US 2001/0036637 A1 · DNA analyzing method

Abstract

The object of the present invention is to provide a method capable of analyzing the presence or absence of a target DNA sequence, the level and sequence characteristics thereof at a high sensitivity, and a device therefor, wherein the overall process from pretreatment to the recovery of DNA information and the analysts thereof can be completed in a speedy fashion by the simple device structure and procedures. Therefore, by preparing a single-stranded DNA fragment of a target DNA region, detecting the change in the absorbance of the single-stranded DNA sample while changing the denaturing condition of the conformation of the single-stranded DNA fragment by a denaturing condition regulatory means, and analyzing the curve of the change in the absorbance over the modification in the denaturing condition, the sequence information of the single-stranded DNA, namely the target DNA, can be generated in a rapid and simple manner.

Claims46

Dependent claims

A DNA analyzing method comprising preparing a single-stranded DNA fragment from a sample double-stranded DNA fragment, denaturing the conformation of the single-stranded DNA fragment under a given denaturing condition, preparing a melting curve data representing the relation between the denaturing condition and the denaturing results, and comparing the melting curve data with the melting curve data of the conformation of a single-stranded DNA fragment from a double-stranded DNA fragment of known DNA sequence when the conformation is denatured under a denaturing condition, wherein the sample double-stranded DNA fragment is represented on the basis of the relation thereof with the double-stranded DNA fragment of known DNA sequence from the comparison results.

A DNA analyzing method comprising preparing a single-stranded DNA fragment from a sample double-stranded DNA fragment, intercalating an intercalating agent capable of emitting fluorescence of a given wave length on receiving excitation beam of another given wave length with the base pairing formed in the complementary sequence of the conformation of the single-stranded DNA fragment if such conformation is formed, irradiating the excitation beam of the given wave length onto the single-stranded DNA fragment intercalated with the intercalating agent, denaturing the conformation of the single-stranded DNA fragment under a given denaturing condition while irradiating the excitation beam, detecting the change in the intensity of the fluorescence of the given wave length due to the denaturing as the denaturing results, preparing a melting curve data representing the relation between the denaturing condition and the denaturing results, and comparing the melting curve data with the melting curve data of the conformation of a single-stranded DNA fragment from a double-stranded DNA fragment of known DNA sequence when the conformation is denatured under a denaturing condition, wherein the sample double-stranded DNA fragment is represented on the basis of the relation thereof with the double-stranded DNA fragment of known DNA sequence from the comparison results.

A DNA analyzer comprising;

a holding means holding a sample solution containing one type of single-stranded DNA fragment or plural types of single-stranded DNA fragments;
a spectroscopic means measuring the UV absorbance of the sample solution containing said single-stranded DNA fragment(s);
a denaturing means having an action depending on the given denaturing condition onto the sample solution so as to denature the conformation formed by the single-stranded fragment(s) under given conditions and a denaturing condition regulatory means regulating the denaturing condition;
and
a signal processing means for inputting the signals for the regulation of the denaturing condition and the signals for the spectroscopic measurement which are then saved therein for processing,
wherein by changing the denaturing condition of the conformation of the single-stranded DNA fragment(s) by the denaturing condition regulatory means to prepare the melting curve data of the single-stranded DNA fragment sample over the change in the denaturing condition and subsequently comparing the melting curve data with the melting curve data of the conformation of a single-stranded DNA fragment from a double-stranded DNA fragment of known DNA sequence when the conformation is denatured under a denaturing condition, the sample double-stranded DNA fragment is represented on the basis of the relation thereof with the double-stranded DNA fragment of known DNA sequence from the comparison results.

3 more elements

A DNA analyzer comprising;

a holding means holding a sample solution containing one type of single-stranded DNA fragment or plural types of single-stranded DNA fragments;
a means for intercalating an intercalating agent capable of emitting fluorescence of a given wave length on receiving excitation beam of another given wave length with the base pairing formed in the complementary sequence forming the conformation of a single-stranded DNA fragment(s) in the sample solution;
and
a means for irradiating excitation beam of the given wave length onto the single-stranded DNA fragment(s) intercalated with the intercalating agent,
wherein by changing the denaturing condition of the conformation of the single-stranded DNA fragment(s) under the irradiation of the excitation beam to prepare the melting curve data of the single-stranded DNA fragment sample over the change in the denaturing condition, and subsequently comparing the melting curve data with the melting curve data of the conformation of a single-stranded DNA fragment from a double-stranded DNA fragment of known DNA sequence when the conformation is denatured under a denaturing condition, the sample double-stranded DNA fragment is represented on the basis of the relation thereof with the double-stranded DNA fragment of known DNA sequence from the comparison results.

2 more elements

A DNA analyzer comprising;

an enzymatic reaction means for effecting the selective amplification of a specific DNA region and simultaneously producing a single-stranded DNA fragment as an analytical subject;
a holding means holding a sample solution containing the single-stranded DNA fragment;
a spectroscopic means measuring the UV absorbance of the sample solution containing said single-stranded DNA fragment, a denaturing means having an action depending on the given denaturing condition onto the sample solution so as to denature the conformation formed by the single-stranded fragment under given conditions and a denaturing condition regulatory means regulating the denaturing condition;
and
a signal processing means for inputting the signals for the regulation of the denaturing condition and the signals for spectroscopic measurement which are then saved therein for processing,
wherein by preparing the melting curve data of the single-stranded DNA fragment sample over the change in the denaturing conditions for the conformation of the single-stranded DNA fragment with the denaturing condition regulatory means, and subsequently comparing the melting curve data with the melting curve data of the conformation of a single-stranded DNA fragment from a double-stranded DNA fragment of known DNA sequence when the conformation is denatured under a denaturing condition, the sample double-stranded DNA fragment is represented on the basis of the relation thereof with the double-stranded DNA fragment of known DNA sequence from the comparison results.

3 more elements

Dependencies are inferred from the claim text.

DNA analyzing method

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DNA analyzing method

Life of this patent

Abstract

Claims46