Arrays of probes for positional sequencing by hybridization

• US 7,319,003 B2
• Filed: 02/24/1998
• Issued: 01/15/2008
• Est. Priority Date: 11/06/1992
• Status: Expired due to Fees

• Current Assignee: Trustees of Boston University
• Original Assignee: Trustees of Boston University
• Inventors: Sano, Takeshi, Cantor, Charles R., Prezetakiewiczr, Marek, Smith, Cassandra L.
• Primary Examiner(s): FORMAN, BETTY J
• Application Number:
  US09/030,571
• Publication Number:
  US 20030118987A1
• Time in Patent Office: 3,612 Days
• Field of Search: 435/6, 435/283.1, 435/287.1, 435/287.2, 435/288.7, 536/23.2, 536/23.1, 536/24.3, 536/26.6, 422/50, 422/61, 422/68.1, 422/82.05, 422/82.08
• US Class Current: 435/6.1
• CPC Class Codes: B01J 19/0046 Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays synthesis methods per se C40B50/00
• CPC Class Codes: B01J 2219/00527 Sheets
• CPC Class Codes: B01J 2219/00529 DNA chips
• CPC Class Codes: B01J 2219/0059 Sequential processes
• CPC Class Codes: B01J 2219/00605 the compounds being directly bound or immobilised to solid supports
• CPC Class Codes: B01J 2219/0061 The surface being organic
• CPC Class Codes: B01J 2219/00612 the surface being inorganic
• CPC Class Codes: B01J 2219/00626 Covalent
• CPC Class Codes: B01J 2219/0063 Other, e.g. van der Waals forces, hydrogen bonding
• CPC Class Codes: B01J 2219/00637 by coating it with another layer
• CPC Class Codes: B01J 2219/00644 the porous medium being present in discrete locations, e.g. gel pads
• CPC Class Codes: B01J 2219/00648 by the use of solid beads
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2521/501 Ligase
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2525/204 specific length of the oligonucleotides
• CPC Class Codes: C12Q 2565/515 characterised by the interaction between or sequential use of two or more arrays
• CPC Class Codes: C12Q 2565/537 : characterised by the capture oligonucleotide acting as a primer
• CPC Class Codes: C40B 40/06 : Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 80/00 : Linkers or spacers specially adapted for combinatorial chemistry or libraries, e.g. traceless linkers or safety-catch linkers

Abstract

This invention is directed to methods and reagents useful for sequencing nucleic acid targets utilizing sequencing by hybridization technology comprising probes, arrays of probes and methods whereby sequence information is obtained rapidly and efficiently in discrete packages. That information can be used for the detection, identification, purification and complete or partial sequencing of a particular target nucleic acid. When coupled with a ligation step, these methods can be performed under a single set of hybridization conditions. The invention also relates to the replication of probe arrays and methods for making and replicating arrays of probes which are useful for the large scale manufacture of diagnostic aids used to screen biological samples for specific target sequences. Arrays created using PCR technology may comprise probes with 5′- and/or 3′-overhangs.

Claims

1. An array of nucleic acid probes, wherein each probe comprises:

a single-stranded first nucleic acid of about 15–

25 nucleotides in length;

a longer single-stranded second nucleic acid of about 20–

30 nucleotides in length, comprising a nucleotide sequence complementary to the first nucleic acid and a random terminal nucleotide sequence of between 3–

10 nucleotides in length; and

an oligonucleotide of 4–

20 nucleotides in length, comprising a random nucleotide sequence, wherein;

the first nucleic acid is hybridized to the second nucleic acid to form a hybrid having a double-stranded portion and a single-stranded portion comprising the random terminal nucleotide sequence of between 3–

10 nucleotides in length; and

the oligonucleotide is ligated to the random nucleotide sequence of the second nucleic acid.

2. The array of claim 1, wherein the nucleic acids in the array are fixed to a solid support selected from the group consisting of plastics, ceramics, metals, resins, gels, membranes, and chips.

3. The array of claim 2, wherein the solid support is a two-dimensional or a three-dimensional matrix with multiple probe binding sites.

4. The array of claim 1, wherein the probes are labelled with a detectable label.

5. The array of claim 4, wherein the detectable label is selected from the group consisting of radioisotope, a stable isotope, an enzyme, an antibody, a fluorescent chemical, a luminescent chemical, a chromatic chemical, and a metal.

6. The array of claim 1, wherein the nucleic acids are DNA, RNA, Protein Nucleic Acid (PNA), or a combination thereof.

7. The array of claim 1, wherein the probes are fixed to a solid support by conjugating to a coupling agent selected from the group consisting of antibody/antigen, biotin/streptavidin, Staphylococcus aureus protein A/IgG antibody F_c fragment, nucleic acid/nucleic acid binding protein, and streptavidin/protein A chimeras.

8. The array of claim 1, wherein the random region is of length R and the array comprises about 4^R different nucleic acid probes.

9. The array of claim 1, wherein the double-stranded portion of each probe includes an enzyme recognition site.