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Biotech / Medical : AFFYMETRIX (AFFX) -- Ignore unavailable to you. Want to Upgrade?

To: jpbrody who wrote (742)	6/30/1998 10:00:00 PM
From: Richard Haugland	Respond to of 1728

JB Brody. Attached versus adsorbed. The specifications of the AFFX patent (5,744,305) give the general details of the preparation of the chips. Using photolithograhy, AFFX first lays down a "linker" sequence, which is primarily designed to get the unique sequence away from the surface of the chip and accessible to its complementary target in the sample. If it did not, it would be difficult for the probe on the chip to efficiently bind to the target. This is described as a covalent linkage between the surface and the target moiety. The AFFX original patent actually concentrates on describing peptides build up this way rather than oligonucleotides but covers both (plus other polymeric probe that can be built up one step at a time such as a polysaccharide, even though they describe no method of doing that class of biomolecules). In this case, peptides are complementary to binding sites on biological cells termed receptors. Such a chip can be used to screen for these receptors or drugs that may interfere with binding of their probe to that receptor, although I am not aware of any big move to use these chips for that purpose. You are probably right that the alternative techniques require absorption of the oligonucleotide onto the surface rather than covalent attachement, as in the case of AFFX. A problem there would be that the oligonucleotide would have to both be "stuck" to the surface and bind to its target in the sample if there is one without being desorbed from the chip. There are often steric and electronic problems induced by this type of binding. For those familiar with it, peptides are built up on the chip by a modified Merrifield method. Bruce Merrifield, who won a Nobel prize for it, attached a single amino acid to a polymeric polystyrene bead by its carboxylic acid group where the amine was temporarily blocked by a "protecting group." In his case, he did a chemical removal of the protecting group to give a free amine, which he coupled to a second protected amino acid of his choice to give a dipeptide (i.e a peptide with two amino acids) attached to the bead. He then removed the protecting group and added a third protected amino acid, etc. etc. to build up the peptide still on the bead. In his final step he removed the peptide from the bead and purified it. On the AFFX chip the linker is first attached to a chip instead of to a bead then the first amino acid is attached. In this case the protecting group is one that can be removed by exposure to ultraviolet light to give the free amine rather than by a chemical reagent. The coupling step of the protected amino acid to give the dipeptide is identical to the Merrifield step followed by a second exposure to light to remove the protecting group again etc, etc. The uniqueness of the AFFX procedure is that they create an optical mask that allows light through only in those "squares" where it wants to do a synthesis. Thus the squares that have NOT been illuminated do not get the next amino acid attached. Instead, for instance, they can come back with a second mask if they wish and put a DIFFERENT amino acid there or they can just go on and the non-illuminated square will have an amino acid deletion. All of this is done in organic solvents, not water, which is partly why they must covalently attach the whole thing to the surface. A problem with the AFFX approach is that not all the chemistry or the light-mediated deprotection step goes 100% at each step so that not quite all of the probes are full, although the vast majority in a single square probably have the "correct" sequence. This becomes more of a problem as one tries to build longer chains and errors build up. There is also the problem that some of the light may leak slightly from one square into an adjacent square and remove a protecting group that was not supposed to be removed. There are ways to determine that this has happened that involve a "parallel synthesis" on a different square and computer analysis so it is probably not a big problem overall. This type of solid phase synthesis is what made Applied Biosystems and their fully automated equipment dominant but ABU used chemical deprotection steps rather than light-mediated steps. Synthesis on the Applied Biosystems (now part of Perkin Elmer) equipment is probably more reliable for purity than on the AFFX chips and Perkin Elmer is now partnering with Hyseq, an AFFX nemesis. HYSQ, of course, would disagree that their DNA probes are "attached" to the chip, at least so as to avoid this type of AFFX patent. They would probably agree, however, that they were "stuck" some way on the chip. HYSQ does NOT use photolithography to address each square (which needs not actually be square, either). I don't know that much about HYSQ's actual chips, however. Synthesis of oligonucleotides using light-addressed protecting groups proceeds similarly but is more difficult to explain. However, it involves only four different bases rather than the 24 or so amino acid building blocks. To address another response to my post: yes, the AFFX patent permits them to cover all "buildings" greater than 4 stories tall, even if they don't know how to build them and anyone building a taller "building" would have to negotiate with the company and pay them for the rights. Its a matter of law! The AFFX patent is one of a series of patents with probably very similar specifications (the body of the patent) and multiple sets of different claims to cover the same thing from various approaches such as materials, methods, uses and variations and improvements.