Patent ID: 6716580
Filing Date: 2004-04-06
Classification: C12N

Abstract:
A method for the identification of a nucleic acid ligand from a candidate mixture of nucleic acids, said nucleic acid ligand being a ligand of a given target, each said nucleic acid in said candidate mixture comprising fixed sequence regions, the method comprising:a) adding the candidate mixture and the target in a predetermined ratio to a reaction vessel at a work station on a work surface using a cartesian robotic manipulator, wherein nucleic acids having an increased affinity to the target relative to the candidate mixture may be partitioned from the remainder of the candidate mixture; b) partitioning increased affinity nucleic acids from the remainder of the candidate mixture using said robotic manipulator; c) PCR-amplifying the increased affinity nucleic acids with primers complementary to said fixed sequence regions to yield a ligand enriched mixture of nucleic acids, wherein the 5â€² ends of said primers are attached to tail sequences having a lower melting temperature (Tm) than said primers, wherein said PCR-amplification comprises: i) adding said primers and PCR reagents to the reaction vessel using said robotic manipulator; ii) thermally-cycling the reaction vessel using a thermal cycler, wherein said thermal cycling comprises performing at a temperature higher than the melting temperature of said tail sequences a denaturation step, a primer annealing step, and a primer extension step, and wherein the amount of amplified product in said reaction vessel is measured during said thermal cycling using a measuring device; and iii) calculating the amount of increased affinity nucleic acids partitioned at step b) using the measured amount of amplified product obtained from said measuring device; d) adjusting the reaction conditions of steps a)-c) in a predetermined manner in response to the amount of nucleic acid ligand calculated at step c) iii); and e) repeating steps a)-d) at least once, wherein the adjusting performed at step d) controls the stringency of each successive repeat; wherein said robotic manipulator, said thermocycler, and said measuring device are automatically controlled by a computer during steps a)-e), and wherein said computer automatically calculates the amount of increased affinity product at step c) and automatically adjusts the reaction conditions at step d); whereby a nucleic acid ligand of said target may be identified.