Hybridization method for the detection of genetic material

• US 4,894,325 A
• Filed: 01/15/1987
• Issued: 01/16/1990
• Est. Priority Date: 04/27/1984
• Status: Expired due to Term

• Current Assignee: Enzo Diagnostics, Inc.
• Original Assignee: Enzo Biochem Incorporated
• Inventors: Englehardt, Dean L., Rabbani, Elazar
• Primary Examiner(s): NOT, DEFINED
• Assistant Examiner(s): Jay, Jeremy M.
• Application Number:
  US07/005,327
• Time in Patent Office: 1,097 Days
• Field of Search: 435/5, 435/6, 435/34, 435/7, 435/803, 436/501, 436/508, 436/518, 436/520, 436/527, 436/533, 935/77, 935/78, 935/81
• US Class Current: 435/6.16
• CPC Class Codes: C12Q 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/682 Signal amplification
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/683 involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
• CPC Class Codes: C12Q 1/6832 Enhancement of hybridisation reaction
• CPC Class Codes: C12Q 2563/149 Particles, e.g. beads
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid
• CPC Class Codes: C12Q 2565/113 based on agglutination/precipitation
• CPC Class Codes: C12Q 2565/519 characterised by the capture moiety being a single stranded oligonucleotide
• CPC Class Codes: Y10S 435/803 Physical recovery methods, e.g. chromatography, grinding

Abstract

This invention provides methods for the detection of a target genetic material having a desired base sequence or gene. Also disclosed are methods for the detection of mutations. Also provided are components for use in such methods.

The methods are based upon techniques which utilize two labeled single stranded polynucleotide segments which are complementary to the same or the opposite strands of the target genetic material. The methods of the invention result in the formation of a double hybrid and/or multihybrid.

Claims

1. A method for the detection of single-stranded target genetic material comprising the steps of:

(a) contacting, under conditions permissive of hybridization, said target genetic material with

(i) a first polynucleotide probe comprising a first partic to which is attached a plurality of first single-stranded polynucleotide segments complementary to a first portion of said target genetic material, and

(ii) a second polynucleotide probe comprising a second particle having attached thereto a plurality of second single-stranded polynucleotide segments complementary to a second portion of said target genetic material;

(b) permitting hybridization of said first polynucleotide segments to said first portion of target genetic material and said second segments to said second portion of target genetic material, said hybridizations resulting in agglutination; and

(c) detecting said agglutination.

2. The method of claim 1, wherein at least one of said first and second particles is a microparticle.

3. The method of claim 1, wherein said second polynucleotide probe further comprises a moiety that is capable of creating a signal.

4. The method of claim 1, wherein said first and second particles are microparticles.

5. A method for the detection of single-stranded target genetic material comprising the steps of:

(a) contacting, under conditions permissive of hybridization.

(i) an unhybridized portion of a first strand of target genetic material with a first polynucleotide probe comprising a first particle to which is attached a plurality of first single-stranded polynucleotide segments complementary to said first single strand of target genetic material;

(ii) an unhybridized portion of a second strand of target genetic material with a second polynucleotide probe comprising a second particle having attached thereto a plurality of second single-stranded polynucleotide segments complementary to said second single strand of target genetic material; and

(iii) an unhybridized portion of said first and said second strand of target genetic material;

with the provisos that;

said first and second single stranded polynucleotide segments are not capable of hybridizing to each other;

hybridization of said first polynucleotide segment to said unhybridized portion of said first strand of target genetic material and hybridization of said second polynucleotide segment to said unhybridized portion of said second strand of target genetic material do not prevent at least a portion of said first strand of target genetic material from being available to hybridize with at least a portion of said second strand of target genetic material,

(b) permitting the hybridization of

(1) said first polynucleotide segment to said unhybridized portion of said first strand of target genetic material,

(2) said second polynucleotide segment to said unhybridized portion of said second strand of target genetic material, and

(3) said unhybridized portion of said first strand of target genetic material and said unhybridized portion of said second strand of target genetic material, the foregoing hybridizations occurring in any order;

said hybridizations resulting in agglutination; and

(c) detecting such hybridizations by means of the agglutination that occurs.

6. The method of claim 5, wherein at least one of said first and second particles is a microparticle.

7. The method of claim 5, wherein said second polynucleotide probe further comprises a moiety that is capable of creating a signal.

8. The method of claim 7, wherein said first and second particles are microparticles.

9. A method for the detection of single-stranded target genetic material which comprises:

(a) Contacting complementary strands of said target genetic material under conditions permissive of hybridization, with an amount, relative to the amount of target genetic material sufficient to prevent substantial reannealing thereof, of a polynucleotide probe wherein said polynucleotide probe comprises a label attached to a single-stranded polynucleotide segment having a first portion complementary to a first strand of the target genetic material and a second portion complementary to a second strand of the target genetic material, said first strand and said second strand of target genetic material being (I) complementary to each other and (ii) such that when said first portion and said second portion are hybridized to said target genetic material, there is at least one sequence of each of the complementary strands of said target genetic material that is available to hybridize to the other strand;

(b) permitting hybridization thereof resulting in the formation of a detectable multihybrid, and

(c) detecting said multihybrid.

10. The method of claim 9 wherein said complementary strands of single-stranded target genetic material are contacted with a polymer formed of repeating units of said polynucleotide probe which comprises a single-stranded polynucleotide segment having a first portion and a second portion wherein said first portion is complementary to a first strand of the target genetic material and said second portion is complementary to a second strand of the target genetic material.

11. The method of claim 9 wherein said polynucleotide probe further comprises a label.

12. The method of claim 11 wherein said label is a particle.

13. The method of claim 12 wherein said particle is a microparticle.

14. The method of claim 13 wherein said microparticle has at least two of said single-stranded polynucleotide segments attached thereto.

15. A method for the detection of a single-stranded target genetic material which comprises

(a) contacting, under conditions permissive of hybridization, a sample suspected of containing said target genetic material with

(i) a plurality of first polynucleotide probes comprising a first entity attached to a first single-stranded polynucleotide segment which is hybridizable with a first portion of said target genetic material;

(ii) a plurality of first particles having attached thereto a second entity which is specifically bindable with said first entity;

(iii) a plurality of second polynucleotide probes comprising a third entity and attached thereto a second single-stranded polynucleotide segment hybridizable with a second portion of said target genetic material; and

(iv) a plurality of second particles having attached thereto a fourth entity which is specifically bindable with said third entity;

(b) permitting hybridization of said first and second probes with said target genetic material, binding of said first entity with said second entity and binding of said third entity with said fourth entity so as to form an agglutination complex; and

(c) detecting the presence of said target by observing said agglutination complex.