Method for nucleic acid quantitation
First Claim
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1. A method for nucleic acid quantitation, comprising the following steps:
- a) preparing a plurality of standard DNA samples each having a single-base substitution in its sequence relative to a sequence in a target DNA sample by carrying out a polymerase chain reaction with the use of primers, at least one of which primers comprises a primer sequence having a single-base substituted relative to a sequence of the target DNA at the 3rd to 10th base position from the 3′
end of the primer and having at least 16 or more bases in its sequence in the 5′
direction from the substituted base, thereby producing the standard DNAs each having the same sequence as that of the target DNA, except each of the standard DNAs has a single-base substitution relative to the sequence of the target DNA, wherein the plurality of standard DNA samples differ in the substituted base;
b) mixing the target DNA sample with a predetermined amount of the plurality of standard DNA samples, wherein the plurality of standard DNA samples differ in concentration;
c) amplifying the mixed sample using a set of primers which are completely complementary to the target DNA and the plurality of DNA standards to thereby amplify a region comprising the single-base substitution site;
d) dissociating the amplified sample into single strands, hybridizing thereto a probe capable of binding to a site immediately before the single-base substitution site, and sequentially adding ddATP, ddGTP, ddCTP, and ddTTP one by one to the hybridization product to perform a complementary strand synthesis reaction, wherein the probe has a sequence completely complementary to the site immediately before the single-base substitution site of the target DNA and the standard DNAs;
e) detecting luciferase reaction-induced luminescence derived from pyrophosphoric acid formed through the complementary strand synthesis reaction; and
f) quantitating the target DNA from the amount of the detected luminescence and the amount of the standard DNA sample.
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Abstract
It is intended to provide a novel convenient approach for DNA quantitative analysis that overcomes the disadvantages of conventional formulations. A standard DNA sample is prepared by introducing a single-base substitution into target DNA, and a predetermined amount thereof is mixed with a target DNA sample. The target and standard DNAs are amplified using the same primers designed to amplify a region comprising the single-base substitution site. To a hybridization product of a probe capable of binding to a site immediately before the single-base substitution site, ddATP, ddGTP, ddCTP, and ddTTP are sequentially added one by one to perform a complementary strand synthesis reaction. Luciferase reaction-induced luminescence derived from the formed pyrophosphoric acid is detected. The target DNA is quantitated from the amount of the detected luminescence and the amount of the added standard DNA sample.
3 Citations
7 Claims
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1. A method for nucleic acid quantitation, comprising the following steps:
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a) preparing a plurality of standard DNA samples each having a single-base substitution in its sequence relative to a sequence in a target DNA sample by carrying out a polymerase chain reaction with the use of primers, at least one of which primers comprises a primer sequence having a single-base substituted relative to a sequence of the target DNA at the 3rd to 10th base position from the 3′
end of the primer and having at least 16 or more bases in its sequence in the 5′
direction from the substituted base, thereby producing the standard DNAs each having the same sequence as that of the target DNA, except each of the standard DNAs has a single-base substitution relative to the sequence of the target DNA, wherein the plurality of standard DNA samples differ in the substituted base;b) mixing the target DNA sample with a predetermined amount of the plurality of standard DNA samples, wherein the plurality of standard DNA samples differ in concentration; c) amplifying the mixed sample using a set of primers which are completely complementary to the target DNA and the plurality of DNA standards to thereby amplify a region comprising the single-base substitution site; d) dissociating the amplified sample into single strands, hybridizing thereto a probe capable of binding to a site immediately before the single-base substitution site, and sequentially adding ddATP, ddGTP, ddCTP, and ddTTP one by one to the hybridization product to perform a complementary strand synthesis reaction, wherein the probe has a sequence completely complementary to the site immediately before the single-base substitution site of the target DNA and the standard DNAs; e) detecting luciferase reaction-induced luminescence derived from pyrophosphoric acid formed through the complementary strand synthesis reaction; and f) quantitating the target DNA from the amount of the detected luminescence and the amount of the standard DNA sample. - View Dependent Claims (2, 3, 4, 5, 6, 7)
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Specification
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Current AssigneeHitachi, Ltd.
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Original AssigneeHitachi, Ltd.
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InventorsTaniguchi, Kiyomi, Kambara, Hideki
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Primary Examiner(s)Benzion, Gary
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Assistant Examiner(s)WILDER, CYNTHIA B
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Application NumberUS12/292,800Publication NumberTime in Patent Office1,560 DaysField of Search435/6, 435/91.2US Class Current435/91.2CPC Class CodesC12Q 1/6851 Quantitative amplificationC12Q 2525/161 incorporating target specif...C12Q 2545/101 with an internal standard/c...
