U.S. patent application number 09/874308 was filed with the patent office on 2001-11-01 for dna analyzing method.
This patent application is currently assigned to Hitachi, Ltd.. Invention is credited to Fujita, Takeshi, Umemura, Shin-Ichiro.
Application Number | 20010036637 09/874308 |
Document ID | / |
Family ID | 17545854 |
Filed Date | 2001-11-01 |
United States Patent
Application |
20010036637 |
Kind Code |
A1 |
Fujita, Takeshi ; et
al. |
November 1, 2001 |
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.
Inventors: |
Fujita, Takeshi;
(Saitama-Ken, JP) ; Umemura, Shin-Ichiro; (Tokyo,
JP) |
Correspondence
Address: |
CROWELL & MORING, L.L.P.
P.O. BOX 14300
Washington
DC
20044-4300
US
|
Assignee: |
Hitachi, Ltd.
|
Family ID: |
17545854 |
Appl. No.: |
09/874308 |
Filed: |
June 6, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09874308 |
Jun 6, 2001 |
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09563454 |
May 3, 2000 |
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09563454 |
May 3, 2000 |
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08552496 |
Nov 9, 1995 |
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6106777 |
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Current U.S.
Class: |
435/6.12 ;
435/287.2; 436/94 |
Current CPC
Class: |
Y10S 436/808 20130101;
C12Q 1/6816 20130101; Y10S 436/80 20130101; Y10S 436/807 20130101;
Y10T 436/143333 20150115; C12Q 2527/107 20130101; C12Q 1/6816
20130101; C12Q 2537/165 20130101 |
Class at
Publication: |
435/6 ;
435/287.2; 436/94 |
International
Class: |
C12Q 001/68; G01N
033/00; C12M 001/34 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 9, 1994 |
JP |
6-274735 |
Claims
What is claimed is:
1. 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.
2. 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.
3. A DNA analyzing method according to claim 1, wherein the
comparison of the melting curve data of the sample double-stranded
DNA fragment with known melting curve data comprises comparing the
data of a freshly measured end input signal curve with one of the
data sets of known template melting curves preliminarily prepared
or with all of the data sets of the curves preliminarily prepared
by linearly binding a plurality of the known template melting
curves in combination, and determining that the data of a template
curve with the least statistical error or the combination of the
data sets of such template curves which in combination form a curve
with the least statistical error is the sequence characteristics of
the measured single-stranded DNA fragment from the sample
double-stranded DNA fragment.
4. A DNA analyzing method according to claim 2, wherein the
comparison of the melting curve data of the sample double-stranded
DNA fragment with known melting curve data comprises comparing the
data of a freshly measured and input signal curve with one of the
data sets of known template melting curves preliminarily prepared
or with all of the data sets of the curves preliminarily prepared
by linearly binding a plurality of the known template melting
curves in combination, and determining that the data of a template
curve with the least statistical error or the combination of the
data sets of such template curves which in combination form a curve
with the least statistical error is the sequence characteristics of
the measured single-stranded DNA fragment from the sample
double-stranded DNA fragment.
5. A DNA analyzing method according to claim 1, wherein the
comparison of the melting curve data of the sample double-stranded
DNA fragment with known melting curve data comprises calculating
the statistical error between the data of a freshly measured and
input signal curve and one of the data sets of known template
melting curves preliminarily prepared or each of the data sets of
the curves preliminarily prepared by linearly binding a plurality
of the known template melting curves in combination, thereby
selecting one curve data with the least error, carrying out the
calculation and selection over each of the data sets of the known
template melting curves or each of the curve data sets
preliminarily prepared by linearly binding a different combination
of the data sets of the known template melting curves, and
representing a given number of the curve data selected from the
group of all of the data sets of the curves in the increasing order
of the statistical error as the sequence characteristics of the
measured single-stranded DNA fragment from the sample
double-stranded DNA fragment.
6. A DNA analyzing method according to claim 2, wherein the
comparison of the melting curve data of the sample double-stranded
DNA fragment with known melting curve data comprises calculating
the statistical error between the data of a freshly measured and
input signal curve and one of the data sets of known template
melting curves preliminarily prepared or each of the data sets of
the curves preliminarily prepared by linearly binding a plurality
of the known template melting curves in combination, thereby
selecting one curve data with the least error, carrying out the
calculation and selection over each of the data sets of the known
template melting curves or each of the curve data sets
preliminarily prepared by linearly binding a different combination
of the data sets of the known template melting curves, and
representing a given number of the curve data selected from the
group of all of the data sets of the curves in the increasing order
of the statistical error as the sequence characteristics of the
measured single-stranded DNA fragment from the sample
double-stranded DNA fragment.
7. A DNA analyzing method according to claim 1, wherein the
denaturing condition is temperature and the melting curve data is
derived from the change in the absorbance of a sample via
temperature.
8. A DNA analyzing method according to claim 2, wherein the
denaturing condition is temperature and the melting curve data is
derived from the change in the fluorescence intensity of a sample
via temperature.
9. A DNA analyzing method according to claim 3, wherein the
denaturing condition is temperature and the melting curve data is
derived from the change in the absorbance of a sample via
temperature.
10. A DNA analyzing method according to claim 4, wherein the
denaturing condition is temperature and the melting curve data is
derived from the change in the fluorescence intensity of a sample
via temperature.
11. A DNA analyzing method according to claim 1, wherein the
sequence information of a single-stranded DNA fragment is obtained
by alternatively changing the denaturing condition for the progress
in denaturing and for the formation of the conformation in a
continuous manner, and measuring and analyzing the hysteresis
characteristics of the change in absorbance depending on the
alternative changing.
12. A DNA analyzing method according to claim 2, wherein the
sequence information of a single-stranded DNA fragment is obtained
by alternatively changing the denaturing condition for the progress
in denaturing and for the formation of the conformation in a
continuous manner, and measuring and analyzing the hysteresis
characteristics of the change in the fluorescence intensity.
13. A DNA analyzing method according to claim 3, wherein the
sequence information of a single-stranded DNA fragment is obtained
by alternatively changing the denaturing condition for the progress
in denaturing and for the formation of the conformation in a
continuous manner, and measuring and analyzing the hysteresis
characteristics of the change in absorbance depending on the
alternative changing.
14. A DNA analyzing method according to claim 5, wherein the
denaturing condition is temperature and the melting curve data is
derived from the change in the absorbance of a sample via
temperature.
15. A DNA analyzing method according to claim 5, wherein the
sequence information of a single-stranded DNA fragment is obtained
by alternatively changing the denaturing condition for the progress
in denaturing and for the formation of the conformation in a
continuous manner, and measuring and analyzing the hysteresis
characteristics of the change in absorbance depending on the
alternative changing.
16. A DNA analyzing method according to claim 4, wherein the
sequence information of a single-stranded DNA fragment is obtained
by alternatively changing the denaturing condition for the progress
in denaturing and for the formation of the conformation in a
continuous manner, and measuring and analyzing the hysteresis
characteristics of the change in the fluorescence intensity
depending on the alternative changing.
17. A DNA analyzing method according to claim 6, wherein the
denaturing condition is temperature and the melting curve data is
derived from the change in the fluorescence intensity of a sample
via temperature.
18. A DNA analyzing method according to claim 6, wherein the
sequence information of a single-stranded DNA fragment is obtained
by alternatively changing the denaturing condition for the progress
in denaturing and for the formation of the conformation in a
continuous manner, and measuring and analyzing the hysteresis
characteristics of the change in the fluorescence intensity
depending on the alternative changing.
19. A DNA analyzing method according to claim 2, wherein the
intercalating agent is ethidium bromide.
20. 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.
21. 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.
22. A DNA analyzer according to claim 20, wherein the comparison of
the melting curve data of the sample double-stranded DNA fragment
with known melting curve data comprises comparing the data of a
freshly measured and input signal curve with one of the data sets
of known melting curves preliminarily prepared or with all of the
combinations of the curve data sets preliminarily prepared by
linearly binding a plurality of template curve data sets, and
determining that the data of a template curve with the least
statistical error or the combination of the data sets of such
template curves which in combination form a curve with the least
statistical error is the sequence characteristics of the measured
single-stranded DNA fragment from the sample double-stranded DNA
fragment.
23. A DNA analyzer according to claim 21, wherein the comparison of
the melting curve data of the sample double-stranded DNA fragment
with known melting curve data comprises comparing the data of a
freshly measured and input signal curve with one of the data sets
of known melting curves preliminarily prepared or with all of the
combinations of the curve data sets preliminarily prepared by
linearly binding a plurality of template curve data sets, and
determining that the data of a template curve with the least
statistical error or the combination of the data sets of such
template curves which in combination form a curve with the least
statistical error is the sequence characteristics of the measured
single-stranded DNA fragment from the sample double-stranded DNA
fragment.
24. A DNA analyzer according to claim 20, wherein the comparison of
the melting curve data of the sample double-stranded DNA fragment
with known melting curve data comprises calculating the statistical
error between the data of a freshly measured and input signal curve
and one of the data sets of known template melting curves
preliminarily prepared or each of the data sets of the curves
preliminarily prepared by linearly binding a plurality of the known
template melting curves in combination, thereby selecting one curve
data with the least error, carrying out the calculation and
selection over each of the remaining data sets of the known
template melting curves or each of the data sets of the curves
preliminarily prepared by linearly binding a plurality of the known
template melting curves in combination, and representing a given
number of the selected curve data in the increasing order of the
statistical error as the sequence characteristics of the measured
single-stranded DNA fragment from the sample double-stranded DNA
fragment.
25. A DNA analyzer according to claim 21, wherein the comparison of
the melting curve data of the sample double-stranded DNA fragment
with known melting curve data comprises calculating the statistical
error between the data of a freshly measured and input signal curve
and one of the data sets of known template melting curves
preliminarily prepared or each of the data sets of the curves
preliminarily prepared by linearly binding a plurality of the known
template melting curves in combination, thereby selecting one curve
data with the least error, carrying out the calculation and
selection over each of the remaining data sets of the known
template melting curves or each of the data sets of the curves
preliminarily prepared by linearly binding a plurality of the known
template melting curves in combination, and representing a given
number of the selected curve data in the increasing order of the
statistical error as the sequence characteristics of the measured
single-stranded DNA fragment from the sample double-stranded DNA
fragment.
26. A DNA analyzer according to claim 20, wherein the denaturing
condition is temperature and the melting curve data is derived from
the change in the absorbance of a sample via temperature.
27. A DNA analyzer according to claim 21, wherein the denaturing
condition is temperature and the data of a melting curve is derived
from the change in the fluorescence intensity of a sample via
temperature.
28. A DNA analyzer according to claim 22, wherein the denaturing
condition is temperature and the melting curve data is derived from
the change in the absorbance of a sample via temperature.
29. A DNA analyzer according to claim 23, wherein the denaturing
condition is temperature and the melting curve data is derived from
the change in the fluorescence intensity of a sample via
temperature.
30. A DNA analyzer according to claim 20, wherein the sequence
information of a single-stranded DNA fragment is obtained by
alternatively changing the denaturing condition for the progress in
denaturing and for the formation of the conformation in a
continuous manner, and measuring and analyzing the hysteresis
characteristics of the change in absorbance depending on the
alternative changing.
31. A DNA analyzer according to claim 21, wherein the sequence
information of a single-stranded DNA fragment is obtained by
alternatively changing the denaturing condition for the progress in
denaturing and for the formation of the conformation in a
continuous manner, and measuring and analyzing the hysteresis
characteristics of the change in the fluorescence intensity
depending on the alternative changing.
32. A DNA analyzer according to claim 22, wherein the sequence
information of a single-stranded DNA fragment is obtained by
alternatively changing the denaturing condition for the progress in
denaturing and for the formation of the conformation in a
continuous manner, and measuring and analyzing the hysteresis
characteristics of the change in absorbance depending on the
alternative changing.
33. A DNA analyzer according to claim 24, wherein the denaturing
condition is temperature and the melting curve data is derived from
the change in the absorbance of a sample via temperature.
34. A DNA analyzer according to claim 24, wherein the sequence
information of a single-stranded DNA fragment is obtained by
alternatively changing the denaturing condition for the progress in
denaturing and for the formation of the conformation in a
continuous manner, and measuring and analyzing the hysteresis
characteristics of the change in absorbance depending on the
alternative changing.
35. A DNA analyzer according to claim 23, wherein the sequence
information of a single-stranded DNA fragment is obtained by
alternatively changing the denaturing condition for the progress in
denaturing and for the formation of the conformation in a
continuous manner, and measuring and analyzing the hysteresis
characteristics of the change in the fluorescence intensity
depending on the alternative changing.
36. A DNA analyzer according to claim 25, wherein the denaturing
condition is temperature and the melting curve data is derived from
the change in the fluorescence intensity of a sample via
temperature.
37. A DNA analyzer according to claim 25 wherein the sequence
information of a single-stranded DNA fragment is obtained by
alternatively changing the denaturing condition for the progress in
denaturing and for the formation of the conformation in a
continuous manner, and measuring and analyzing the hysteresis
characteristics of the change in the fluorescence intensity
depending on the alternative changing.
38. A DNA analyzer according to claim 21, wherein the intercalating
agent is ethidium bromide.
39. 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.
40. A DNA analyzer according to claim 39, wherein the
single-stranded DNA fragment is a specific single-stranded DNA
fragment prepared from a specific DNA region fragment preliminarily
amplified and enriched by PCR in a manner specific to the
region.
41. A DNA analyzer according to claim 40, wherein the
single-stranded DNA fragment is a specific single-stranded DNA
fragment preliminarily amplified in a manner specific to the region
by asymmetric PCR capable of replicating an excess amount of the
objective single-stranded DNA fragment by setting the volume ratio
of a pair of primers to be used for PCR at an uneven ratio.
42. A DNA analyzer according to claim 40, wherein the
single-stranded DNA fragment is a specific single-stranded DNA
fragment prepared by removing either one of a single-stranded DNA
fragment not immobilized on a support or a single-stranded DNA
fragment immobilized on the support from the PCR products amplified
with one primer preliminarily immobilized on the support or the
other primer not immobilized on the support.
43. A DNA analyzing method according to claim 1, wherein the
comparison of the melting curve data of the sample double-stranded
DNA fragment with known melting curve data-comprises comparing the
data of a freshly measured and input signal curve as the defined
combination of given functions with known template melting curve
data preliminarily prepared as the defined combination of given
functions and determining that a template curve data with the least
statistical error is defined as the sequence characteristics of the
measured single-stranded DNA fragment of the sample double-stranded
DNA fragment.
44. A DNA analyzing method according to claim 2, wherein the
comparison of the melting curve data of the sample double-stranded
DNA fragment with known melting curve data comprises comparing the
data of a freshly measured and input signal curve as the defined
combination of given functions with known template melting curve
data preliminarily prepared as the defined combination of given
functions, and determining that a template curve data with the
least statistical error is defined as the sequence characteristics
of the measured single-stranded DNA fragment of the sample
double-stranded DNA fragment.
45. A DNA analyzing method according to claim 21, wherein the
comparison of the melting curve data of the sample double-stranded
DNA fragment with known melting curve data comprises comparing the
data of a freshly measured and input signal curve as the defined
combination of given functions with known template melting curve
data preliminarily prepared as the defined combination of given
functions, and determining that a template curve data with the
least statistical error is defined as the sequence characteristics
of the measured single-stranded DNA fragment of the sample
double-stranded DNA fragment.
46. A DNA analyzing method according to claim 22, wherein the
comparison of the melting curve data of the sample double-stranded
DNA fragment with known melting curve data comprises comparing the
data of a freshly measured and input signal curve as the defined
combination of given functions with known template melting curve
data preliminarily prepared as the defined combination of given
functions, and determining that a template curve data with the
least statistical error is defined as the sequence characteristics
of the measured single-stranded DNA fragment of the sample
double-stranded DNA fragment.
Description
BACKGROUND OF THE INVENTION
[0001] The present invention relates to a method for analyzing DNA
information in the fields of clinical diagnosis and life science,
and a device therefor.
[0002] As the progress in the technology of DNA analysis, recently,
significant attention has been focused on the analysis of
information of DNA/RNA sequence in the fields of clinical diagnosis
and life science. In the field of life science, for example,
advances have been made for determining the whole nucleotide
sequence of a variety of animal and plant DNAs, as illustrated by
the Human Genome Project. Thus, the coding region of a novel
protein and the regulatory site of the expression thereof have been
analyzed gradually, involving also the elucidation of pathogenic
genes such as oncogenes and the like.
[0003] In the field of clinical diagnosis, alternatively, the
introduction of the technology of DNA analysis has been accelerated
toward the identification of a variety of etiology and laboratory
tests, on the basis of the fruitful results of these research
works. The diagnosis of infectious diseases including viral
hepatitis type C and AIDS (acquired immunodeficiency syndrome) due
to HIV (human immunodeficiency virus) infection is one example of
the fields for which the introduction of DNA diagnosis has been
highly desired because of the high detection sensitivity required
therefor and because of the relation between the infectious
performance of these viruses (retroviruses) and the DNA/RNA
polymorphism. For the laboratory tests of tumor cells, which are
now depending on empirical pathological diagnosis, and for the
tests of HLA (human leukocyte antigen) type with the sample number
rapidly increasing from the demand of the registration at the
myeloid bank, the introduction of the technology of DNA analysis
has been expected, which can give accurate and precise
information.
[0004] A great progress has been made recently in the DNA analysis
technology desired in such fields, wherein a method for separating
slight difference in DNA sequence utilizing the difference in the
conformation of a single-stranded DNA during electrophoresis has
been developed, in addition to the conventional DNA sequencing and
hybridization methods. As introduced in Genomics, Vol. 5, pp.
874-879, 1989, for example, the method designated as SSCP (Single
Strand Conformation Polymorphisms) has been drawing attention as a
technique for detecting even a single base substitution at a high
sensitivity. The separating method detects the difference in
sequence by detecting the difference in the conformation as the
difference in the mobility on gel electrophoresis, with attention
focused on the finding that leaving DNA, normally composed of a
pair of complementary double strands, in the state of a single
strand, typically, the single-stranded DNA autonomously associates
by itself within the molecule under appropriate conditions (ion
strength, temperature and the like) and form certain conformation
specific to the sequence, which conformation varies depending on
the sequence.
[0005] The method detects the difference in DNA at a high
sensitivity, but because electrophoresis is employed in the process
of separation, such a long period of time should be required for
the separation that a high throughput is realized with much
difficulty. The selection of the conditions for efficiently
reflecting the difference in DNA sequence over the difference in
the mobility on electrophoresis involves tough works. Still
furthermore, the method is hardly automated, and additionally, the
method involves another drawback in requiring the separation in
some case under a plurality of conditions so as to thoroughly
separate the whole polymorphism.
[0006] A method called denaturant gradient gel electrophoresis for
detecting slight difference in DNA sequence is also proposed, but
because the method also employs electrophoresis, it has the same
drawbacks as described above.
[0007] It has been known that the difference between the denaturing
conditions of a double-stranded DNA with a completely complementary
sequence and the conditions of a double-stranded DNA with an almost
complementary but not completely complementary sequence can be
detected as the difference in absorbance change when such
double-stranded DNA is denatured into a single-stranded DNA
(melting curve) (for example, see I. V. Razlutuskii, L. S.
Shlyakhtenko and Yu. L. lyubchenko: Nucleic Acids Research, Vol.
15, No. 16, pp. 6665-6676 (1987)). Furthermore, it has been known
that the type of a single-stranded RNA forming conformation (hair
pin, stem, loop structure, etc.) can be detected and identified by
the change in the absorbance when the base pairing of the
single-stranded RNA is denatured (melting curve) (for example, see
L. G. Laing and D. E. Draper: J. Mol. Biol. (1994) 237,
560-576).
[0008] According to these methods, no electrophoresis procedure is
required after a sample is collected, so that these methods are
advantageous in that the procedures are simple and the measurements
are easily carried out as optical measurement, with higher
reliability.
[0009] Additionally, a technique is proposed, comprising optically
measuring and detecting the phenomenon that when a double-stranded
DNA having a completely complementary sequence and a
double-stranded DNA having a nearly completely but not completely
complementary sequence are denatured, the fluorescent energy
transfer induced by two types of fluorophors individually labeling
each of the complementary strands is eliminated, thereby detecting
the difference in the sequences of the two types of DNAs not
completely complementary (Japanese Patent Laid-open No. Hei
7-31500). Because no electrophoresis procedure is then required
after a sample is collected, the same advantage is brought about as
described in the aforementioned example.
[0010] However, it is only sequence compositions (GC contents,
etc.) or deletion/insertion of bases that these methods can detect.
The identification of detailed difference in sequence, particularly
DNA polymorphism including single-base substitution, is
substantially difficult by these methods. These methods require to
carry out the regulation of denaturing conditions at such an
extremely low rate that the methods have been hardly applicable to
the detection and determination at a high throughput.
[0011] Furthermore, no examination has been made about direct
optical analysis of DNA polymorphism including single-base
substitution in a single-stranded DNA.
SUMMARY OF THE INVENTION
[0012] In order to overcome the problem, in accordance with the
present invention, the melting curve of the conformation of a
single-stranded DNA is directly detected, whereby a more precise
and practical method of signal processing is provided along with a
device therefor with a simplified structure.
[0013] For separation and analysis of a single-stranded DNA of a
target DNA region including a heterozygote type having more than
two DNA types in one cell, the method of the present invention
comprises memorizing the melting curves of all known types of
polymorphism (template curves), comparing the signal curve of a
sample with such single template curve or with all the curves
prepared via linear binding of a plurality of the template curves
in combination, and determining that the DNA type, namely the
sequence characteristics of the measured single-stranded DNA
fragment, is defined as a combination of the template curves
providing that the RMS between the signal curve and the combination
is the smallest below a given value.
[0014] For the DNA analysis by PCR for clinical diagnosis, the
sequence information of the target DNA fragment together with the
amount of the PCR product, should be obtained. Therefore, the
present invention is designed advantageously so as to bring about
simultaneously all the information mentioned above via the
quantitative measurement of the melting curve.
[0015] By designing that a sample holding means which hold the
sample and regulates the sample temperature should be a means with
a larger surface/volume ratio, the denaturing rate gets faster for
measurement. Consequently, it has been found that the melting curve
of a single-stranded DNA during the temperature elevation for
resolving the conformation draws a different curve from the melting
curve during the temperature decrease for forming the conformation,
which indicates the presence of hysteresis. By processing the data
with a signal processing means identical to what has been described
above, DNA polymorphism with single-base substitution can be
analyzed.
[0016] It has been found that, as an intercalating agent, such as
ethidium bromide which can sift the fluorescent wave length after
intercalating to the DNA base pairing, is intercalated, the
intensity of the fluorescence emitted from the interaction of the
single-stranded DNA with the intercalating agent during the
irradiation of the excitation beam changes corresponding to the
denaturation of the single-stranded DNA. Therefore, by measuring
the intensity and processing the data with a signal processing
means identical to what has been described above, the sequence
information of the single-stranded DNA fragment can be yielded.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] FIG. 1 is a block diagram of the structure of a detector of
a first example in accordance with the present invention;
[0018] FIGS. 2(a) and (b) are graphs of the absorbance data and
differential absorbance data obtained from the temperature
differentiation of the absorbance data, representing the melting
curve of a single-stranded DNA of the known type DQA1*0101 of DNA
in the exon 2 of the HLA-DQA1 region;
[0019] FIGS. 3(a) and (b) are graphs of the absorbance data and
differential absorbance data obtained from the temperature
differentiation of the absorbance data, representing the melting
curve of a single-stranded DNA of the known type DQA1*0102 DNA in
the exon 2 of the HLA-DQA1 region;
[0020] FIGS. 4(a) and (b) are graphs of the absorbance data and
differential absorbance data obtained from the temperature
differentiation of the absorbance data, representing the melting
curve of a single-stranded DNA of the known type DQA1*0103 DNA in
the exon 2 Of the HLA-DQA1 region;
[0021] FIGS. 5(a) and (b) are graphs of the absorbance data and
differential absorbance data obtained from the temperature
differentiation of the absorbance data, representing the melting
curve of a single-stranded DNA of the known type DQA1*0301 DNA in
the exon 2 of the HLA-DQA1 region;
[0022] FIGS. 6(a) and (b) are graphs of the absorbance data and
differential absorbance data obtained from the temperature
differentiation of the absorbance data, representing the melting
curve of a single-stranded DNA of the known type DQA1*0401 DNA in
the exon 2 of HLA-DQA1 region;
[0023] FIGS. 7(a) and (b) are graphs of the absorbance data and
differential absorbance data obtained from the temperature
differentiation of the absorbance data, representing the melting
curve of a single-stranded DNA of the known type DQA1*0601 DNA in
the exon 2 of HLA-DQA1 region;
[0024] FIG. 8 is a graph of the derivative of the melting curve of
the type DQA1*0301, after fitting with the Gaussian distribution
curves.
[0025] FIG. 9 is a graph of the derivative of the melting curve of
a heterozygote sample of the types DQA1*0102 and DQA1*0301,
representing the characteristic parameters of the curve;
[0026] FIG. 10 is graphs of another analysis example of a
heterozygote DNA;
[0027] FIG. 11 depicts a measurement example of the hysteresis
curve of a melting curve;
[0028] FIG. 12 depicts the schematic chart of the process flow of
an example in accordance with the present invention;
[0029] FIG. 13 depicts the schematic view of the structure of the
detector of the first example in accordance with the present
invention;
[0030] FIG. 14 depicts the detailed structure of the spectroscopic
cell of an example in accordance with the present invention;
[0031] FIG. 15 depicts the block diagram of an example of the
structure of a detector for detecting the fluorescence of the DNA
and the intercalating agent;
[0032] FIG. 16 is a graph of the melting curve via the fluorescence
from HLA-DQA1*0101 and HLA-DQA1*0102.
DESCRIPTION OF THE PREFERRED EMBODIMENT
[0033] The present invention will now be illustrated hereinbelow in
one example.
[0034] FIG. 1 is a block diagram of the fundamental structure of
the DNA analyzer in accordance with the present invention.
Ultraviolet ray (of a wave length of 260 nm) from light source 1 is
divided into two beams at optical system 2, which are then
individually incident into sample part 4 and control part 5, both
being placed in cell holder 6. Thereafter, the individual beams
collimated with optical systems (not shown) are detected with
photomultiplier 7, and are then passed through amplifier 9 and
processed with analytical signal processing device 10. The cell
holder 6 can control the temperatures of the sample part 4 and the
control part 5, following the temperature profiles programmed
optionally with temperature regulator 8. Temperature control can be
done at an optional rate of temperature increase or decrease. The
temperature in the cell holder 6 is measured with a temperature
sensor (not shown), and input to the feedback temperature regulator
8 and the signal processing device 10 simultaneously. Because it is
required that the temperature of some sample should be controlled
within the range from -20.degree. C. to 100.degree. C., the cell
holder 6 should have such a structure that dry air flows from the
bottom of each sample holding cell 4 and 5 to the top thereof so as
to prevent the occurrence of bedewing on the surface of the both
cells.
[0035] The control part 5 is arranged, so as to correct the
absorption of a buffer solution dissolving a sample at the sample
part 4 and the absorption of the sample cell to determine the net
DNA absorption. This is a routine technique in the spectrometry,
and the data in examples described below are all through the
correction.
[0036] Using the device described above, DNA polymorphism analysis
will be illustratively described hereinbelow.
[0037] In the present Example, single-stranded DNA of HLA (human
leukocyte antigen) class II: DQA1 region was amplified using
asymmetric-PCR method from genomic DNA extracted from human blood
cells. Then, the DNA polymorphism analysis (DNA typing) of the
region was carried out.
[0038] By the standard procedure, a DNA sample solution with the
extracted genomic DNA, a PCR buffer solution containing a final 10
mM concentration of Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM
MgCl.sub.2, 0.02% gelatin, and 200 .mu.M of each of
deoxyribonucleotide triphosphates (dATP, dCTP, dGTP, and dTTP), 2.5
U Taq polymerase, 20 pmol of each of the two types of primers GH 26
and GH 27 corresponding to the HLA-DQA1 region to be analyzed (Ulf
B. Gyllensten and Henry A. Erlich; Proceedings of the National
Academy of Sciences of USA, Vol. 85, pp. 7652-7656, October 1988)
were mixed together in a test tube, followed by overlaying mineral
oil on the mixture. The PCR cycling condition was 27 cycles of
94.degree. C. (1 minute), 57.degree. C. (1.2 minute) and 72.degree.
C. (1 minute) in this order.
[0039] Using {fraction (1/100)} of the reaction product, asymmetric
PCR was done. The asymmetric PCR solution was almost the same as
described for PCR, except that the amounts of the primers were
modified such that GH 26 was 10 pmol and GH 27 was 1 pmol. The PCR
cycling condition was 15 cycles of 94.degree. C. (1 minute),
57.degree. C. (1.2 minute) and 72.degree. C. (1 minute) in this
order.
[0040] The reaction product was desalted and concentrated with a
microfilter (Microcon.TM.30, manufactured by Grace Japan), which
was then dissolved in the TNE buffer (10 mM Tris-HCl, 1 mM EDTA (pH
8.3), 30 mM NaCl). The resulting product was defined as a sample
solution.
[0041] In the present Example, asymmetric PCR was used for
preparing a single-stranded DNA, but other methods may be used as
well, including a method comprising PCR amplifying a
double-stranded DNA and thereafter digesting one single-stranded
DNA with .lambda. exonuclease (.lambda. exonuclease method) and a
method comprising PCR amplification in the state where either one
of the PCR primers is immobilized on membrane and thereafter
washing off the single-stranded DNA not immobilized on the membrane
while elevating the temperature to the melting temperature
(membrane method). Although the membrane method requires to
immobilize a predetermined primer on membrane, the method can
prepare a single-stranded DNA at a high purity in a simple manner.
Additionally, the method is suitable for automation.
[0042] Placing the sample solution at the sample part 4 while
placing the TNE buffer as the control solution at the control part
5, the sample temperature was once decreased to 0.degree. C.
Subsequently, the temperature was elevated to 60.degree. C. at an
elevation rate of 1.degree. C./min.
[0043] FIGS. 2 to 7(a) and (b) depict the melting curves of the
single-stranded DNAs of the known six types (DQA1*0101, DQA1*0102,
DQA1*0103, DQA1*0301, DQA1*0401, DQA1*0601; The WHO Nomenclature
Committee for Factors of the HLA System, 1989, Immunogenetics, 31:
131-140, 1990) of the HLA-DQA1 region DNA (242 bp or 239 bp), as
absorbance data and differential absorbance data by temperature
(the data of the other two types, i.e. DQA1*0201 and DQA1*0501,
were not shown herein because the inventors could not obtain their
homozygote samples). In the figures (b), only differential
absorbance data are shown. One base is different between the types
DQA1*0101 and DQA1*0102; three bases are different between the
types DQA1*0101 and DQA1*0103; and two bases are different between
the types DQA1*0102 and DQA1*0103. Twenty-seven bases are different
between the types DQA1*0101 and DQA1*0301. The types DQA1*0401 and
DQA1*0601, by three bases shorter than the other types, have the
sequences difference in 20 bases or more from the sequence of
DQA1*0101, but only one base is different between these types
DQA1*0401 and DQA1*0601.
[0044] Still further, the individual figures (a) and (b) depict the
results of the analysis of the same samples, but the figure (a)
depicts the results of the analysis obtained until the application
of the priority of the present invention, while the figure (b)
depicts the results of the analysis relatively recently obtained.
The reason that the two figures do not completely agree with each
other although the figures depict the analysis results of the same
samples resides in the difference in the skill of analytical
procedures and the data correction adopted for making up for the
unskilled analytical procedures. The fact does not mean that the
analysis has no reproducibility.
[0045] The figures show that groups with very different sequences,
for example, a group of DQA1*0101-DQA1*0103 (type 1), a group
DQA1*0301 (type 3) and a group of DQA1*0401 and DQA1*0601 (short
type), have distinctively different characteristics in their
melting curves. It is also shown that the type difference such as
the difference in one base or two bases (for example,
DQA1*0101-DQA1*0103, DQA1*0401, DQA1*0601), can be detected as a
marked difference.
[0046] FIG. 8 depicts the derivative of the melting curve of the
type DQA1*0301, fitting with the Gaussian distribution functions.
As apparently shown in FIG. 8, the melting curve of the type
DQA1*0301 can be satisfactorily fitted with the superposition of
two types of Gaussian curves.
[0047] In other words, this indicates that by preparing the melting
curves of the known DNA-type and comparing the melting curve of a
sample DNA with the melting curves of these known DNA type as the
templates, the type of sample DNA can be identified. Furthermore,
by fitting the Gaussian curve to the melting curve, these
identification can be carried out numerically and more
efficiently.
[0048] Table 1 collectively shows the amplitude (a), peak location
(mean; .mu.) and range (standard deviation; .sigma.) of a plurality
of the Gaussian curves fitted to the derivatives of the melting
curves of the known six types depicted in FIGS. 2 to 7.
1TABLE 1 Number of DNA type terms a .mu. .sigma. DQA1*0101 1 0.0026
18 10 2 0.0012 37 9 DQA1*0102 1 0.0026 19.0 11 2 0.001 36.0 9
DQA1*0103 1 0.0025 20 12 2 0.0013 34 39 DQA1*0301 1 0.0017 17 7 2
0.0012 29 11.5 DQA1*0401 1 0.0037 26 10 2 0.0015 29 5.5 DQA1*0601 1
0.0037 26 10 2 0.0014 33 5.5
[0049] The number of terms in the Gaussian functions used for
fitting is defined as a number where fitting error is saturated at
minimum. As shown in the Table, the derivative of a melting curve
corresponding to one of the types can be represented by its
characteristic parameters (a, .mu., .sigma.). Comparing the melting
curve of an unknown DNA (type) with the melting curves of these
known types of DNA polymorphism as the templates via the comparison
with the parameters shown in Table 1, not only the procedure can be
done through automatic computer processing but also strict
comparison can be realized through the mathematical process.
[0050] For the mathematical process, comparing a freshly measured
and input signal curve with one of the known melting curves
preliminarily prepared or with all the curves preliminarily
prepared by linearly binding a plurality of the template curves in
combination, a template curve with the least statistical error or a
combination of the template curves that give the linearly bound
curve with the least statistical error should be defined as the
sequence characteristics of a single-stranded DNA fragment prepared
from the sample double-stranded DNA fragment.
[0051] FIG. 9 depicts the derivative of the melting curve of a
heterozygote sample of 0102 and 0301(representing DQA1*0102 and
DQA1*0301), together with the characteristic parameters of the
curve. In such heterozygote sample, the derivative is represented
as the superposition of templete curves of the individual DNA type,
which indicates that typing can be carried out on the principle of
spectral analysis.
[0052] On the basis of the results of FIG. 9, Table 2 summarizes
the parameters of the individual DNA types. More specifically, if a
heterozygote DNA has the parameter values of .mu. (peak location)
and .sigma.(standard deviation), being almost the same as those
represented as the regression values shown in Table 2, the
heterozygote DNA is of a heterozygote of two DNA types deduced from
the values.
2TABLE 2 Regression Regression DNA .mu. values .sigma. values types
.mu.1 16.9 .sigma.1 6.89 0301 .mu.2 19.0 .sigma.2 11.0 0102 .mu.3
28.9 .sigma.3 11.5 0301 .mu.4 35.9 .sigma.4 9.0 0102
[0053] In the present Example, a satisfactorily accurate melting
curve could be generated in a practical sense, at a temperature
elevation rate of 5.degree. C./min at maximum. In this case, the
analysis time is about 10 minutes per sample, achieving speeding up
by 20 fold or more compared with the conventional DNA sequencing
and SSCP method (4 hours or more).
[0054] FIG. 10 depicts another analysis example of a heterozygote
DNA based on FIGS. 2(a) to 7(b). In the present Example, the
melting curves of the eight types of DNA type in the HLA-DQA1 exon
2 region (242 p or 239 bp (3-bp depletion)) as the templates for
individual sense strands should be measured preliminarily. The
measured heterozygote curve from a sample agrees well with the
synthetic curve of the template 0101 represented to the DQA1*0101
and the template 0102 represented to the DQA1*0102 (RMS=0.00008).
Consequently, it is indicated that the sample can be identified as
the heterozygote DNA of the two.
[0055] For the polymorphism analysis, it is necessary to determine
to which type the DNA type of an analytical sample belongs and/or
whether the DNA type is novel or not. Also, essentially, a
heterozygote sample having a plurality of DNA types should be
isolated and analyzed. In the present Example, however, accurate
identification of a DNA type can be carried out in a smooth manner
using a signal processor comprising memorizing the melting curves
corresponding to all known types of polymorphism (template curves),
comparing a freshly measured and input signal curve with one of a
plurality of the template curves or with all the curves
preliminarily prepared by linearly binding a plurality of the
template curves in combination, and determining that a combination
of the template curves that give the least RMS below a given value
is the DNA type (namely, sequence characteristics) of the measured
single-stranded DNA fragment. Furthermore, combinations with larger
probabilities can be output in the order of smaller RMS.
[0056] As to the results shown in FIG. 10, the merit of this
procedure is shown in Table 3.
[0057] The combination with the least final error represents an
accurate heterozygote combination, and the value of the error is
almost the same as (rather smaller than) the reproducibility error
in the measurement of the melting curve 5 times. Table 3 shows the
reproducibility error and RMS of an accurate combination (the
combination marked with double circles in the Table), along with
the RMSs of some of the combinations with less error among the
remaining combinations. For a heterozygote combination of sequences
different by one base from each other, the DNA type with the
highest probability of erroneous judgment is a homozygote type of
each of the individual DNA types originally constituting the
heterozygote type. As apparently shown in Table 3, it is indicated
that significant difference is present between accurate and
inaccurate judgments.
3TABLE 3 cf. Hetero (DQA1*0101/0102) DNA types Error (RMS) .times.
10.sup.-4 .circleincircle. 0101/0102 hetero 0.74 0101 homo 1.28
0102 homo 1.55 0101/0103 hetero 2.56 0101/0201 hetero 4.5
Reproducibility error 0.8
[0058] By substantially uniformly regulating the temperature of
samples at a high speed, the dynamic response of the conformation
of a single-stranded DNA fragment of some sample to the temperature
change was identified in a range above 10.degree. C./min of
temperature elevation or decrease. More specifically, it was
identified that during the denaturing and forming of the
conformation (during temperature elevation and decrease,
respectively), the melting curve drew a hysteresis curve
(1.fwdarw.2.fwdarw.3.fwdarw.4.fwdarw.5.fwdarw.6.fwdarw.2.fwdarw.3.fwdarw.-
4.fwdarw.5.fwdarw. . . . ) as shown in FIG. 11. Specific to the
difference in sequence such as the substitution, depletion or
insertion of bases, the hysteresis curve varies depending on the
sample. This indicates that the method is not only applicable to
the change in the absorbance but also to the hysteresis curve of
the absorbance, wherein more accurate determination of such type at
a higher speed can be done by comparing a measured hysteresis curve
with the template hysteresis curves in the same manner as in the
case of the signal processing method.
[0059] In such manner, the measurement of a sample was completed
within one minute (for 50 seconds) at maximum speed, to generate a
hysteresis curve at two cycles of temperature elevation and
decrease. The hysteresis curve varies depending on the rate of
temperature elevation. Therefore, if the rate of temperature
elevation is set at an appropriate level depending on the type, an
effective hysteresis curve corresponding to the DNA type can be
produced. Thus, the DNA analysis can be effected on the basis of
the dependency of the hysteresis curve on the rate of temperature
elevation.
[0060] Then, examples of a device for DNA analysis are shown in
FIGS. 12 and 13, for continuously carrying out a flow system from
PCR as a preliminary treatment to the melting curve
measurement.
[0061] FIG. 12 depicts the schematic chart of the process flow;
FIG. 13 depicts the schematic view of the device structure; and
FIG. 14 depicts the detailed structural view of the spectroscopic
cell.
[0062] The reaction process progresses through the processes (a) to
(g) shown in FIG. 12. As shown in (a), PCR is carried out in PCR
cell 600 immobilizing oligonucleotide A 602 as a PCR primer on
porous filter membrane 601 on the bottom of the PCR cell. In the
PCR cell 600, extracted and purified genomic DNA is placed as a
sample, which is then mounted in the device of FIG. 13. As shown in
(b) as the PCR progresses, a double-stranded DNA (PCR product)
corresponding to the sample DNA is generated in the manner such
that the single strand on the solid phase is fixed at one end on
the filter membrane 601. The denaturing of the double-stranded DNA
separates a free single-stranded DNA in the liquid phase as shown
in (d) from the single-stranded DNA fixed at one end on the filter
membrane (solid phase) as shown in (c). Dissolving the
single-stranded DNA of the liquid phase in a buffer solution for
measurement, and transferring the DNA solution into a spectroscopic
cell, the temperature of the spectroscopic cell is controlled to
regulate the state of a single-stranded DNA in the liquid phase (in
the denatured state) as shown in (e) and the formation of the
conformation as shown in (f). As shown in (g), the absorbance is
measured through the spectroscopic cell, to prepare a melting
curve.
[0063] FIG. 13 depicts the schematic view of the device structure.
As described below, after transferring a PCR solution, a washing
buffer and a spectroscopic buffer through gates 705, 706 of sample
pretreatment cell 700 into PCR cell 600, the PCR cell 600 is
regulated at a given heat cycle. Porous filter 601 immobilizing
primer A is placed in the PCR cell 600. In the sample pretreatment
cell 700, the treatments (a) to (d) described in FIG. 12 are
carried out.
[0064] On the porous filter 601 inside the PCR cell 600 is
immobilized oligonucleotide A (10 pmol) as a PCR primer, and then,
the extracted and purified genomic DNA (100 ng) is placed as a
sample in PCR solution tank 701. Gas is transferred through gas
source 707 and valves 723, 722, 721 into the PCR solution tank 701,
while the PCR solution (50 .mu.l) is transferred through valves
725, 724 into the PCR cell 600. In this case, the PCR solution was
made of a mixture solution of primer (oligonucleotide ) B (10
pmol), 10 nmol each of deoxyribonucleotide triphosphates (DNTP:
dATP, dCTP, dGTP, dTTP) and a heat-resistant DNA polymerase (Taq
polymerase) (1 unit) in a buffer solution containing 50 mM KCl, 10
mM Tris-HCl (pH 8.3), 1.5 mM MgCl.sub.2, gelatin of 0.001% (as a
final concentration).
[0065] In this state, PCR is performed while regulating the
temperature of the PCR cell 600 in hot air or cold air. The PCR
cycling condition is 25 cycles of 94.degree. C. for 30 minutes,
55.degree. C. for 1 minute 72.degree. C. for 30 seconds and in this
order. The total reaction time was 50 minutes. The time period
required for the PCR is possibly shortened as short as about 20
minutes, by making the cell shape into a thinner form and enlarging
the surface/volume ratio. By closing the valves 724, 728, the
vaporization of the reaction solution at higher temperatures could
be made substantially zero.
[0066] After the termination of the reaction, gas is fed from gas
source 707 through valves 722, 723 into a washing or spectroscopic
buffer tank, while the washing buffer flows through valves 726,
725, 724 onto the filter 601 for washing the filter 601 several
times, to wash off the remaining primer and the residual dNTP. The
liquid waste is disposed through valve 728 into liquid waste tank
703. PCR product remains on the filter 601. If unwanted matters
which cannot pass through the filter 601 may possibly remain, gas
may be fed through valve 728 from gas source 708, while liquid
waste is disposed through valve 724 into liquid waste tank 709. As
shown in FIG. 12(b), all of these motions are driven under the
conditions where double strands are on the porous filter 601 in the
solid phase state.
[0067] Introducing subsequently a final washing solution (serving
as the spectroscopic buffer) into the PCR cell, and then inducing
the inside of the PCR cell 600 into a melting state by raising the
temperature, the solution flows from the PCR cell 600 toward the
outlet 705 by using the gas source 708. More specifically, as shown
in FIG. 12(d), a single-stranded DNA in the liquid phase is
collected and then transferred into spectroscopic cell 300. In the
present Example, as a washing buffer, use was made of TNE buffer
(20 .mu.l; 10 mM Tris-HCl, 1 mM EDTA (pH 8.3), 30 mM NaCl). After
introducing and measuring the sample in the spectroscopic cell 300,
the sample is disposed in the liquid waste tank 704 by the flow of
the washing buffer through valves 726, 727 into the cell.
Consequently, the sample is disposed after such measurement while
the inside of the spectroscopic cell is washed. Instead of the
liquid waste tank 704, then, a fraction collector may be placed to
recover the sample after the measurement.
[0068] In the device structure aforementioned, the ratio of the
numbers of the PCR cell 600 and the number of the spectroscopic
cell 300 was 1:1, but attaching a plurality of PCR cells through
valves to a single spectroscopic cell, the reaction products may be
introduced sequentially into the spectroscopic cell for
measurement.
[0069] Alternatively, by directly supplying a biological cell
sample such as blood as a sample material into PCR cell 600,
carrying out the extraction of a sample DNA by a known method, and
subsequently carrying out the aforementioned DNA typing, the system
from extraction to analysis may be made consistent. However, the
overall structure of such system may possibly be more complex.
[0070] FIG. 14 is the figure of a structural example of the
spectroscopic cell 300 employed in FIG. 13, wherein the side view
is on the left side and the cross sectional view on the right side
is a view in line A-A of the side view in the arrow direction.
[0071] The spectroscopic micro-cell 300 in the present Example is
made of quartz glass and black quartz glass; the window of the
light path is made of quartz (transparent) glass, and the remaining
parts of the cell box in a rectangular parallelepiped with the
upper top open are made of black quartz glass. On the upper top are
internally arranged spacers of black quartz glass, while leaving
the flow gates 303 on both the sides. Therefore, a sample solution
holding part of a square shape, with a light path of a 10 mm length
and a cross section of a 1.4 mm.times.1.4 mm-square shape, is
formed below the spacers. A temperature sensor 301 is slightly
projected toward the sample solution holding part at the central
part of the spacers. As shown (inserted) with the broad arrow in
the figure, the spectroscopic micro-cell 300 is placed internally
inside temperature regulator 302 to regulate the temperature of a
sample solution.
[0072] In the present embodiment, the cell wall is made of black
quartz glass with a thickness of about 1 mm, because the glass has
far less reflection stray light with a relatively high thermal
conductivity and a substantially great strength; for the cell
material, a material with less reflection stray light and an
excellent thermal conductivity is suitable. Another example is
aluminum alloy coated with platinum black and TiN (titanium
nitride). In the cell of the present Example, temperature sensor
301 is embedded in temperature regulator 302 to measure the sample
temperature at the central location of the light path. Therefore,
the cell can regulate the temperature of samples at a high
efficiency. Additionally, the cell can measure the temperature at a
high precision. Furthermore, compared with general
commercially-available spectroscopic cells, the cell of the present
Example has such a larger surface/volume ratio of 2800 that the
cell can realize the temperature increase or decrease at the rate
of from 0.1.degree. C./min to 5.degree. C./sec.
[0073] Finally, description will now be made of an example by means
of ethidium bromide, wherein the fluorescence from a sample DNA and
the intercalating agent is detected to prepare the melting
curve.
[0074] FIG. 15 depicts the block diagram of an example of a
detector structure to detect the fluorescence from the DNA and the
intercalating agent. Ultra-violet ray (of a wave length of 260 nm)
from light source 801 passes through a filter and optical system
802 such as lens, to be incident into sample 804 placed in sample
holder 805. Via the presence of ethidium bromide intercalated with
the sample DNA, fluorescence of 590 nm is emitted, which is then
collimated with the optical system 807 followed by detection with
photoelectric converter 808. The signal is thereafter processed
through amplifier 805 at analytical signal processing means 810.
The sample holder 805 can regulate the sample temperature following
the temperature profile optionally programmed with temperature
regulator 806. Temperature regulation can be preset optionally at a
rate of temperature increase or decrease from 0.1.degree. C./min to
2.degree. C./sec. Temperature can be regulated within the range of
-20.degree. C. to 100.degree. C. by an electronic heating-cooling
means using Peltier effect.
[0075] In the present Example, the cell may be adapted for signal
processing and preventing the occurrence of bedewing on the cell
surface, as is described in the foregoing examples.
[0076] When determining the melting curve by means of the
fluorescence from the intercalating agent, the fluorescence
intensity decreases as the conformation of a single-stranded DNA is
denatured. This is because the fluorescence emitted from the
ethidium bromide intercalated with the base pairs forming the
conformation is not any more emitted since the intercalation is
eliminated as the conformation is denatured. The problem of
reproducibility was noticed at an earlier stage, including the
change of the fluorescence intensity depending on the concentration
of ethidium bromide, but using the melting curve standardized on
the fluorescence intensity at the lowest limit temperature, the
reproducibility between samples could be secured.
[0077] FIG. 16 depicts the melting curve of HLA-DQA1*0101 and
HLA-DQA1*0102 by means of fluorescence. In the same manner as in
the case of absorbance, a single-base substitution could be
identified. The data in FIG. 16 are measured at a concentration
{fraction (1/50)} fold that of the case of absorbance. By using
fluorescence, sensitivity was improved by 10 fold to 100 fold
(precision in measuring melting curve=improvement of S/N
ratio).
[0078] As in the case of absorbance, signal processing is carried
out on the comparison with template curves. Consequently, all
samples were accurately analyzed.
[0079] In accordance with the present invention, some examples have
been described insofar, but the applicable range of the present
invention is not limited to these examples. A method comprising
analyzing the melting curve of a single-stranded DNA, thereby
producing the information of the DNA sequence, as well as a device
therefor, is within the scope of the present invention.
[0080] By using the method and device in accordance with the
present invention as has been mentioned insofar, DNA information at
least at minimum required for clinical diagnosis and DNA tests,
namely the presence or absence of a target sequence, the level
thereof if present and the sequence characteristics thereof, can be
obtained. The overall process from the pretreatment to the recovery
of DNA information and the analysis thereof can be completed, for a
short period, by the simple device structure and procedures.
* * * * *