Method for the automated generation of nucleic acid ligands

• US 6,716,580 B2
• Filed: 03/22/2001
• Issued: 04/06/2004
• Est. Priority Date: 06/11/1990
• Status: Expired due to Fees

• Current Assignee: Somalogic Incorporated
• Original Assignee: Somalogic Incorporated
• Inventors: Jenison, Robert D., Schneider, Daniel J., Zichi, Dominic A., Gold, Larry
• Primary Examiner(s): Forman, B. J.
• Application Number:
  US09/815,171
• Publication Number:
  US 20020064780A1
• Time in Patent Office: 1,111 Days
• Field of Search: 435/91.2, 435/6, 435/91.1, 435/283.1, 435/287.2, 435/288.3, 536/25.4, 536/24.3, 536/231, 753/041.2
• US Class Current: 435/6.12
• CPC Class Codes: C12N 15/1048 SELEX

Abstract

The present invention includes a method and device for performing automated SELEX. The steps of the SELEX process are performed at one or more work stations on a work surface by a robotic manipulator controlled by a computer. The invention also includes methods and reagents to obviate the need for size-fractionation of amplified candidate nucleic acids before beginning the next round of the SELEX process.

Claims

1. 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 5′

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.

2. The method of claim 1 wherein said target is attached to a solid support, and wherein step b) is accomplished by automatically partitioning said solid support from said candidate mixture using said robotic manipulator.

3. The method of claim 2 wherein said solid support is a multi-well microtitre plate.

4. The method of claim 3 wherein said plate is comprised of polystyrene.

5. The method of claim 4 wherein said target is attached to said plate by hydrophobic interactions.

6. The method of claim 2 wherein said solid support is a paramagnetic bead and wherein the partitioning of said paramagnetic bead is performed automatically by a magnetic bead separator controlled by said computer.

7. The method of claim 1 wherein each nucleic acid in said candidate mixture of nucleic acid ligands has a tail sequence at both the 5′

terminus and the 3′

terminus, wherein each tail sequence has a lower melting temperature (Tm) than said fixed sequence regions.

8. The method of claim 1 wherein step c) is performed in the presence of a fluorescent nucleic acid-intercalating dye, said dye undergoing a change in fluorescence intensity upon binding double-stranded DNA, and wherein the amount of amplified product is measured by quantitating a fluorescence signal from said dye.

9. 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) contacting the candidate mixture with the target 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; and

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 5′

ends of said primers are attached to tail sequences having a lower melting temperature (Tm) than said primers, wherein the polymerase chain reaction comprises a denaturation step, a primer annealing step, and a primer extension step, wherein said primer annealing step and said primer extension step are performed at a temperature higher than the melting temperature of said tail sequences;

d) repeating steps a) through c) using the ligand enriched mixture of each successive repeat as many times as required to yield a desired level of increased ligand enrichment;

wherein said candidate mixture comprises the sequence set forth in SEQ. ID. NO;

7, and wherein said primers comprise the sequences set forth in SEQ. ID NO;

16 and SEQ. ID. NO;

25;

whereby a nucleic acid ligand may be identified.