U.S. patent application number 09/842791 was filed with the patent office on 2002-07-25 for method for assaying dna fragments in mixture.
This patent application is currently assigned to Hitachi, Ltd.. Invention is credited to Kambara, Hideki, Okano, Kazunori, Uematsu, Chihiro.
Application Number | 20020098490 09/842791 |
Document ID | / |
Family ID | 17702418 |
Filed Date | 2002-07-25 |
United States Patent
Application |
20020098490 |
Kind Code |
A1 |
Uematsu, Chihiro ; et
al. |
July 25, 2002 |
Method for assaying DNA fragments in mixture
Abstract
The inventive method for assaying DNA fragments in mixture
comprises step 1 of ligating different oligomers hybridizable to
primers of the same melting temperature and the same length to
individual groups of DNA fragments in a set of DNA fragments; step
2 of mixing together the groups of DNA fragments ligated with the
oligomers; step 3 of simultaneous PCR of the groups of DNA
fragments ligated with the oligomers in one receptacle by using the
primers being complementary to the oligomers and corresponding to
the individual groups; and step 4 of detecting PCR amplified DNA
fragments; characterized in that the method enables the comparison
of plural samples under no influence of PCR reproducibility.
Inventors: |
Uematsu, Chihiro;
(Kawasakii, JP) ; Kambara, Hideki; (Hachiouji,
JP) ; Okano, Kazunori; (Shiki, JP) |
Correspondence
Address: |
MATTINGLY, STANGER & MALUR
104 East Hume Avenue
Alexandria
VA
22301
US
|
Assignee: |
Hitachi, Ltd.
|
Family ID: |
17702418 |
Appl. No.: |
09/842791 |
Filed: |
April 27, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09842791 |
Apr 27, 2001 |
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09413814 |
Oct 7, 1999 |
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6225064 |
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Current U.S.
Class: |
435/6.12 ;
435/6.16; 435/91.2 |
Current CPC
Class: |
C12Q 1/6809 20130101;
C12Q 1/6809 20130101; C12Q 1/6855 20130101; C12Q 2525/173
20130101 |
Class at
Publication: |
435/6 ;
435/91.2 |
International
Class: |
C12Q 001/68; C12P
019/34 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 8, 1998 |
JP |
10-286286 |
Claims
What is claimed is:
1. A method for assaying DNA fragments in mixture comprising step 1
of ligating different oligomers hybridizable to primers of + the
same melting temperature and the same length to individual groups
of DNA fragments in a set of DNA fragments; step 2 of mixing
together the groups of DNA fragments ligated with the oligomers;
step 3 of simultaneous PCR of the groups of DNA fragments ligated
with the oligomers in one receptacle by using the primers being
complementary to the oligomers and corresponding to the individual
groups; and step 4 of detecting PCR amplified DNA fragments.
2. A method for assaying DNA fragments in mixture according to
claim 1, wherein the primers corresponding to the individual groups
are labeled with fluorophores different from each other in a
corresponding manner to the individual groups, to detect PCR
amplified DNA fragments labeled with the fluorophores by
electrophoresis.
3. A method for assaying DNA fragments in mixture according to
claim 1, wherein the PCR amplified DNA fragments are detected by
using a DNA probe array immobilizing plural types of DNA probes of
nucleotide sequences complementary to the individual groups of the
DNA fragments thereon.
4. A method for assaying DNA fragments in mixture according to
claim 1, wherein the primers corresponding to the individual groups
are labeled with different fluorophores, correspondingly to the
individual groups.
5. A method for assaying DNA fragments in mixture according to
claim 1, wherein the primers comprise plural module sequences, each
module sequence being composed of 4 to 6 nucleotides, wherein the
order of the plural module sequences varies, depending on each of
the individual groups.
6. A method for assaying DNA fragments in mixture according to
claim 5, wherein the plural modules comprise the same nucleotide
species at the 3' terminus and 5' terminus thereof.
7. A method for assaying DNA fragments in mixture according to
claim 1, wherein the primers corresponding to the individual groups
are composed of a 10- to 25-nucleotide common nucleotide sequence
in common to the individual primers of the individual groups and a
selective nucleotide sequence being composed of one to 3
nucleotides and recognizing the DNA fragments of the individual
groups, wherein the common nucleotide sequence comprises plural
module sequences in orders varying, depending on the individual
groups, each module sequence being composed of 4 to 6 nucleotides
and wherein the selective nucleotide sequence includes all
nucleotide sequences of combinations of one to 3 nucleotides.
8. Primers for use for a method for assaying DNA fragments in
mixture according to claim 1, wherein the primers comprise plural
module sequences in orders varying, depending on the individual
groups, each module sequence being composed of 4 to 6
nucleotides.
9. Primers according to claim 8, wherein the plural modules
comprise the same nucleotide species at the 3' terminus and 5'
terminus thereof.
10. Primers for use for a method for assaying DNA fragments in
mixture according to claim 1, wherein the orimers corresponding to
the individual groups characteristically comprise a 10- to
25-nucleotide common nucleotide sequence in common to the primers
of the individual groups, and a selective nucleotide sequence being
composed of one to 3 nucleotides and recognizing the DNA fragments
of the individual groups, wherein the common nucleotide sequence
comprises plural module sequences in orders varying, depending on
the individual groups, each module sequence being composed of 4 to
6 nucleotides and wherein the selective nucleotide sequence
includes all nucleotide sequences of combinations of one to 3
nucleotides.
11. Primers according to claim 10, wherein the plural modules
comprise the same nucleotide species at the 3' terminus and 5'
terminus thereof.
12. Plural primers with different nucleotide sequences for use in
PCR, wherein the primers comprise different orders of plural module
sequences composed of plural nucleotides and the plural primers
thus comprising such different orders of plural module sequences
are of the same melting temperature.
13. Plural primers according to claim 12, wherein the plural
modules comprise the same nucleotide species at the 3' terminus and
5' terminus thereof.
14. Plural primer sets of plural primers with different nucleotide
sequences for use in PCR, wherein each primer set is composed of a
10- to 25-nucleotide common nucleotide sequence in common to the
primers of each primer set and a selective nucleotide sequence
being composed of one to 3 nucleotides and recognizing DNA
fragments derived from sample DNA, wherein the common nucleotide
sequence comprises plural module sequences in orders varying,
depending on each primer set, each module sequence being Composed
of 4 to 6 nucleotides and wherein the selective nucleotide sequence
includes all nucleotide sequences of combinations of one to 3
nucleotides.
15. Plural primer sets according to claim 14, wherein the plural
modules comprise the same nucleotide species at the 3' terminus and
5' terminus thereof.
16. Plural primer sets according to claim 14, wherein the plural
primers of the plural primer sets are of the same melting
temperature.
Description
BACKGROUND OF THE INVENTION
[0001] The present invention relates to a method for amplifying
nucleic acid, a method for assaying DNA fragments in mixture, a
method for assaying RNA fragments in mixture, and an expression
profiling process of a group of expressed genes; more specifically,
the invention relates to a method for assaying DNA fragments by gel
electrophoresis and by using fluorophore label.
[0002] The nucleotide sequence of a gene or a DNA can be compared
with other nucleotide sequences by DNA sequencing, but it is very
difficult to determine the sequence of a long DNA or a mixture
sample of a great number of DNA fragments. So as to determine the
sequence of such long DNA, therefore, the long DNA is first
fragmented; then, the resulting DNA fragments are assayed on gel
electrophoresis patterns. Due to the recent progress of various
assay methods and apparatuses therefor, numerous expressed genes
can simultaneously be detected and assayed.
[0003] The expression profiling method includes for example a
scanning process based on gel electrophoresis. The scanning process
includes FDD [Fluorescent Differential Display; FEBS Letters 351,
231-236 (1994)] and a process using terminal nucleotide selective
primer [Nucleic Acids Research, 24, 2616-2617 (1996); Nucleic Acids
Symposium Series, No. 35, 257-258 (1996)].
[0004] All these scanning processes comprise PCR (Polymerase Chain
Reaction) using plural primers in common to plural DNA fragments
along with a sample CDNA or iRNA as template and recovering gene
expression information based on the electrophoresis patterns of the
resulting PCR products. Because the expression can be detected with
no use of any gene-specific probe according to the scanning
processes, advantageously, the expression profile of a gene with a
nucleotide sequence not yet identified can be yielded.
[0005] The process using terminal nucleotide selective primer
comprises digesting a double-stranded cDNA with a restriction
enzyme and ligating an oligonucleotide with a known nucleotide
sequence at the terminus of a DNA fragment as a digestion product.
PolyA tail is generally present at the 3' terminus of cDNA. Because
a nucleotide sequence between the polyA tail at the 3' terminus of
the cDNA and the ligated oligonucleotide is specific to the cDNA, a
DNA fragment carrying the nucleotide sequence is assayed as a
fragment representing (or identifying) the cDNA.
[0006] Because gene species of several thousands to several ten
thousands in number are expressed in living organisms, the types of
such cDNA representative fragments are so numerous that these
representative fragments cannot be separated and assayed in one
lane by electrophoresis. Thus, all these types of such
representative fragments are divided in plural groups, whereby the
number of such DNA fragments included in each one group is
sufficiently reduced such that the DNA fragments in each one group
can be separated and assayed satisfactorily by gel electrophoresis.
So as to divide all the representative fragments in plural groups,
PCR primers with a selective nucleotide sequence composed of two
nucleotides at the 3' terminus thereof are used. By PCR, DNA
fragments with terminal two nucleotides complementary to each
selective nucleotide sequence are amplified. From two primer sets
with two selective nucleotide sequences is selected each one
primer; subsequently, a group of a combination of appropriate two
primers is prepared.
[0007] By PCR with primers of the individual sets, the resulting
amplified products are assayed by electrophoresis. Via combinations
of DNA fragments expressed on the electrophoresis pattern recovered
by using the primers of the individual sees, the whole information
of each expressed gene can be recovered. Consequently, the type of
each expressed gene and the expression level thereof can be
identified.
[0008] For fragment assay, at least one primer of oligo dT primer
and a primer complementary to the oligomer ligated at the terminal
digestion site of a DNA fragment is labeled with fluorophore; using
the primer labeled with the fluorophore, the DNA fragment is
amplified in such a number above the detection sensitivity of an
assay apparatus such as fluorescent DNA sequencer and the like.
SUMMARY OF THE INVENTION
[0009] Conventional expression profiling processes based on
electrophoresis have various drawbacks as described below in
practical sense. By the processes on the basis of electrophoresis,
the resulting comparative results are at serious error if the
reproducibility of PCR amplification is low.
[0010] For comparing the difference in expression profile between
plural samples, the samples each are independently subjected to PCR
and the resulting PCR products are then electrophoresed. The
results are compared to each other. Plural samples labeled with the
same fluorophore species are subjected to PCR in different reaction
tubes; and the resulting PCR products are assayed in different
electrophoresis lanes. Plural samples labeled with different
fluorophore species are independently subjected to PCR in different
tubes; and the resulting products are assayed in one
electrophoresis lane. According to the conventional processes,
sample preparation for electrophoresis requires PCR in different
tubes, so PCR reproducibility influences the precision of the
comparison of the electrophoretic assay results of PCR products.
Currently, nevertheless, PCR reproducibility is disadvantageously
insufficient.
[0011] It is an object of the present invention to provide a method
for assaying DNA fragments in mixture, comprising PCR in one
reaction tube, thereby enabling an expression profiling process of
comparing plural samples together, under no influence of PCR
reproducibility.
[0012] According to the inventive method for assaying DNA fragments
in mixture, each sample requires a PCR primer with a different
nucleotide sequence but of the same length and the same melting
temperature (T.sub.m), for reaction of plural samples in one tube.
Each primer corresponding to each of plural samples is of a
nucleotide sequence, with no chance of secondary structure
formation between these primers, so that these primers can
independently function and never influence the reaction of other
primers. Plural DNA samples are placed in one reaction tube for
PCR, whereby variation of each PCR can be eliminated.
[0013] So as to amplify a first DNA sample with a primer of a first
primer set and amplify a second DNA sample with a primer of a
second primer set, an oligonucleotide with a complementary
nucleotide sequence to each primer is ligated to the primer. So as
to identify the primers serving for amplification, the primer of
the first primer set and the primer of the second primer set are
labeled with different fluorophore species. Because plural DNA
samples are amplified by using the primers of plural primer sets in
one tube, no problem concerning PCR reproducibility occurs.
[0014] In order to compare together DNA fragments amplified with
the primers of plural primer sets, the difference in reaction
reactivity between these primers should be eliminated. T.sub.m
value of primer, T.sub.m value of sample DNA, and T.sub.m value of
PCR amplified product determine the PCR reaction efficiency.
[0015] Expression of one gene in plural sample DNAs means that the
sample DNAs with relation to the gene and the PCR amplified
products thereof are identical; thus, the T.sub.m values thereof
are equal. Hence, the PCR reaction efficiency depends on the
T.sub.m value of primer. By setting the T.sub.m values of plural
primers at an equal value, the resulting PCR reaction efficiencies
can be retained equally.
[0016] The T.sub.m value of DNA can be calculated, approximately,
depending on the nucleotide species composing the DNA sequence
[Biopolymers, 3, 195-208 (1965)]. The T.sub.m value can be
calculated by using the difference in stacking between a nucleotide
species and a nucleotide adjacent thereto. By using a nucleotide
sequence composed of an interesting nucleotide and one nucleotide
adjacent thereto, the T.sub.m value can be more accurately
calculated [Proc. Natl. Acad. Sci. USA, 83, 3746-3750 (1986)].
[0017] So as to prepare plural primers at an equal T.sub.m value,
the individual primers are allowed to comprise a nucleotide
sequence of several species of modules, each module being composed
of 4 to 6 nucleotides. For example, 5 modules (A, B, C, D, and E),
each module being composed of 4 nucleotides, are aligned
sequentially in the order A-B-C-D-E to prepare a primer with the
nucleotide sequence or in the order C-D-B-A-E to prepare a primer
with the latter nucleotide sequence. Herein, each of the individual
modules comprises the same nucleotide species at both the termini
thereof. Even if these modules that have same nucleotide species at
the both termini are shuffled together in order, the nucleotide
sequence in the linking region between the modules is never
modified because the nucleotides at both the termini are identical.
Thus, no effect of the change of the sequence order of these
modules is reflected on the T.sub.m value.
[0018] The nucleotide composition composing each primer in its
entirety is never changed because only the order of these modules
is modified in each primer. As has been described above, almost no
difference is found in the T.sub.m values of individual primers
with modified sequence orders of modules or in PCR reaction
efficiency. Additionally because module-shuffling primers are not
complementary to each other, these primers together never form a
double strand to suppress PCR.
[0019] By aligning several modules in various fashions wherein each
of the individual modules comprises the same nucleotide at both the
termini thereof, plural primers with no difference in PCR reaction
efficiency can be prepared. Because PCR is effected in one reaction
tube, the variation of PCR reaction efficiency can be
eliminated.
[0020] The inventive method for assaying DNA fragments in mixture
comprises PCR o plural samples in one reaction tube by using
primers with nucleotide sequences different from each other and at
the same length and the same melting temperature, namely the same
T.sub.m value, under no influence of the variation of PCR reaction
efficiency, to quantitatively compare the ratio of DNA fragments
present in plural samples.
[0021] The inventive method for assaying DNA fragments in mixture
comprises
[0022] step 1 of ligating different oligomers hybridizable to
primers of the same melting temperature and the same length to
individual groups of DNA fragments in a set of DNA fragments,
[0023] step 2 of mixing together the groups of DNA fragments
ligated with the oligomers;
[0024] step 3 of simultaneous PCR of the groups of DNA fragments
ligated with the oligomers in one receptacle by using the primers
being complementary to the oligomers and corresponding to the
individual groups; and
[0025] step 4 of detecting PCR amplified DNA fragments.
[0026] The inventive method for assaying DNA fragments in mixture
is characteristic as follows.
[0027] 1. The primers corresponding to the individual groups are
labeled with fluorophores different from each other in a
corresponding manner to the individual groups, to serve for the
detection of electrochoresed DNA fragments labeled with the
fluorophores after PCR amplification.
[0028] 2. The PCR amplified DNA fragments are detected by using a
DNA probe array immobilizing plural types of DNA probes of
nucleotide sequences complementary to the individual groups of DNA
fragments thereon.
[0029] 3. The primers corresponding to the individual groups are
labeled with different fluorophores, correspondingly to each of the
individual groups.
[0030] 4. The primers comprise plural module sequences, each module
sequence being composed of 4 to 6 nucleotides, wherein the order of
plural module sequences varies, depending on each of the groups,
while the plural module sequences comprise the same nucleotide
species at the 3' terminus and 5' terminus thereof
[0031] 5. The primers corresponding to the individual groups are
composed of a 10- to 25-nucleotide common nucleotide sequence in
common to the individual primers for the individual groups and a
selective nucleotide sequence being composed of one to 3
nucleotides and recognizing the DNA fragments of the individual
groups, wherein the common nucleotide sequence comprises plural
module sequences in orders varying, depending on the individual
groups, each module sequence being composed of 4 to o nucleotides
and wherein the selective nucleotide sequence includes all
nucleotide sequences of combinations of one to 3 nucleoutides.
[0032] The inventive primers are used for the method for assaying
DNA fragments in mixture; the primers comprise plural module
sequences in orders varying, depending on the individual groups,
each module sequence being composed of 4 to 6 nucleotides, while
the plural module sequences comprise the same nucleotlde species at
the 3' terminus and 5' terminus thereof.
[0033] According to the method for assaying DNA fragments in
mixture, the inventive primers corresponding to the individual
groups characteristically comprise the 10- to 25-nucleotlde common
nucleotide sequence in common to the primers of the individual
groups, and the selective nucleotide sequence being comoosed of one
to 3 nucleotides and recognizing the DNA fragments of the
individual groups, wherein the common nucleotide sequence comprises
plural module sequences in orders varying, depending on the
individual groups, each module sequence being composed of 4 to 6
nucleotides and wherein the selective nucleotide sequence includes
all nucleotide sequences of combinations of one to 3 nucleotides
and the plural module sequences comprise the same nucleotide
species at the 3' terminus and 5' terminus thereof.
[0034] Furthermore, the inventive primers are plural primers of
different nucleotide sequences for use in PCR, wherein the primers
comprise different orders of plural module sequences composed of
plural nucleotides and the plural primers thus comprising such
different orders of plural module sequences are of the same melting
temperature and wherein the plural module sequences comprise the
same nucleotide species at the 3' terminus and 5' terminus
thereof.
[0035] Still furthermore, the inventive primer sets are plural
primer sets of plural primers with different nucleotide sequences
for use in PCR, wherein each primer set is composed of a 10- to
25-nucleotide common nucleotide sequence in common to the primers
of each primer set and a selective nucleotide sequence being
composed of one to 3 nucleotides and recognizing DNA fragments
derived from sample DNA, wherein the common nucleotide sequence
comprises plural module sequences in orders varying, depending on
each primer set, each module sequence being composed of 4 to 6
nucleotides and wherein the selective nucleotide sequence includes
all nucleotide sequences of combinations of one to 3 nucleotides;
the plural module sequences comprise the same nucleotide species at
the 3' terminus and 5' terminus thereof and the plural primers of
the plural primer sets are of the same melting temperature.
[0036] In accordance with the invention, plural primers comprising
different orders of plural module sequences are used to prepare
plural primers at the same T.sub.m value. For PCR amplification of
plural sample DNAs and comparison of the resulting PCR products,
consequently, DNAs or RNAs extracted from plural samples can be PCR
amplified in one reaction tube and she resulting PCR products can
be assayed in one electrophoresis lane. Because laborious reaction
procedures can be saved, with no PCR variation in various reaction
tubes, the precision of comparison can be improved with no concern
of PCR reproducibility in the conventional art.
[0037] With reference to FIG. 1, a representative composition of
the invention will be schematically described below. The inventive
method for assaying DNA fragments in mixture and capable of
expression profiling, comprises
[0038] step 1 of ligating different oligomers 11, 12 hybridizable
to primers 21, 22 of the same melting temperature and the same
length to groups of DNA fragments 3, 4 in a set of DNA
fragments;
[0039] step 2 of mixing together the groups of DNA fragments 5, 6
ligated with the oligomers 11, 12;
[0040] step 3 of simultaneous PCR of the groups of DNA fragments 5,
6 ligated with the oligomers 11, 12 in one receptacle 29 by using
the primers 21, 22 being complementary to the oligomers 11, 12 and
corresponding to the individual groups; and
[0041] step 4 of detecting PCR amplified DNA fragments by
electrophoretic separation. The above composition enables
comparison of plural samples, under no influence of PCR
reproducibility.
BRIEF DESCRIPTION OF THE DRAWINGS
[0042] FIG. 1 is a flowchart of the procedures of the expression
profiling of Example 1 in accordance with the invention;
[0043] FIGS. 2A and 2B are figures depicting the structures of the
primers of Example 1 of the invention and also showing the
structures of oligonucleotides of known nucleotide sequences;
[0044] FIGS. 2C and 2D are figures depicting the hybridization of
the primers of Example 1 of the invention and DNA fragments;
[0045] FIG. 3 shows parts of electropherograms of expression
profiling by using the primers of Example 1 of the invention;
[0046] FIG. 4 is a flowchart of the procedures of expression
profiling in Example 2 of the invention;
[0047] FIG. 5 is a flowchart of the procedures of expression
profiling by using DNA probe array in Example 2 of the
invention;
[0048] FIG. 6 shows the mechanistic composition of a fluorescent
microscope detecting a sample on DNA probe array in Example 2 of
the invention; and
[0049] FIG. 7 shows fluorescent intensities in bar graph from a DNA
probe array for expression profiling using the primers of Example 2
of the invention.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
EXAMPLE 1
[0050] FIG. 1 is a flowchart depicting the procedures of the
expression profiling of Example 1 in accordance with the invention.
The sample cDNAs were prepared from the RNAs extracted from rat
liver and rat kidney. From the cells homogenized in a conventional
manner were extracted RNAs by the guanidium thiocyanate method. The
extracted RNAs were converted to cDNAs by using reverse
transcriptase, which were designated as sample cDNAs (rat
liver-derived cDNA and rat kidney-derived cDNA).
[0051] As shown in FIG. 1, rat liver-derived sample cDNA 1 (in a
mixture of various cDNAs; one cDNA is shown in the figure) was
digested with a class II restriction enzyme in reaction tube 27 to
prepare cDNA fragment 3. In the same manner, rat kidney-derived
sample cDNA 2 was digested with the same restriction enzyme as used
for the digestion of the sample cDNA 1 to prepare cDNA fragment 4
(in a mixture of various cDNAs; one cDNA is shown in the figure).
Sau3A I recognizing the nucleotide sequence 5'-C-ATC-3' to digest
the site GATC was used as the class II restriction enzyme. The
class II restriction enzyme recognizing four nucleotides is
preferable and includes for example Nla III and Hha I. Restriction
enzymes recognizing six nucleotides may also be satisfactory,
including Hind III and EcoR I.
[0052] By ligating oligonucleotide 11 with a known nucleotide
sequence at the digestion site of the cDNA fragment 3 by ligation,
cDNA fragment 5 with the oligonucleotide 11 with the known
nucleotide sequence ligated at the 3' terminus thereof is
recovered. By ligating oligonucleotide 12 with a known nucleotide
sequence at the digestion site of the cDNA fragment 4 in another
reaction tube 28 by ligation, CDNA fragment 6 with the
oligonucleotide 12 with the known nucleoitide sequence ligated at
the 3' terminus thereof is recovered.
[0053] By subsequently mixing together the cDNA fragment 5 and the
cDNA fragment 6 to prepare a mixture solution, a part of the
solution is placed in PCR tube 29 for use as template DNA fragment
for PCR.
[0054] Primer 21 composed of four module sequences, namely I. TCAT,
II. CACC, III. TTCT and IV. CCAC, the restriction enzyme
recognition nucleotide sequence GATC, and selective nucleotide
sequence NN 15 being composed of two nucleotides NN (independently
corresponding to any one of A, C, G and T) and recognizing two
nucleotides subsequent to the 5' terminus (G) of the restriction
enzyme recognition nucleotide sequence GATC is prepared.
[0055] Among the nucleotides composing the sequence of each module,
the nucleotide species at both the termini of each of the
individual modules are the same so as to avoid the modification of
the nucleotide sequence at the module-linking parts even if the
order of the modules that have same nucleotide sequence at both the
termini is modified. The nucleotide sequence of the primer 21 (SQ
ID No. 1) is composed of common nucleotlde sequence 13 composed of
the module sequence in the order of I-II-III-IV and the restriction
enzyme recognition nucleotide sequence GATC, and the selective
nucleotide sequence NN 15 composed of two nucieotides.
[0056] SQ ID No. 1
[0057] 5'-TCATCACCTTCTCCACGATCNN-3'
[0058] NN corresponds to any one of A, C, G and T; the primer 21
composes primer set 23 composed of 16 primers.
[0059] Furthermore, primer 22 composed of , the common nucleotide
sequence 14 composed of four module sequences aligned in the order
of III-IV-I-II and restriction enzyme recognition nucleotide
sequence GATC, and selective nucleotide sequence NN 16 composed of
two nucleotides NN (NN corresponding to any one of A, C, G and T)
is prepared as shown as the nucleotide sequence of SQ ID No. 2. NN
composes primer set 24 composed of 16 primers, like the primer
11
[0060] SQ ID No. 2
[0061] 5'-TTCTCCATTCATCACCGATCNN-3'
[0062] The oligonucleotide 11 of a known nucleotide sequence is
complementary to the sequence of the modules in the order of
I-II-III-IV in the primer 21 belonging to the primer set 23. The
oligonucleotide 11 is ligated in the digestion size of the cDNA
fragment 3 as a digestion product with the restriction enzyme Sau3A
I recognizing the nucleotide sequence 5'-GATC-3' to digest the site
CATC. The oligonucleotide 11 is ligated in the 3' terminus of the
sequence of the 4 nucleotides, namely 5'-GATC-3' at the 3' terminus
of the - strand of the CDNA fragment 3 and in the 3' terminus of
the sequence of the 4 nucleotides, namely 5'-GATC-3' at the 3'
terminus of the + strand of the CDNA fragment.
[0063] The primer set 23 hybridizes to the restriction enzyme
recognition sequence and the oligonucleotide 11 at the 3' terminus
of the - strand of the cDNA fragment 3 and to the restriction
enzyme recognition sequence and the oligonucleotide 11 at the 3'
terminus of the + strand of the CDNA fragment 3. Thus, the primer
set 23 amplifies DNA fragment 5 with the oligonucleotlde 11 ligated
at the 3' terminus thereof.
[0064] The oligonucleotide 12 of a known nucleotide sequence is
complementary to the sequence of the modules in the order of
III-IV-I-II in the primer 22 belonging to the primer set 24. The
oligonucleotide 12 is ligated in the digestion site of the cDNA
fragment 4 as a digestion product with the restriction enzyme Sau3A
I recognizing the nucleotide sequence 5'-GATC-3' to digest the site
IGATC. The oligonucleotide 12 is ligated in the 3' terminus of the
sequence of the 4 nucleotides, namely 5'-GATC-3' at the 3' terminus
of the - strand of the cDNA fragment 4 and in the 3' terminus of
the sequence of the 4 nucleotides, namely 5'-GATC-3' at the 3'
terminus of the + strand of the CDNA fragment.
[0065] The primer set 24 hybridizes to the restriction enzyme
recognition sequence and the oligonucleotide 12 at the 3' terminus
of the - strand of the CDNA fragment 4 and to the restriction
enzyme recognition sequence and the oligonucleotide 12 at the 3'
terminus of the +strand of the cDNA fragment 4. Thus, the primer
set 24 amplifies DNA -fragment 6 with the oligonucleotide 12
ligated at the 3' terminus thereof.
[0066] The individual primers of the primer sets 23, 24 are
independently labeled with fluorophores FA25 and F326,
respectively.
[0067] FIG. 2A depicts the structure of primer 41. The primer 41 is
composed of the sequence I-II-III-IV of 4 modules in the order
starting from the 5' terminus, each module being composed of 4
nucleotides, restriction enzyme recognition nucleotide sequence 35
and selective nucleotide sequence 36 composed of 2 nucleotides,
wherein the sequence I-II-III-IV is the sequence 31-32-33-34 FIG.
2B depicts the structure of primer 42. The primer 42 is composed of
the sequence III-IV-I-II of 4 modules in the order starting from
the 5' terminus, each module being composed of 4 nucleotides,
restriction enzyme recognition nucleotide sequence 35 and selective
nucleotide sequence 36 composed of 2 nucleotides, wherein the
sequence III-IV-I-II is the sequence 33-34-31-32.
[0068] FIG. 2C depicts the state of the complex of primer 41/DNA
fragment 45; and FIG. 2D depicts the state of the complex of primer
42/DNA fragment 46. In FIGS. 2C and 2D, the nucleotide sequence of
the modules 31, 32, 33 and 34 in this order is complementary to the
oligonucleotide 43 and the nucleotide sequence of the modules 33,
34, 31 and 32 in this order is complementary to the oligonucleotide
44. The primers 41 and 42 are composed of the same modules 31, 32,
33, 34, the restriction enzyme recognition nucleotide sequence 35
and the selective nucleotide sequence 36 composed of two
nucleotides; although the orders of the modules in these sequences
are different from each other, these primers are at the same
T.sub.m value. The primer 41 hybridizes to the 3' terminus of the
DNA fragment 45 with the oligonucleotide 43 complementary to the
primer 41, the oligonucleotide 43 being ligated to the 3' terminus
of the restriction enzyme-digested site 48. The primer 42
hybridizes to the 3' terminus of the DNA fragment 46 with the
oligonucleotide 44 complementary to the primer 42, the
oligonucleotide 44 being ligated to the 3' terminus of the
restriction enzyme-digested site 48.
[0069] Because the primers 41, 42 are at the same T.sub.m value,
these primers hybridize to the oligonucleotides 43, 44 at the same
reaction efficiency, with no hybridization to each other because
these primers are not complementary to each other, the primers
never suppress each other's reaction, whereby PCR can be
facilitated in one reaction tube. Because the primers 41, 42 are
labeled with the different fluorophores 37, 38, respectively, the
DNA fragments amplified by PCR with the primers 41, 42 are
therefore labeled then with the different fluorophores, which are
detected by electrophoresis based on the difference in fluorescent
wave length.
[0070] FIG. 1 shows a forward primer and a reverse primer selected
from the primer set 23 so as to amplify the DNA fragment 5, as well
as a forward primer and a reverse primer selected from the primer
set 24 so as to amplify the DNA fragment 6. The sample DNA
fragments 5,6 are divided in a reaction tube. The selected 4
primers in total are placed in the reaction tube containing the
sample DNA fragments 5,6, followed by addition of a reaction
solution of thermostable DNA polymerase, substrate dNTP and a
reaction buffer, for PCR. By PCR, amplification reaction is
promoted at a temperature cycle composed of three steps, namely
heat denaturation, re-annealing and elongation.
[0071] At the heat denaturation step, the reaction solution is
heated at about 94.degree. C., so that the CDNA fragments
(double-stranded DNAs) 5, 6 are separated as cDNA fragment +
strands 7, 8 and cDNA fragment - strands 9, 10 in the form of
single-stranded DNA fragment. At the re-annealing step, the
reaction solution is kept at about 60.degree. C. Because the
concentrations of the primers are higher than the concentrations of
the cDNA fragments + stands 7, 8 and the cDNA fragments - strands
9, 10, the annealing of the primers with the cDNA fragments
progresses more preferentially than the re-annealing of the cDNA
fragment + strand 7 with the cDNA fragment - strand 9 or the
re-annealing of the cDNA fragment + strand 8 with the cDNA fragment
- strand 10. The annealing of the primer 21 with the cDNA fragment
+ strand 7 and the annealing of the primer 21 with the cDNA
fragment - strand 9 generates a double-stranded DNA; and the
annealing of the primer 22 with the cDNA fragment +-strand 8 and
the annealing of the primer 22 with the cDNA fragment --strand 10
generates a double-stranded DNA. By the DNA polymerase, the primers
21, 22 generating the double strands together with the cDNA
fragments are elongated at the elongation step, so that the DNA
fragments are amplified. By PCR, amplification progresses when such
oligonucleotide is elongated, starting from both the termini of the
cDNA fragments. When only the + strand or - strand of the DNA
fragment is elongated, the thermal cycle repeated at a number n
induces amplification only by n-fold, below the detection
sensitivity. When both the +and - strands are elongated,
amplification by 2.sup.n fold occurs.
[0072] Because the primers 21,22 are labeled with the fluorophores,
PCR products 17, 18 generated by amplification with the primers 21,
22 are also labeled with the fluorophores. Accordingly, the PCR
products can be assayed by using electrophoresis systems of
fluorophore detection type and the like. On laser irradiation of
the PCR products separated by electrophoresis, the fluorophores 25,
26 can emit fluorescence at different wave lengths, which are
detected through image splitting prisms and filters with a
two-dimensional detector so as to identify which fluorophore emits
the fluorescence. The resulting detected signals are derived from
the PCR amplified products of the cDNA fragments recovered by
restriction digestion of the sample cDNA. Hence, the fragment
length and fluorescent intensity of an amplified DNA fragment can
identify each type of various mRNAs.
[0073] FIG. 3 depicts electropherograms (electrophoretic patterns)
of expression profiling recovered by using the primers of Example 1
of the invention, wherein the parts 51, 52 of the electropherograms
are shown in a manner corresponding to nucleotide length 53. In
Example 1, genes expressed in rat liver and kidney are compared to
each other The electropherogram 51 can be recovered by
electrophoresis of the PCR amplified products with the primer 21,
while the electropherogram 52 can be recovered by electrophoresis
of the PCR amplified products with the primer 22.
[0074] Most of the peaks in the electropherograms 51, 52 are
detected at the same positions, indicating that the peaks represent
genes commonly expressed in both the tissues. In one of the
electropherograms, namely 52, peak 55 is detected, which is derived
from a gene specifically expressed at an expression state different
from the other tissue. The genes commonly expressed in the two
tissues are detected at good reproducibility on the two
electropherograms. PCR in one reaction tube by using primers of
plural primer sets readily enables the comparison of PCR products
in an accurate manner, with no requirement of PCR in separate
manners by using primers of individual primer sets.
EXAMPLE 2
[0075] In Example 2, PCR products are assayed by using a probe
array immobilizing numerous types of cDNA probes with nucleotide
sequences complementary to gene sequences.
[0076] FIG. 4 shows the flowchart of the procedures for expression
profiling of Example 2 of the invention. In the same manner as in
Example 1, CDNA mixtures 61, 62 as samples prepared from RNAs
extracted from different yeast strains are placed in reaction tubes
91, 92 (in the FIG. 4, only one cDNA mixture is shown) and are
digested with a class II restriction enzyme. The class II
restriction enzyme was Mbo I recognizing the sequence 5'-GATC-3'
and digesting the site GATC. Oligomers 71, 72 are ligated to the
resulting cDNA fragments 63, 64, to prepare cDNA fragments 65, 66
with the oligomers 71, 72 ligated at the termini thereof. Then, the
cDNA fragments 65, 66 are mixed together; a part of the resulting
mixture solution is placed in PCR tube 93, which is used as
template DNA fragments for PCR.
[0077] As a PCR primer, primer 81 composed of common nucleotide
sequence 73 composed of 4 module sequences, I. ACAA, II. GACG, III.
ATCA and IV. GCAG, and the restriction enzyme recognition
nucleotide sequence GATC, and selective nucleotide sequence NN 75
being composed of two nucleotides NN and recognizing two
nucleotides subsequent to the restriction enzyme recognition
sequence GATC (NN corresponds to any one of A, C, G and T) is
prepared. The primer 81 is of the nucleotide sequence of SQ ID No.
3.
[0078] SQ ID No. 3
[0079] 5'-ACAAGACGATCAGCAGGATCNN-3'
[0080] NN corresponds to any one of A, C, G and T; the primer 81
composes primer set 83 composed of 16 primers. Furthermore, primer
82 is prepared, which is composed of common nucleotide sequence 74
composed of 4 module sequences in the order of I-IV-III-II and the
restriction enzyme recognition nucleotide sequence GATC, and
selective nucleotide sequence NN 76 composed of two nucleotides (NN
corresponds to any one of A, C, G and T). The primer 82 is of the
nucleotide sequence of SQ ID No. 4. NN corresponds to any one of A,
C, G and T; the primer 82 composes the primer set 84 composed of 16
primers, like the primer 81. SQ ID No. 4
[0081] 5'-ACAAGCAGATCAGACGGATCNN-3',
[0082] The oligonucleotide 71 of a known nucleotide sequence is
complementary to the sequence of the modules in the order of
I-II-III-IV in the primer 81 belonging to the primer set 83. The
oligonucleotide 72 of a known nucleotide sequence is complementary
to the sequence of the modules in the order of I-IV-III-II in the
primer 82 belonging to the primer set 84. Thus, the primer set 83
amplifies DNA fragment 65 with the oligonucleotide 71 ligated at
the 3' terminus thereof, while the primer set 84 amplifies DNA
fragment 66 with the oligonucleotide 72 ligated at the 3' terminus
thereof.
[0083] The individual primers of the primer sets 83, 84 are
independently labeled with fluorophores FA85 and FB86,
respectively.
[0084] A forward primer and a reverse primer are selected from the
primer set 83 so as to amplify the DNA fragment 65; a forward
primer and a reverse primer are selected from the primer set 84 so
as to amplify the DNA fragment 66. The selected 4 primers in total
are placed in reaction tube 93 containing the sample DNA fragments
65,66 divided therein, followed by addition of a reaction solution
of thermostable DNA polymerase, substrate dNTP and a reaction
buffer, for PCR. Subsequently, PCR products 67, 68 are assayed by
using a DNA probe array with numerous types of cDNA probes
immobilized on a glass plate.
[0085] FIG. 5 is a flowchart depicting the procedures of expression
profiling by using the DNA probe array. In FIG. 5, 191 is glass
plate; 192, 193 are DNA probes individually immobilized on the
glass plate. Within a zone of several square centimeters on the
glass plate 191 are two-dimensionally immobilized several tens to
several hundreds of DNA probes in groups divided, depending on the
types. On the glass plate are arranged PCR products 101, 102
labeled with fluorophores FA111, FB112, for hybridization to the
DNA probes. Thereafter, the plate was rinsed, to wash off DCR
products with no hybridization. On a glass plate section with DNA
probe 192 immobilized thereon, PCR products with no hybridization
to the DNA probe 192 are rinsed off among the PCR products, while
the complementary PCR products 103, 104 hybridize to the DNA probe
192. In the same manner, PCR products with no hybridization to the
DNA probe 193 are rinsed off among the PCR products, while the
complementary PCR product 105 hybridizes to the DNA probe 193.
Among PCR products 101, PCR products 103, 105 are DNA fragments
hybridizing to the DNA probes 192, 193 on the DNA probe array and
being labeled with the fluorophore FA111. Alternatively, the PCR
produce 104 is a DNA fragment hybridizing to the DNA probe 192 on
the DNA probe array and being labeled with the fluorophore
FB112.
[0086] FIG. 6 is a figure depicting the mechanistic composition of
a fluorescent microscope for detecting the samples on the DNA probe
array in Example 2 of the invention. By using the laser microscope
of FIG. 6, PCR products hybridizing to the DNA probe array can be
detected. Laser beam 132 from laser source 131 is reflected on
dichroic mirror 134, to irradiate DNA probe array 135 arranged on
microscope stage 133. Fluorescence 136 emitted from the fluorophore
arranged on the microscope stage 133 is passed through filter 137
to be detected with detector 138. In Example 2, two types of
fluorophores FA and FB are used, so two types of filters are
prepared for different wave lengths to detect fluorescence at the
individual fluorescent wave lengths.
[0087] FIG. 7 shows fluorescent intensities in bar graph from a DNA
probe array, depicting expression profiling recovered by using the
primers of Example 2 of the invention. As shown in FIG. 7, plural
types of DNA fragments can be compared to each other, by comparing
the fluorescent intensities detected at positions of individual
immobilized DNA probes on the DNA probe array. In FIG. 7, the
longitudinal axes 141, 142 represent fluorescent intensities from
PCR products labeled with different fluorophores. The crosswise
axis represents the number of the position of each DNA probe
immobilized on the DNA probe array. The comparison of the
fluorescent intensity at each wave length at the position of one
DNA probe on the DNA probe array enables the comparison of DNA
fragments extracted from plural samples. Because cDNA fragments are
simultaneously PCR amplified in one reaction tube, these fragments
are readily assayed in a comparative manner with no concern about
PCR reproducibility.
[0088] In accordance with the invention, at least two or more sets
of primers are used for PCR in one reaction tube. Individual
primers are labeled with different fluorophores and thus function
independently. So as to amplify one sample DNA with a first set of
primers and to amplify the other sample DNA with a second set of
primers, oligonucleotides with complementary nucleotide sequences
to the individual primers are ligated to the individual sample
DNAs, which are then placed in one reaction tube for PCR. Plural
primers for use in PCR should have the same T.sub.m value to
eliminate the difference in reaction efficiency among the
individual primers. So as to allow plural primers to have the same
T.sub.m value, different sequence orders of plural modules are
ligated in the individual primers, each module being composed of 4
to 6 nucleotides, to prepare plural primers. So as to avoid the
modification of the nucleotide sequence in the linking regions
between individual modules even after exchange of the sequence
order of the modules that have same nucleotide species, both the
termini of each module are composed of the same nucleotide species.
By using plural modules at almost the same T.sub.m value and with
no difference in PCR reaction efficiency, plural sample DNAs are
subjected to PCR in one reaction tube, with no variation of
PCR.
[0089] Reference numbers in the individual figures are now
described below.
[0090] 1, 2, 61, 62: sample cDNAs in mixture
[0091] 3,4,63,64: cDNA fragments digested by restriction enzyme
[0092] 5,6: cDNA fragment with an oligonucleotide of a known
nucleotide sequence being ligated at the terminus thereof 7,8: cDNA
fragment + strand
[0093] 9,10: cDNA fragment - strand
[0094] 11, 12: oligonucleotide of a known nucleotide sequence
[0095] 13, 14, 73, 74: common nucleotide sequence
[0096] 15, 16, 36, 75, 76: selective nucleotide sequence
[0097] 17, 18, 67, 68, 101, 102: PCR amplified products
[0098] 21, 22, 41, 42, 81, 82: primer
[0099] 23, 24, 83, 84: primer set
[0100] 25, 26, 37, 38, 85, 86, 111, 112: fluorophore
[0101] 27, 28, 29, 91, 92, 93: reaction tube
[0102] 31, 32, 33, 34: sequence of modules (module sequence)
[0103] 35: recognition site of restriction enzyme
[0104] 43, 44: oligonucleotide
[0105] 45, 46: DNA fragment
[0106] 48: restriction enzyme digested site
[0107] 51; 52: electropherogram
[0108] 53: base length
[0109] 55: peak derived from specifically expressed gene
[0110] 65, 66: cDNA fragment with an oligomer of a known nucleotide
sequence being ligated at the terminus thereof
[0111] 71, 72: oligomer of a known nucleotide sequence
[0112] 103, 104, 105: PCR products hybridizing to DNA probe
[0113] 131: laser source
[0114] 132: laser beam
[0115] 133: microscope stage
[0116] 134: dichroic mirror
[0117] 135: DNA probe array
[0118] 136: fluorescence
[0119] 137: filter
[0120] 138: detector
[0121] 141, 142: fluorescent intensity of PCR products
[0122] 191: glass plate
[0123] 192, 193: DNA probe
Sequence CWU 1
1
4 1 22 DNA Artificial Sequence Description of Artificial Sequence
Primer 1 tcatcacctt ctccacgatc nn 22 2 22 DNA Artificial Sequence
Description of Artificial Sequence Primer 2 ttctccactc atcaccgatc
nn 22 3 22 DNA Artificial Sequence Description of Artificial
Sequence Primer 3 acaagacgat cagcaggatc nn 22 4 22 DNA Artificial
Sequence Description of Artificial Sequence Primer 4 acaagcagat
cagacggatc nn 22
* * * * *