Gene testing method

• US 20030087272A1
• Filed: 06/26/2002
• Published: 05/08/2003
• Est. Priority Date: 11/06/2001
• Status: Abandoned Application

• Current Assignee: Hitachi, Ltd.
• Original Assignee: Hitachi, Ltd.
• Inventors: Kambara, Hideki, Okano, Kazunori, Nagai, Keiichi, Zhou, Guohua
• Application Number:
  US10/179,208
• Publication Number:
  US 20030087272A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/485 involving kinase
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 2537/101 Homogeneous assay format, e.g. one pot reaction
• CPC Class Codes: C12Q 2563/101 radioactivity, e.g. radioactive labels
• CPC Class Codes: C12Q 2563/103 luminescence
• CPC Class Codes: C12Q 2565/301 Pyrophosphate (PPi)

Abstract

A method tests a gene and includes the steps of amplifying a target region to be analyzed of a double-stranded DNA using primers and dNTPs, enzymatically decomposing pyrophosphate formed in the former step and the primers and dNTP used therein, repeatedly synthesizing a complementary strand using primers specific to the sequence and mutation of the target region to be analyzed, and converting a pyrophosphate formed in the step of synthesizing into ATP, allowing the ATP to react with a chemiluminescent reagent to generate chemiluminescence, and monitoring the generated chemiluminescence to thereby detect the presence of the target to be analyzed. This method can easily and simply be performed with high sensitivity at low cost, in which a series of reactions can be performed in a homogenous measuring system.

Claims

1. A gene testing method comprising the steps of:

enzymatically synthesizing a DNA strand or RNA strand repeatedly, the DNA strand or the RNA strand being complementary to a sequence of a target region to be analyzed of a single-stranded or double-stranded DNA as a target;

converting a pyrophosphate being formed in the former step into ATP;

allowing the ATP to react with a chemiluminescent reagent to generate chemiluminescence, and

monitoring the generated chemiluminescence to thereby detect the presence of the target to be analyzed.

2. The gene testing method according to claim 1 for detecting the presence of a mutation region in a base sequence in the target, the method further comprising the steps of:

allowing a DNA probe to hybridize with the target, the DNA probe being specific to the sequence of the target region to be analyzed and having an 3′

-end capable of hybridizing with a base of the mutation region, if any, of the base sequence; and

repeatedly synthesizing a complementary strand to thereby detect the presence of the mutation region.

3. The gene testing method according to claim 1, further comprising the steps of:

preparing a testing DNA strand having a sequence of the target DNA and a promoter sequence; and

synthesizing an RNA strand using the testing DNA strand as a template to thereby yield the pyrophosphate.

4. The gene testing method according to claim 3 wherein the step of synthesizing a DNA strand having a promoter region specific to a base mutation comprises the step of controlling the synthesis of the DNA strand depending on whether or not a mutation of the target base sequence to be analyzed is present.

5. The gene testing method according to claim 1, comprising the steps of:

simultaneously synthesizing respective types of DNA strands including respective target regions to be analyzed of the target;

performing complementary strand synthesis reactions in partitioned regions or reaction subcells of a reactor, the respective reactions being specific to the respective target regions to be analyzed;

converting the resulting pyrophosphate to ATP; and

allowing the ATP to react with the chemiluminescent reagent to thereby generate chemiluminescence.

6. The gene testing method according to claim 1, further comprising the step of decomposing pyrophosphate, nucleotide substrates, DNA oligomers and other components which may adversely affect the step of detecting chemiluminescence in a liquid containing the testing template DNA, prior to the step of synthesizing the complementary strand for producing pyrophosphate for use in chemiluminescence.

7. A method for testing single nucleotide polymorphisms, the method comprising the step of controlling the synthesis of a complementary strand of a DNA strand depending on the type of a target base mutation, the DNA strand having a promoter sequence.

8. The method for testing single nucleotide polymorphisms, further comprising the steps of:

synthesizing an RNA from the DNA strand having a promoter sequence;

generating chemiluminescence using pyrophosphate formed during the RNA synthesis; and

optically detecting the chemiluminescence.

9. A gene testing method comprising the steps of:

amplifying a target region to be analyzed of a double-stranded DNA using a primer and dNTPs;

enzymatically decomposing pyrophosphate formed in the amplification step, and the primer and dNTPs used in the step of amplifying;

repeatedly synthesizing a complementary strand using a primer specific to the sequence and mutation of the target region to be analyzed;

converting a pyrophosphate formed in the step of synthesizing into ATP;

allowing the ATP to react with a chemiluminescent reagent to generate chemiluminescence; and

monitoring the generated chemiluminescence to thereby detect the presence of the target to be analyzed.