Method and apparatus for the automated generation of nucleic acid ligands

• US 7,629,151 B2
• Filed: 11/18/2003
• Issued: 12/08/2009
• Est. Priority Date: 01/19/1999
• Status: Expired due to Fees

• Current Assignee: Somalogic Incorporated
• Original Assignee: Somalogic Incorporated
• Inventors: Zichi, Dominic A., Gold, Larry, Jenison, Robert D., Schneider, Daniel J.
• Primary Examiner(s): KAPUSHOC, STEPHEN THOMAS
• Application Number:
  US10/717,105
• Publication Number:
  US 20040106145A1
• Time in Patent Office: 2,212 Days
• Field of Search: None
• US Class Current: 435/91.2
• 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 identifying nucleic acid ligands of a target molecule from a candidate mixture of nucleic acids, the method comprising the steps:

a) preparing a candidate mixture of nucleic acids, said nucleic acids comprising photoreactive groups and fixed sequence regions;

b) contacting said candidate mixture with said target molecule, wherein nucleic acid sequences having increased affinity to the target molecule relative to the candidate mixture form nucleic acid-target molecule complexes;

c) irradiating said candidate mixture, wherein said nucleic acid-target molecule complexes photocrosslink;

d) partitioning the crosslinked nucleic acid-target molecule complexes from free nucleic acids in the candidate mixture; and

e) PCR-amplifying the nucleic acids that crosslinked to the target molecule to yield a mixture of nucleic acids enriched in sequences that are capable of photocrosslinking the target molecule, wherein the PCR amplification is performed with primers complementary to said fixed sequence regions to yield amplified increased affinity 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 tail sequences are selected from the group consisting of AAAAAAAA, TTTTTTTT and ATATATAT, wherein the polymerase chain reaction comprises a denaturation step, a primer annealing step, and a primer extension step, and wherein said primer annealing step and said primer extension step are performed at a temperature higher than the melting temperature of said tail sequences;

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

wherein a nucleic acid ligand is identified.

2. The method of claim 1 wherein steps a)-e) are performed at one or more work stations on a work surface by a robotic manipulator controlled by a computer.

3. A method for identifying a photocrosslinking nucleic acid ligand of a protein from a candidate mixture of nucleic acids comprising fixed sequence regions, said method comprising:

a) contacting said candidate mixture with said protein, wherein nucleic acids having increased affinity to the protein relative to the candidate mixture form nucleic acid-protein complexes with the protein;

b) partitioning the complexed increased affinity nucleic acids from the remainder of the candidate mixture;

c) PCR-amplifying the increased affinity nucleic acids using primers complementary to said fixed sequence regions to yield amplified increased affinity 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 tail sequences are selected from the group consisting of AAAAAAAA, TTTTTTTT and ATATATAT, wherein the polymerase chain reaction comprises a denaturation step, a primer annealing step, and a primer extension step, and 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) incorporating photoreactive groups into the amplified increased affinity nucleic acids;

e) repeating step a;

f) irradiating said increased affinity nucleic acids, wherein said nucleic acid-protein complexes photocrosslink;

g) repeating steps c) and d); and

h) identifying a photocrosslinking nucleic acid ligand of the protein.

4. The method of claim 3 wherein steps a)-g) are carried out by automated machines controlled by a computer.