Use of continuous/contiguous stacking hybridization as a diagnostic tool

• US 5,908,745 A
• Filed: 01/16/1996
• Issued: 06/01/1999
• Est. Priority Date: 01/16/1996
• Status: Expired due to Term

• Current Assignee: U Chicago Argonne, LLC
• Original Assignee: University of Chicago
• Inventors: Yershov, Gennadiy Moseyevich, Mirzabekov, Andrei Darievich, Parinov, Sergei Valeryevich, Barski, Victor Evgenievich, Kirillov, Eugene Vladislavovich, Lysov, Yuri Petrovich
• Primary Examiner(s): Horlick, Kenneth R.
• Application Number:
  US08/587,332
• Time in Patent Office: 1,232 Days
• Field of Search: 435/6, 536/22.1, 536/25.32, 536/26.6, 536/26.74, 436/94
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2521/101 DNA polymerase
• CPC Class Codes: C12Q 2525/151 repeat or repeated sequences, e.g. VNTR, microsatellite, concatemer
• CPC Class Codes: C12Q 2525/197 incorporating a spacer/coupling moiety
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

A method for detecting disease-associated alleles in patient genetic material is provided whereby a first group of oligonucleotide molecules, synthesized to compliment base sequences of the disease associated alleles is immobilized on a predetermined position on a substrate, and then contacted with patient genetic material to form duplexes. The duplexes are then contacted with a second group of oligonucleotide molecules which are synthesized to extend the predetermined length of the oligonucleotide molecules of the first group, and where each of the oligonucleotide molecules of the second group are tagged and either incorporate universal bases or a mixture of guanine, cytosine, thymine, and adenine, or complementary nucleotide strands that are tagged with a different fluorochrome which radiates light at a predetermined wavelength. The treated substrate is then washed and the light patterns radiating therefrom are compared with predetermined light patterns of various diseases that were prepared on identical substrates.

Claims

1. A method for detecting disease associated alleles in patient genetic material comprising:

a) immobilizing oligonucleotide molecules from a first group, at a predetermined position on a substrate, each of the molecules having a first end, a second end, and a known length, whereby the first end is attached to the substrate said oligonucleotide molecules synthesized to complement base sequences of the disease associated alleles;

b) contacting the patient genetic material with said first group of oligonucleotides to form duplexes;

c) positioning oligonucleotide molecules from a second group of oligonucleotide molecules at the second end of the immobilized oligonucleotide molecules of the first group to noncovalently extend the length of the oligonucleotide molecules of the first group, where each of the oligonucleotide molecules of the second group are tagged with a fluorochrome which radiates light at a predetermined wavelength, and where each of the oligonucleotide molecules of the second group consist of "n" number of bases and wherein just one of the bases of the oligonucleotide molecules of the second group is guanine or cytosine or thymine or adenine or uracil with the remaining bases each comprised of a universal base selected from the group consisting of 5-nitroindole, 3-nitropyrrole, and inosine; and

d) comparing the light patterns radiating from the predetermined positions on the substrate with predetermined light patterns emitted from identical substrates which were contacted with the disease associated alleles.

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

3. The method as recited in claim 1 wherein the oligonucleotide molecules of the first group of oligonucleotides consists of different oligonucleotide sequences and are of equal length.

4. The method as recited in claim 1 wherein the length of the oligonucleotides in the first group is between 8 and 20 bases.

5. The method as recited in claim 1 wherein the oligonucleotide molecules of the second group of oligonucleotides consist of different base sequences and are of equal length.

6. The method as recited in claim 1 wherein the predetermined length of the oligonucleotides in the second group is between 4 and 7 bases.

7. The method as recited in claim 1 wherein the fluorochrome which radiates light at a predetermined wavelength is selected from the group consisting of tetramethylrodamine, fluoresceine, Texas Red, Cascade Blue, rhodamine, HEX and combinations thereof.

8. The method as recited in claim 1 wherein the oligonucleotide molecules of the second group are tagged with the same fluorochrome and step c is repeated 4n times.

9. The method as recited in claim 1 wherein the oligonucleotide molecules of the second group are tagged with different fluorochromes with each fluorochrome specific for only one of guanine or cytosine or thymine or adenine, or uracil and step c is repeated n times.

10. A method for determining nucleotide sequences from patient genetic material comprising:

a) immobilizing oligonucleotide molecules from a first group, at a predetermined position on a substrate, each of the molecules having a first end, a second end, and a known length, whereby the first end is attached to the substrate, said oligonucleotide molecules synthesized to complement base sequences of the disease associated alleles;

b) contacting the patient genetic material with said first group of oligonucleotides to form duplexes;

c) positioning oligonucleotide molecules from a second group of oligonucleotide molecules at the second end of the immobilized oligonucleotide molecules of the first group to noncovalently extend the length of the oligonucleotide molecules of the first group, where each of the oligonucleotide molecules of the second group are tagged with a fluorochrome which radiates light at a predetermined wavelength, and where each of the oligonucleotide molecules of the second group consist of "n" number of bases and wherein just one of the bases of the oligonucleotide molecules of the second group is guanine or cytosine or thymine or adenine and the remaining bases are each comprised of a universal base selected from the group consisting of 5-nitroindole, 3-nitropyrrole, and inosine; and

d) repeating step c to determine the complete sequence of the patient genetic material duplexed with both the oligonucleotide molecules from the first group and the oligonucleotide molecules from the second group.

11. The method as recited in claim 10 wherein all of the nucleotide molecules of the second group contain the same fluorochrome and step c is repeated 4n times.

12. The method as recited in claim 11 wherein the number of nucleotide molecules in the second group is 4n.

13. The method as recited in claim 10 wherein the fluorochrome is specific for only one of guanine or cytosine or thymine or adenine and step c is repeated n times.

14. The method as recited in claim 13 wherein the number of nucleotide molecules in the second group is n.

15. A method for determining nucleotide sequences from patient genetic material comprising:

a) immobilizing oligonucleotide molecules from a first group, at a predetermined position on a substrate, each of the molecules having a first end, a second end, and a known length, whereby the first end is attached to the substrate, said nucleotide molecules synthesized to complement base sequences of the disease associated alleles;

b) contacting the patient genetic material with said first group of oligonucleotides to form duplexes;

c) positioning oligonucleotide molecules from a second group of oligonucleotide molecules at the second end of the immobilized oligonucleotide molecules of the first group to noncovalently extend the length of the oligonucleotide molecules of the first group, where each of the oligonucleotide molecules of the second group are tagged with a fluorochrome which radiates light at a predetermined wavelength, and where each of the oligonucleotide molecules of the second group is a pentamer containing four universal bases and just one base is guanine or cytosine or thymine or adenine; and

d) repeating step c to determine the base sequence of the patient genetic material that extends five bases beyond the second end of the immobilized oligonucleotide molecule.

16. The method as recited in claim 15 wherein the universal base is 5-nitroindole, 3-nitropyrrole or inosine.

17. The method as recited in claim 15 wherein the second group of oligonucleotide molecules consist of five subgroups with the first subgroup consisting of

A-N-N-N-N, T-N-N-N-N, C-N-N-N-N, and G-N-N-N-N, the second group consisting of

N-A-N-N-N, N-T-N-N-N, N-C-N-N-N, and N-G-N-N-N, the third group consisting of

N-N-A-N-N, N-N-T-N-N, N-N-C-N-N, and N-N-G-N-N, the fourth group consisting of

N-N-N-A-N, N-N-N-T-N, N-N-N-C-N, and N-N-N-G-N, and the fifth group consisting of

N-N-N-N-A, N-N-N-N-T, N-N-N-N-C, and N-N-N-N-G

where N is the universal base, A is adenine, T is thymine, C is cytosine and G is guanine.

18. The method as recited in claim 17 wherein each of the molecules in each of the subgroups are tagged with a fluorochrome specific for only one of guanine, or cytosine or thymine or adenine and only one subgroup at a time is positioned at the second end of the immobilized oligonucleotide molecules.

19. The method as recited in claim 15 wherein the pentamers are derived from five structures, the first structure consisting of

X-N-N-N-N, the second structure consisting of

N-X-N-N-N, the third structure consisting of

N-N-X-N-N, the fourth structure consisting of

N-N-N-X-N, and the fifth structure consisting of

N-N-N-N-X,

wherein N designates the universal base, X designates the same base, each of the five structures contain the same fluorochrome that is specific for that base, and only one structure at a time is positioned at the second end of the immobilized oligonucleotide molecules.

20. The method as recited in claim 19 wherein a different base and a fluorochrome specific for that base is utilized.