Method and apparatus for detecting disorders in genomic substances

• US 5,194,372 A
• Filed: 07/06/1990
• Issued: 03/16/1993
• Est. Priority Date: 07/10/1989
• Status: Expired due to Term

• Current Assignee: Hitachi, Ltd.
• Original Assignee: Hitachi, Ltd.
• Inventors: Tokita, Jiro, Nagai, Keiichi
• Primary Examiner(s): NOT, DEFINED
• Assistant Examiner(s): Chambers, Scott A.
• Application Number:
  US07/548,798
• Time in Patent Office: 984 Days
• Field of Search: 435/6, 435/18, 436/800, 436/151, 935/78, 935/76, 536/27
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 2521/30 Phosphoric diester hydrolysing, i.e. nuclease
• 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 436/80 Fluorescent dyes, e.g. rhodamine

Abstract

Disorders of the base sequences in genomic substances such as DNA and RNA are detected by changing the state of aggregation of fine particles by cleavaging using a nuclease. A single-stranded denatured product of the objective genomic substance is added to first and second fine particles each attached to plural pieces of first and second single-stranded nucleic acid probes, respectively. The first and second single-stranded nucleic acid probes are complementary to a first region and a second region, respectively, on the objective genomic substance, which are exclusive of each other and contiguous from each other. Aggregations of the first and second fine particles are formed by double or multiple hybridization reaction of the denatured objective genomic substance added with the first and second single-stranded nucleic acid probes. The aggregations are then digested with a nuclease which cleaves the non-complementary-mismatch-containing portion of the double strand of each hybrid permitting the formation of the aggregations, in the vicinity of a mismatch-localized region, but substantially not the completely complementary portion of the double strand of the hybrid. The size of the aggregations in the digested solution is first measured and then the degree of mismatch between the complementary base sequences of the objective genomic substance and the first or second single-stranded nucleic acid probe is measured using the size of the aggregations.

Claims

1. A method for detecting disorders in genomic substances comprising the steps of:

preparing a solution containing first particles each having plural pieces of a first single-stranded nucleic acid probe attached thereto and a large number of second particles each having plural pieces of a second single-stranded nucleic acid probe attached thereto, said first and second single-stranded nucleic acid probes being complementary to a first region and a second region, respectively, on an objective genomic substance, which are exclusive of each other and contiguous to each other;

adding a single-stranded denatured product of the target nucleic acid to the solution;

forming aggregations of the first and second particles in the solution by hybridization of the denatured target nucleic acid added with particles with attached first and second single-stranded nucleic acid probes;

digesting the solution containing the aggregations with a nuclease which cleave the non-complementary-mismatch-containing portion of the double strand of each hybrid which had permitted the formation of the aggregations, in the vicinity of a mismatch-localized region;

measuring the size of the aggregations in the digested solution; and

determining the degree of mismatching between the complementary base sequences of the target nucleic acid and the first or second single-stranded nucleic acid probe using the size information of the aggregations in the digested solution.

2. A method according to claim 1, wherein the difference of the sizes of the aggregations in the solutions before and after the digestion is measured.

3. A method according to claim 1, wherein the measurement of the size of the aggregations in the solution is conducted by measuring the intensity of scattered light generated by irradiation of the solution with laser beams.

4. A method according to claim 1, wherein the measurement of the size of the aggregations in the solution is conducted by detecting a photoacoustic signal given by irradiation of the solution with modulated laser beams.