Polymers of oligonucleotide probes as the bound ligands for use in reverse dot blots

• US 5,683,872 A
• Filed: 12/23/1994
• Issued: 11/04/1997
• Est. Priority Date: 10/31/1991
• Status: Expired due to Fees

• Current Assignee: University Of Pittsburgh (University of Pittsburgh of The Commonwealth System of Higher Education)
• Original Assignee: University Of Pittsburgh (University of Pittsburgh of The Commonwealth System of Higher Education)
• Inventors: Trucco, Massimo, Rudert, William A.
• Primary Examiner(s): Campbell, Eggerton A.
• Application Number:
  US08/363,585
• Time in Patent Office: 1,047 Days
• Field of Search: 435/6, 435/91.2, 935/77, 935/78
• US Class Current: 435/6.13
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6881 for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
• CPC Class Codes: C12Q 2525/151 repeat or repeated sequences, e.g. VNTR, microsatellite, concatemer
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers

Abstract

A method of detecting nucleic acid sequences in which polymers of selected oligonucleotide probes which are complementary to a region in a nucleic acid sequence that is to be detected are bound to a substrate. The polymers bound to the substrate contain multiple randomly repeated copies of a specific oligonucleotide probe and may be synthesized using enzymatic amplification techniques.

Claims

1. A method of detecting the presence of nucleic acid sequences, comprising:

binding polymers of at least one oligonucleotide probe to a substrate wherein said polymeric probes are comprised of monomeric sequence units that are complementary to a target sequence which exists as a single copy within a region of a nucleotide sequence contained in a DNA test sample wherein said monomeric sequence units are selceted from the group consisting of Sequence I.D. Nos. 1-112;

contacting said substrate to which said polymeric probes are bound with amplification products of said test DNA sample containing uncharacterized oligonucleotide sequences, wherein a reporter moiety has been incorporated, such that individual complementary sequences that are contained in the amplified test sample are able to anneal to any monomeric sequence that is contained in any polymeric probe bound to said substrate;

washing said substrate to remove unannealed amplified DNA test sample from said substrate; and

detecting the presence or absence of reporter moieties retained by said polymeric probes on said substrate.

2. The method of claim 1, wherein said substrate is a nylon membrane.

3. The method of claim 1, wherein said substrate is a nitrocellulose membrane.

14. The method of claim 1, wherein entire vectors containing said polymeric probes are bound to said substrate.

15. The method of claim 1, where said polymers are single-stranded DNAs.

16. The method of claim 1 wherein said reporter moiety is selected from among ³² p, biotin, and digoxigenin, wherein said reporter moiety is attached to one or both of said primers prior to synthesis of said amplification products.

17. The method of claim 1, wherein said amplification products are modified by attaching a reporter moiety after said products are synthesized.

18. The method of claim 1, wherein said reporter moiety is an enzyme which is covalently attached to the test DNA and wherein said enzyme functions as part of the detection system.

4. A method of synthesizing oligonucleotides probes consisting of repeated monomeric sequence units by the steps of:

(a) annealing two oligonucleotide primers which are oppositely oriented and which are complementary at their 3'"'"' ends and which together either directly or in the form of complementary nucleotides define the sequence of a complete monomeric unit which incorporates the selected oligonucleotide probe sequence as well as a sufficient number of the nucleotides of the adjacent monomeric units such that the primers anneal at their 3'"'"' ends and that the 5'"'"' ends of the primers are not annealed and function as templates for synthesis of first extension products;

(b) treating the primers with a DNA polymerase in the presence of deoxyribonucleotides such that a first extension product of each primer is synthesized which is complementary to each template;

(c) separating the complementary first extension products by denaturation to produce single-stranded molecules;

(d) annealing complementary strands of said first extension products at their 3'"'"' ends in a staggered arrangement such that the 5'"'"' ends are not annealed and function as templates for further extension of the previous extension products;

(e) treating the annealed strands using a DNA polymerase and deoxyribonucleotides such that double-stranded extension products are synthesized;

(f) separating the complementary extension products by denaturization; and

(g) repeating steps (d) through (f) using the extension products of step (f) to produce successively longer extension products with each repetition without the addition of any new primer sequences not generated by the initial two oligonucleotide primers.

5. The method of claim 4, further comprising after step (c) the steps of:

(c'"'"') treating the single-stranded first extension products generated in step (c) with the primers of step (a) under conditions such that additional molecules of the first extension products are synthesized using single strands of the first extension products as templates; and

(c") optionally repeating steps (c) and (c'"'"') to further increase the quantity of the first extension products.

6. The method of claim 4, wherein said denaturing is caused by heating.

7. The method of claim 4, wherein longer starting oligonucleotide primers are used which are substantially equivalent to said first extension products such that steps (a)-(c) are deleted.

8. The method of claim 5, wherein the products of each step are not purified such that the step (g) repetition occurs simultaneously with the repetitions of steps (c) and (c'"'"') when there is a sufficient concentration of first extension products that step (d) annealing occurs.

9. The method of claim 4, wherein said DNA polymerase is selected from the group consisting of E.coli DNA polymerase I, Klenow fragment of E.coli DNA polymerase I, T4 DNA polymerase, and T7 DNA polymerase.

10. The method of claim 4, wherein said DNA polymerase is a thermostable DNA polymerase selected from the group consisting of Thermus aqaticus DNA polymerase, Thermococcus litoralis DNA polymerase, and Pyrococcus furiosus DNA polymerase, and the repetitive cycles of step (g) are achieved by cycling the reaction temperature successively from temperatures suitable for annealing, extension, and denaturing.

11. The method of claim 4 further comprising the step of:

cloning the final double-stranded primer extension product in a vector.

12. The method of claim 11, wherein said cloned primer extension product is repetitiously duplicated within said vector by directional cloning to further increase the quantity of probe sequence inserted in the vector.

13. The method of claim 11 wherein said vector has been derived from pUC18, pUC19, or the bacteriophage M13mp series.

19. A substrate to which selected long polymers of at least one oligonucleotide probe are bound wherein said polymeric probes are comprised of monomeric sequence units that are complementary to a target sequence which exists as a single copy within a region of a nucleotide sequence contained in a DNA test sample wherein said monomeric sequence units are selected from the group consisting of Sequence I.D. Nos. 1-112.

20. The substrate of claim 19 wherein said monomeric sequence unit of each said polymeric probes contains about 12 to 20 nucleotides and each polymer contains about 200-2000 base pairs.

21. The substrate of claim 20 wherein said polymeric probes are comprised of specific oligonucleotides selected from the group consisting of Class II HLA DQα

, HLA DQβ

, HLA DRα

, HLA DRβ

, HLA DPα

, and HLA DPβ

alleles.

22. A method of detecting the presence of nucleic acid sequences, comprising:

contacting the substrate of claim 21 with an amplified test DNA sample containing unknown oligonucleotide sequences, wherein a reporter moiety has been incorporated, such that complementary nucleotide sequences anneal;

washing said substrate to remove unannealed amplified test DNA from said substrate; and

detecting the presence or absence of reporter moieties retained by said polymeric probes on said substrate.

23. The method of claim 22 further comprising the step:

analyzing and interpreting the results of said detection.