TARGETED MOLECULAR BAR CODES AND METHODS FOR USING THE SAME

• US 20020142344A1
• Filed: 12/10/1999
• Published: 10/03/2002
• Est. Priority Date: 12/11/1998
• Status: Active Grant

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: AKESON, MARK, DEAMER, DAVID W., BRASLAU, REBECCA, SINGARAM, BAKTHAN, STEINER, DEREK, VERCOUTERE, WENONAH, OLSEN, HUGH E., CAPPUCCUI, FRANK
• Granted Patent: US 6,465,193 B2
• Time in Patent Office: Days
• US Class Current: 435/7.1
• CPC Class Codes: C12Q 1/6823 Release of bound markers
• CPC Class Codes: C12Q 2523/319 Photocleavage, photolysis, photoactivation
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid
• CPC Class Codes: C12Q 2565/607 being a sensor, e.g. electrode
• CPC Class Codes: G01N 33/48721 Investigating individual macromolecules, e.g. by translocation through nanopores Coulter counters in general G01N15/12; fabrication methods for nanoscale apertures B81B1/00; sequencing of nucleic acids C12Q1/68
• CPC Class Codes: G01N 33/532 Production of labelled immunochemicals
• CPC Class Codes: G01N 33/5438 Electrodes
• CPC Class Codes: G01N 33/54386 Analytical elements

Abstract

Targeted molecular bar codes and methods for using the same are provided. The subject targeted molecular bar codes include a molecular bar code and a member of a specific binding pair, where the specific binding pair member is generally bonded to the bar code through a linking group. The subject molecular bar code may be read during translocation through a single nano-meter scale pore. The subject targeted molecular bar codes find use in a variety of different applications involving analyte detection, such as screening and diagnostic applications.

Claims

1. A targeted molecular bar code comprising:

(a) a molecular bar code, wherein said molecular bar code is a charged polymer capable of generating a reproducible signal upon passage through a nanopore; and

(b) a member of a specific binding pair, wherein said specific binding pair member is joined directly or through a linking group to said molecular bar code.

2. The targeted molecular bar code of claim 1, wherein said charged polymer is negatively charged.

3. The targeted molecular bar code according to claim 2, wherein said negatively charged polymer is made up of monomeric units that comprise a moiety selected from the group consisting of a phosphate group or a phosphorothioate group.

4. The targeted molecular bar code according to claim 2, wherein said polymer is a block copolymer of a plurality of blocks, wherein said plurality of blocks are selected from two or more different blocks.

5. The targeted molecular bar code according to claim 4, wherein said block copolymer comprises three different blocks.

6. The targeted molecular bar code according to claim 1, wherein said molecular bar code comprises a linking group.

7. The targeted molecular bar code according to claim 6, wherein said linking group is a photocleavable linking group.

20. A method for detecting the presence of an analyte in a sample, said method comprising:

(a) contacting said sample with at least one targeted molecular bar code according to claim 1 under conditions sufficient for said specific binding pair to bind to said analyte in said sample;

(b) separating unbound targeted molecular bar code from analyte-bound targeted molecular bar code;

(c) treating said analyte bound targeted molecular bar code in a manner sufficient to release said molecular bar code from said analyte-bound targeted molecular bar code and produce free molecular bar code;

(e) detecting the presence of said free molecular bar code; and

(f) relating the presence of said free molecular bar code to the presence of said analyte in said sample.

21. The method according to claim 20, wherein said detecting step comprises translocating said free molecular bar code through a nanopore.

22. The method according to claim 21, wherein said detecting step further comprises observing the current blockade effect of said translocation on said nanopore.

23. The method according to claim 20, wherein said method comprises contacting a plurality of different targeted molecular bar codes with said sample.

24. The method according to claim 23, wherein said sample size does not exceed the size of a biological cell.

25. A kit for use in the detection of an analyte in a sample, said kit comprising:

a targeted molecular bar code according to claim 1.

26. The kit according to claim 25, wherein said kit comprises a plurality of different targeted molecular bar codes.

27. The kit according to claim 25, wherein said kit comprises at least 4 different types of targeted molecular bar codes.

28. The kit according to claim 25, wherein said kit comprises at least 64 different types of targeted molecular bar codes.

29. The kit according to claim 25, wherein said kit comprises at least 100 different types of targeted molecular bar codes.

8. A targeted molecular bar code comprising:

(a) a negatively charged block copolymer of from one to twenty blocks, wherein said blocks are selected from two or more different blocks, wherein each block consists of monomeric units comprising a phosphate group; and

(b) a member of a specific binding pair, wherein said member of a specific binding pair is joined to said negatively charged block copolymer through a linking group.

9. The targeted molecular bar code according to claim 8, wherein said blocks are selected from two to four different blocks.

10. The targeted molecular bar code according to claim 8, wherein each block is a homopolymer of monomeric units selected from the group consisting of phosphates and sugar phosphates.

11. The targeted molecular bar code according to claim 8, wherein said sugar phosphates are selected from the group consisting of ribose phosphates and deoxyribose phosphates.

12. The targeted molecular bar code according to claim 11, wherein said sugar phosphates may optionally comprise a heterocyclic nitrogenous base.

13. The targeted molecular bar code according to claim 12, wherein said heterocyclic nitrogenous base is a purine or a pyrimidine.

14. The targeted molecular bar code according to claim 8, wherein the length of each block of said block copolymer ranges from 15 to 25 nm.

15. The targeted molecular bar code according to claim 14, wherein said linker is a photocleavable linker.

17. The targeted molecular bar code according to claim 16, wherein said three different blocks are:

polyethylene glycol-phosphodiesters;

oligodeoxyribosephosphates; and

oligonucleotides modified to prevent Watson-Crick base pairing.

18. The targeted molecular bar code according to claim 16, wherein each of said blocks is about 20 nm long.

19. The targeted molecular bar code according to claim 16, wherein said linker is a photocleavable linker.

16. A targeted molecular bar code comprising:

(a) a negatively charged block copolymer of from two to twenty blocks, wherein said blocks are selected from a group of three different blocks, wherein each block is a homopolymer of monomeric units selected from the group consisting of polyphosphates, oligonucleotides, oligodeoxyribosephosphates; and

polyethylene glycol-phosphodiesters;

(b) a member of a specific binding pair, wherein said member of a specific binding pair is joined to said negatively charged block copolymer through a linking group.