Degree of hybridization detection method

Details Degree of hybridization detection method Degree of hybridization detection method

1999-12-13

2004-08-10

Marschel, Ardin H. (Department: 1631)

Data processing: measuring, calibrating, or testing

Measurement system in a specific environment

Biological or biochemical

C435S006120

Reexamination Certificate

active

06775621

ABSTRACT:

CROSS-REFERENCES TO RELATED APPLICATIONS
The present application, under 35 USC §119, claims the benefit of foreign priority applications, Japanese patent application serial number 355956/1998, filed Dec. 15, 1998 and Japanese patent application serial number 328352/1999, filed Nov. 18, 1999. These applications are explicitly incorporated herein by reference in their entirety and for all purposes.
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a hybridization detection method for the analysis of the presence or absence of a sequence of interest in a biopolymer sample by utilizing the hybridization between the sample biopolymer and a probe biopolymer, and also relates to a biochip applicable for the method.
2. Related Background Art
Heretofore, for the detection or fractionation of a molecule in the living body, particularly the detection of DNA of interest or the detection of the presence or absence of a genomic DNA, hybridization methods have been often utilized which uses a nucleic acid or protein having a known sequence as a probe. In such hybridization methods, a sample DNA labeled with a fluorescent material is hybridized to a probe DNA immobilized onto a substrate. When the sample DNA is bound to the probe DNA, the sample DNA is in turn immobilized on the substrate together with the probe DNA. The fluorescent material attached to the sample DNA is fluorescently excited by irradiation with excitation light from a light source to emit fluorescence, and the fluorescence is then detected. In this manner, the hybridization between the sample DNA and the probe DNA can be detected.
The principle of the prior art hybridization detection method as described above is illustrated in
FIGS. 7
,
8
and
9
.
As illustrated in
FIG. 7
, a given amount of probe DNA
1
a
is immobilized on a substrate (e.g., a glass plate)
4
as a spot
3
a
. Another probe DNAs
1
b
,
1
c
, . . . are also immobilized on the substrate
4
as spots
3
b
,
3
c
, . . . , respectively. In this case, however, it is impossible to immobilize all of the probe DNAs in equal amounts on the respective spots.
As illustrated in
FIG. 8A
, each of sample DNAs
5
a
,
5
b
,
5
c
, . . . is labeled with a fluorescent material
6
. As illustrated in
FIG. 8B
, the substrate
4
spotted with the probe DNAs
5
a
,
5
b
,
5
c
, . . . is placed in a hybridization solution
7
and then the fluorescently labeled sample DNAs
5
a
,
5
b
,
5
c
, . . . are added thereto to cause the hybridization between the probe DNAs and the sample DNAs. The hybridization solution
7
is a mixed solution comprising, for example, formaldehyde, SSC (sodium chloride/trisodium citrate), SDS (sodium dodecyl sulfate), EDTA (ethylenediaminetetraacetic acid), distilled water, and the like, in which the mixing ratio between the components may vary depending on the nature of the DNAs employed.
As illustrated in
FIG. 8C
, if any of the sample DNAs is complementary to any of the probe DNAs, the sample DNA is hybridized to the probe DNA to form a double-stranded structure (see the illustrations for the probe DNAs
1
a
and
1
b
). If not, the sample DNA remains unbound (see the illustration for the probe DNA
1
c
). As illustrated in
FIG. 9
, the detection of the hybridization can be performed by irradiating the substrate
4
with excitation light from a lamp
9
(i.e., an excitation light source) to excite the fluorescent material
6
, cutting off the light having wavelengths out of the emission wavelength range of the fluorescent material
6
with an optical filter
10
, and then detecting the light emitted from each spot with a two-dimensional photosensor
8
(e.g., a CCD camera).
In this case, in the spots where the hybridization takes place (e.g., spots
3
a
and
3
b
), the fluorescent material
6
is present, and therefore fluorescent emission can be detected by exciting the fluorescent material
6
by irradiation with excitation light from the lamp
9
. In contrast, in the spots where the hybridization does not take place (e.g., spot
3
c
), no fluorescent material
6
is present, and therefore no fluorescent emission is observed by irradiation with excitation light from the lamp
9
. In this manner, a light or dark spot is observed depending on the presence or absence of the hybridization event. The image data detected by the two-dimensional photosensor
8
is transferred to a computer
13
via a controller
12
and indicated on a display.
However, in this method, upon the immobilization of probe DNAs to a substrate, it is impossible to spot all of the probe DNAs on the substrate uniformly or in equal amounts, resulting in an undesirable variation in the amount of probe DNA between the spots with a larger amount of probe DNA and the spots with a smaller amount of probe DNA. Therefore, in the detection of the hybridization, although the presence of the hybridization event between the sample DNAs and the probe DNAs can be detected, it is impossible to determine quantitatively the amount of each sample DNA hybridized to any of the probe DNAs relative to the amount of the probe DNA.
SUMMARY OF THE INVENTION
The present invention has been made for improving the above-mentioned drawbacks of the prior art methods. Accordingly, the object of the present invention is to provide a detection method which can quantitatively determine the degree of the hybridization between a probe DNA and a sample DNA.
According to the present invention, to achieve the above-mentioned object, a probe biopolymer and a sample biopolymer are labeled with different fluorescent materials, so that the probe biopolymer and the sample biopolymer present on spots deposited on a substrate can be detected separately for each spot utilizing the difference of the emission wavelength of the fluorescent materials. In the detection of the hybridization between the probe biopolymer and the sample biopolymer, the emission wavelength of the fluorescent material labeling the probe biopolymer and the emission wavelength of the fluorescent material labeling the sample biopolymer are detected separately, so that it becomes possible to separately detect and, therefore, quantitatively determine the amount of the probe biopolymer and the amount of the sample biopolymer hybridized to the probe biopolymer for each spot.
Specifically, a fluorescent material that has labeled a probe biopolymer is caused to emit fluorescence to determine the amount of the probe biopolymer immobilized on a spot deposited on a substrate (e.g., a glass plate). Another type of fluorescent material that has labeled a sample biopolymer is also caused to emit fluorescence to determine the amount of the sample biopolymer hybridized to the probe biopolymer. Then, the difference between the amount of the probe biopolymer and the amount of the sample biopolymer is normalized with the amount of the probe biopolymer. Based on the normalized value, the amount of the sample biopolymer relative to the amount of the probe biopolymer spotted on the substrate can be determined. As used herein, the term “biopolymer” refers to any polymeric material constituting a living body, such as DNA, RNA and a protein.
That is, one aspect of the present invention is a hybridization detection method for detecting the hybridization between a probe and a sample, which comprising detecting both the amount of the probe and the amount of the sample bound to the probe. As used herein, the term “probe” refers to any biopolymer to be immobilized onto a substrate, such as DNA, and the term “sample” refers to any biopolymer to be hybridized to the probe, such as DNA.
Another aspect of the present invention is a hybridization detection method for detecting the hybridization between a probe and a sample, which comprising detecting a value produced by normalizing the difference between the amount of the probe and the amount of the sample bound to the probe with the amount of the probe.
In the detection of the amounts of the probe and the sample bound to the probe, the amount of the probe may be detected prior to the hybrid

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