cuts the DNA
Graphic: Jason Soffe
An enzyme locks onto the DNA and cuts off a short tag
Graphic: Jason Soffe
Two tags are linked together
Graphic: Jason Soffe
Enzymes cut off the "Docking Molecules"
Graphic: Jason Soffe
Di-Tags are combined into large concatemers
Graphic: Jason Soffe