Microchip method for the enrichment of specific DNA sequences

• US 5,851,772 A
• Filed: 01/29/1996
• Issued: 12/22/1998
• Est. Priority Date: 01/29/1996
• Status: Expired due to Term

• Current Assignee: U Chicago Argonne, LLC
• Original Assignee: University of Chicago
• Inventors: Dubiley, Svetlana Alekseevna, Shick, Valentine Vladimirovich, Mirzabekov, Andrei Darievich, Lysov, Yuri Petrovich
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): SHOEMAKER, DEBRA GUNNERSEN
• Application Number:
  US08/593,345
• Time in Patent Office: 1,058 Days
• Field of Search: 435/6, 536/24.3, 536/25.32, 935/77, 935/78
• US Class Current: 435/6.14
• CPC Class Codes: B01J 2219/00529 DNA chips
• CPC Class Codes: B01J 2219/00608 DNA chips
• CPC Class Codes: B01J 2219/0061 The surface being organic
• CPC Class Codes: B01J 2219/00612 the surface being inorganic
• CPC Class Codes: B01J 2219/00619 using hydrophilic or hydrophobic regions
• CPC Class Codes: B01J 2219/00626 Covalent
• CPC Class Codes: B01J 2219/00644 the porous medium being present in discrete locations, e.g. gel pads
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00662 Two-dimensional arrays within two-dimensional arrays
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: C12Q 1/6811 Selection methods for production or design of target specific oligonucleotides or binding molecules
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C40B 40/06 Libraries containing nucleotides or polynucleotides, or derivatives thereof

Abstract

A method for enriching specific genetic material sequences is provided, whereby oligonucleotide molecules complementary to the desired genetic material is first used to isolate the genetic material from a first source of genomic material. Then the genetic material is used as a label to isolate similar genetic sequences from other sources.

Claims

1. A method for enriching specific genetic sequences, comprising:

a) immobilizing a first set of oligonucleotide molecules at a plurality of positions on a first gel substrate, said first set of oligonucleotide molecules synthesized to complement base sequences of the genetic sequences;

b) contacting genetic material with said first set of oligonucleotides, said genetic material containing the specific genetic sequences, so as to allow formation of first duplexes of said first set of oligonucleotides and fragments of the specific genetic sequences;

c) simultaneously isolating the fragments of the specific genetic material sequences from the duplexes formed via diffusion;

d) labeling the now-isolated fragments with a fluorochrome;

e) simultaneously contacting each now-labeled fragment with a second separate set of sequencing oligonucleotide molecules contained on a sequencing gel substrate, said sequencing set of oligonucleotide molecules which are complementary to subsequence regions of each now-labelled fragment, so as to allow for second duplexes to form between the subsequence regions and said sequencing set of oligonucleotide molecules; and

f) isolating the subsequence regions, thereby enriching the specific genetic sequences.

2. The method as recited in claim 1 wherein the genetic material is selected from the group consisting of deoxyribonucleic acid and ribonucleic acid.

3. The method as recited in claim 1 wherein the first set and second set of oligonucleotide molecules contain bases selected from the group consisting of guanine, cytosine, adenine, thymine and uracil.

4. The method as recited in claim 1 wherein the first set of oligonucleotide molecules consist of different base sequences and are of equal length.

5. The method as recited in claim 1 wherein the oligonucleotide molecules range in length from approximately 6 and 12 bases.

6. The method as recited in claim 1 wherein the fluorochrome is selected from the group consisting of tetramethylrodamine, fluoresceine, Texas Red, Cascade Blue, Rhodamine, HEX, and combinations thereof.

7. The method as recited in claim 1 wherein the substrate is selected from the group consisting of polyacrylamide, polyamide, agarose, silicate glass matrix, and combinations thereof.

8. The method as recited in claim 1 wherein the second set of oligonucleotide molecules further comprises a universal base.

9. The method as recited in claim 8 wherein the universal base is selected from the group consisting of 5-nitroindole, 3-nitropyrrole, inosine, and combinations thereof.

10. The method as recited in claim 1 wherein said genetic material is fragmented into lengths of between approximately 250 and 30,000 bases prior to contacting said genetic material with said first set of oligonucleotides.

11. The method as recited in claim 1 wherein said now isolated fragments are fragmented into lengths of between approximately 10 to 50 bases prior to contacting said now-isolated fragments with the second set of oligonucleotides.

12. A method for enriching specific genetic sequences contained in genetic material, comprising:

a) fractionating genetic material to lengths of between approximately 250 and 30,000 bases;

b) labeling the now fractionated genetic material with a fluorochrome;

c) immobilizing a first set of oligonucleotide molecules at a plurality of positions on a first porous substrate, said first set of oligonucleotide molecules synthesized to complement base sequences of the genetic material;

d) contacting the now-labeled genetic material with said first set of oligonucleotides so as to allow formation of duplexes of said first set of oligonucleotides and the specific genetic sequences;

e) simultaneously isolating each of the specific genetic material sequences from the duplexes formed via diffusion;

f) fragmenting the now-isolated specific genetic material sequences into lengths of between approximately 10 and 50 bases;

g) labeling the now-isolated specific genetic material with a fluorochrome;

h) simultaneously contacting each now-labeled fragment with a separate set of sequencing oligonucleotide molecules contained on a sequencing gel substrate, said sequencing set of oligonucleotide molecules which are complementary to subsequence regions of each now-labelled fragment, so as to allow for second duplexes to form between the subsequence regions and said sequencing set of oligonucleotide molecules; and

i) isolating the subsequence regions thereby enriching the specific genetic sequences.

13. The method as recited in claim 12 wherein the genetic material is selected from the group consisting of deoxyribonucleic acid and ribonucleic acid.

14. The method as recited in claim 12 wherein the first and second set of oligonucleotide molecules contain bases selected from the group consisting of guanine, cytosine, adenine, thymine and uracil.

15. The method as recited in claim 12 wherein the first set of oligonucleotide molecules consist of different base sequences and are of equal length.

16. The method as recited in claim 12 wherein the length of the oligonucleotides is between 6 and 12 bases.

17. The method as recited in claim 12 wherein the genetic material is labeled with a fluorochrome selected from the group consisting of tetramethylrodamine, fluoresceine, Texas Red, Cascade Blue, Rhodamine, HEX, and combinations thereof.

18. The method as recited in claim 12 wherein the substrate is selected from the group consisting of polyacrylamide gel, polyamide gel, agarose, silicate glass matrix and combinations thereof.

19. The method as recited in claim 12 wherein the second set of oligonucleotides are further comprised of a universal base.