U.S. patent application number 17/115740 was filed with the patent office on 2021-06-24 for method for detecting target nucleic acid sequence using cleaved complementary tag fragment and a composition therefor.
The applicant listed for this patent is GENEMATRIX, INC.. Invention is credited to Ae Ri CHO, Woo Jae CHO, Hyun Jae CHUNG, Seong Soo HONG, Sun Pyo HONG, Sun Young JEONG, Jae Il KIM, Jeong Woo KIM, Soo Ok KIM, Suk Joon KIM, Seung Min YANG.
Application Number | 20210189456 17/115740 |
Document ID | / |
Family ID | 1000005436360 |
Filed Date | 2021-06-24 |
United States Patent
Application |
20210189456 |
Kind Code |
A1 |
KIM; Soo Ok ; et
al. |
June 24, 2021 |
METHOD FOR DETECTING TARGET NUCLEIC ACID SEQUENCE USING CLEAVED
COMPLEMENTARY TAG FRAGMENT AND A COMPOSITION THEREFOR
Abstract
The present invention relates to a method and a composition for
detecting a target nucleic acid sequence using a cleaved
complementary tag fragment. Specifically, the present invention
relates to a method for linking a complementary tag sequence to a
PCR primer so that a tagging can be produced by a restriction
enzyme during a PCR reaction, diversifying the complementary tag
sequence to be linked to each primer by utilizing factors such as
length and nucleic acid combination, etc., and distinguishing the
target sequence using the same. According to the present invention,
a cleaved complementary tag fragment (CCTF) under stringent
conditions is a complementary sequence to any sequence at the 5'
end linked to the primer and cannot be formed unless a PCR reaction
and a restriction enzyme reaction occur, and the cleaved single
strand is formed only when hybridization to the target sequence
occurs and a primer extension product complementary to the target
sequence is formed, so as to have a higher degree of accuracy
secured by reading the cleaved single strand. In addition, the CCTF
can be used to identify a plurality of target nucleic acid
sequences by selecting various analytical techniques and analysis
equipment according to a user's intention. For example, a result
can be confirmed rapidly and accurately in genetic testing,
identification of organisms in a sample, diagnosis of microbial or
viral infection, etc.
Inventors: |
KIM; Soo Ok; (Seoul, KR)
; KIM; Suk Joon; (Seongnam-si, KR) ; HONG; Sun
Pyo; (Seoul, KR) ; CHUNG; Hyun Jae; (Gunpo-si,
KR) ; CHO; Woo Jae; (Anyang-si, KR) ; KIM; Jae
Il; (Seoul, KR) ; YANG; Seung Min;
(Gwangju-si, KR) ; CHO; Ae Ri; (Seongnam-si,
KR) ; HONG; Seong Soo; (Seoul, KR) ; KIM;
Jeong Woo; (Yongin-si, KR) ; JEONG; Sun Young;
(Hwaseong-si, KR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
GENEMATRIX, INC. |
Seongnam-si |
|
KR |
|
|
Family ID: |
1000005436360 |
Appl. No.: |
17/115740 |
Filed: |
December 8, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16095695 |
Oct 23, 2018 |
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PCT/KR2017/004297 |
Apr 21, 2017 |
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17115740 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q 1/6816 20130101;
C12Q 1/686 20130101; C12Y 207/07007 20130101; C12Q 1/705 20130101;
C12Q 1/6806 20130101; C12Q 1/6853 20130101; C12Q 1/683 20130101;
C12Q 1/6872 20130101; C12Q 1/708 20130101 |
International
Class: |
C12Q 1/683 20060101
C12Q001/683; C12Q 1/6806 20060101 C12Q001/6806; C12Q 1/686 20060101
C12Q001/686; C12Q 1/6816 20060101 C12Q001/6816; C12Q 1/70 20060101
C12Q001/70; C12Q 1/6853 20060101 C12Q001/6853; C12Q 1/6872 20060101
C12Q001/6872 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 25, 2016 |
KR |
10-2016-0050313 |
Claims
1. A method for forming and identifying a tag used in classifying
and analyzing kinds of the target sequences amplified in the
Polymerase Chain Reaction, which comprises: a) hybridizing a target
sequence with a primer comprising a template of a tag for
generating the tag, which is a cleaved complementary tag fragment;
b) generating the complementary tag fragment cleaved from the
primer by an activity of a restriction enzyme when the
amplification procedure is proceeded by the hybridization of a)
step and releasing and introducing it into a reaction solution; and
c) identifying the generated cleaved complementary tag fragment
through an analyzer to confirm the presence of the target nucleic
acid sequence wherein said primer of a) step comprises a random
nucleic acid sequence noncomplementary to a target sequence and has
a structure sequentially comprising a restriction enzyme
recognition sequence and a nucleic acid sequence complementary to
the target sequence.
2. The method according to claim 1, characterized in that the
restriction enzyme recognition sequence is the recognition sequence
for the restriction enzyme selected from the group consisting of
Pho I, PspGI, BstNI, TfiI, ApeKI, TspMI, BstBI, BstEII, BstNI,
BstUI, BssKI, BstYI, TaqI, MwoI, TseI, Tsp45I, Tsp509I, TspRI,
Tth111I, Nb.BsmI, Nb.BsrDI, NLBspQI, Nt.BstNBI restriction enzymes
and Nick restriction enzyme.
3. The method according to claim 1, characterized in that the
modified dNTP is inserted in a region of the primer cleaved by a
restriction enzyme in order to prevent cleaved by-products other
than the cleaved complementary tag fragment from participating in
the reaction.
4. The method according to claim 3, characterized in that the
modified dNTP inserted in the cleaved region comprises
Phosphorothioated dNTP, dNTP comprising 7-Deazapurine, or
2'-O-methyl nucleotide(2'-OMeN) in DNA template.
5. The method according to claim 1, characterized in that the said
method analyzes the mass of the cleaved complementary tag fragment
to identify the cleaved complementary tag fragment.
6. The method according to claim 5, characterized in that the
instrument used for the mass spectrometry is a matrix-assisted
laser desorption-ionization-time-of-flight mass spectrometer
(MALDI-TOF MS), a Liquid Chromatography Mass Spectrometer, or a
High Performance Liquid Chromatography Mass Spectrometer.
7. The method according to claim 6, characterized in that the mass
per unit electric charge (m/z) of the cleaved tag fragment used for
mass spectrometry is present in the range of from greater than 0 to
10000 Da or less.
8. The method according to claim 7, characterized in that DNA
polymerase that the function of adenine addition elongation effect
(A tailing) at the 3' end, being an inherent property of the
polymerase is inhibited, is used in order to preserve the mass of a
cleaved complementary tag fragment used in mass analysis during the
amplification process.
9. The method according to claim 1, characterized in that the
fluorescence signal is analyzed by using the oligonucleotide that
is tagged by fluorescence and Quencher and has the complementary
sequence of the cleaved complementary tag fragment, as the
identification method of the cleaved complementary tag
fragment.
10. The method according to claim 9, characterized in that the said
method analyzes the dissociation temperature and melting peak by
varying the inherent dissociation temperature at which the double
strand of the oligonucleotide and the cleaved complementary tag
fragment are dissociated into a single strand, and identifies the
cleaved complementary tag fragment to confirm the presence of the
target sequence.
11. The method according to claim 9, characterized in that the said
method is made to have different dissociation temperatures to
simultaneously analyze two or more kinds of targets through a
melting peak analysis in the case of that two or more targets are
detected.
12. The method according to claim 9, characterized in that the
oligonucleotide is from 5 or more to 50 or less in length.
13. The method according to claim 9, characterized in that the
nucleotide at the 3'end of the oligonucleotide is blocked in order
to prevent elongation of the base sequence from the
oligonucleotide.
14. The method according to claim 13, characterized in that the
said method attaches Spacer C3, Phosphat, ddC, or Inverted END to
the nucleotide at the 3'end of the oligonucleotide in order to
prevent elongation of the base sequence from the
oligonucleotide.
15. The method according to claim 13, characterized in that the
said method attaches the quencher to the nucleotide at the 3'end of
the oligonucleotide is blocked in order to prevent elongation of
the base sequence from the oligonucleotide.
16. The method according claim 1, characterized in that the method
identifies a causative organism of the sexually transmitted
disease, the causative organism of gastrointestinal tract disease,
a Human Papilloma virus, a causative organism of the respiratory
disease, or a gene type of a single nucleotide polymorphism
(SNP).
17. The method according to claim 9, characterized in that the
method identifies the complementary tag fragment cleaved by
analyzing the cycle threshold (Ct) value of the fluorescence signal
of the oligonucleotide.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a divisional application of the U.S.
application Ser. No. 16/095,695, filed Oct. 23, 2018, the
disclosure of which is incorporated herein in its entirety by
reference.
TECHNICAL FIELD
[0002] The present invention relates to a method for detecting
target nucleic acid sequence using cleaved complementary Tag
fragment and a composition therefor, and specifically relates to a
method of identifying the amplified product by ligating a template
of tag capable of producing a marked substance to a primer that
specifically reacts with the target sequence, thereby synthesizing
and releasing the tag by restriction enzyme activity in the PCR
reaction and introducing it into the reaction solution. Also, the
present invention relates to a method for generating and
identifying a tag characterized in that the tag generated during a
reaction using only one kind of template sequence of a tag for one
target sequence is analyzed in various analyzing apparatuses to
identify the tag, and a composition used in the method.
BACKGROUND ART
[0003] Polymerase Chain Reaction (PCR) is one of techniques very
usefully utilized in detecting and analyzing low concentration
nucleic acids. The detection of the nucleic acid is based on the
complementarity of the double strand oligonucleotide sequences and
the extension reaction of each DNA polymerase, and the target
nucleic acid sequence can be detected using this (Barry et al.,
Current Opinion in Biotechnology, 12; 21, 2001).
[0004] Multiple PCR is a method that can simultaneously amplify
nucleic acids of multiple target sequences, and is relatively fast
and simple compared to other methods, and thus plays a very large
role in diagnosis field such as genetic test, identification of
organisms in samples, and microbial or viral infection, etc.
[0005] The most common method for confirming the results of such
multiplex PCR is to design primers by varying an amplification
product size of the target sequence as desired in PCR, and to
analyze the size of the amplified product by electrophoresis of the
PCR result, and then to confirm as to whether amplification of the
target sequence is made. In this case, the number of genes that can
be amplified at one time is limited to 3 to 4 experimentally
because there is a restriction that the size of the amplification
product should be limited within a narrow range, due to that the
efficiency of amplification depends on the size of the
amplification product that can be generated during the PCR reaction
and thus a uniform amplification efficiency cannot be guaranteed.
In this case, it also occurs the case that the size of the desired
gene amplification product may overlap. Therefore, there is a limit
to the interpretation of the detection method when the multiple PCR
is analyzed depending on the size.
[0006] Real-time PCR guarantees a confirmation of a rapid PCR
result in confirming the PCR results, and it can identify as to
whether the amplification is made by marking fluorescent material
regardless of the size of the amplified product. The methods
performing and detecting Real-time PCR can be divided into
intercalating method and probe method, wherein the intercalating
method is referred to a method of confirming fluorescence intensity
by inserting fluorescent substance between double-stranded base
sequences. Since this method cannot distinguish the amplification
products forming the double strands, and can observe all of them as
the fluorescence of the same wavelength. Therefore, it has a limit
on identifying the amplification product by each target sequence to
detect and identify at least one amplified product simultaneously.
The probe method is a method of detecting the amplified product by
reading the fluorescence value of the probe designated for each
target sequence and, in the case of using this method, since the
amplification product can be detected only in the number of
analyzable fluorescence channels of a device to be used, the
multiple analysis over the number of fluorescent channels is not
suitable for this.
[0007] Therefore, studies were continuously carried out to insert
the tag during PCR to enable the maximum number of multiple
analysis.
[0008] In the case of Luminex's xTAG technology, a constant base
sequence comprised of a random array of thymine (T), adenine (A),
and guanine (G), which constitutes the nucleic acid, was set and
named xTAG. It is a method comprising inserting xTAG sequence into
the primer to be located the xTAG sequence at the end in the
amplification of the target sequence to be observed, so that the
xTAG was inserted into the amplification product during the PCR
procedure, and secondarily joining the xTAG with a bead to which
the complementary sequence to xTAG attached to form a complementary
bond between the two base sequences, detecting the target using the
same, and analyzing the target sequence with fluorescence of the
bead. In this method, even though the xTAG does not participate in
the amplification, if the primer is not completely removed after
the amplification, it has problems that there is a possibility that
it binds to the complementary xTAG of the bead to recognize the
mark, and an error occurs that the complementary sequence of xTAG
forms non-specific reaction by PCR and thus non-specific target is
detected (U.S. Pat. Nos. 7,645,868 and 8,624,014).
[0009] In order to solve this problem, studies has been
continuously performed that a tag is constructed during the PCR
reaction, the tag does not affect the PCR reaction, the maximum
numbers of multiple detections are possible.
DISCLOSURE
Technical Problem
[0010] The present invention is derived to solve the above problems
and to meet the above needs and the object of the present invention
is to provide a method for solving the uncertainty which can be
occurred when the results are determined depending on the length of
the generated product in amplifying and analyzing a target sequence
using an amplification reaction such as PCR, and for solving the
restriction to the maximum numbers of amplification that can be
identified in multiple detection.
[0011] The another object of the present invention to provide a
method for improving accuracy by solving errors due to non-specific
amplification which can be caused by the use of artificial sequence
as a tag itself in identifying a target sequence amplification by
forming the tag.
Technical Solution
[0012] In order to accomplish the above object, the present
invention provides a primer with the structure comprising a target
sequence and a non-complementary random nucleic acid sequence and
sequentially comprising a restriction enzyme recognition sequence
and a nucleic acid sequence complementary to the target
sequence.
[0013] In one embodiment of the present invention, the restriction
enzyme recognition sequence is preferably one selected from the
group consisting of Pho I, PspGI, BstNI, TfiI, ApeKI, TspMI, BstBI,
BstEII, BstNI, BstUI, BssKI, BstYI, TaqI, MwoI, TseI, Tsp45I,
Tsp509I, TspRI, Tth111I, Nb.BsmI, Nb.BsrDI, Nt.BspQI, Nt.BstNBI
restriction enzymes and Nick restriction enzymes, but is not
limited thereto.
[0014] In another embodiment of the present invention, the said
primer is preferably one that a modified dNTP inserted at the
cleavage site of the restriction enzyme recognition sequence of the
primer, for the purpose of that a cleaved by-product other than the
cleaved complementary tag fragment allow not to participate in the
reaction, and the modified dNTP to be inserted into the cleavage
site is phosphorothioated dNTP, dNTP containing 7-deazapurine, or a
2'-O-methyl nucleotide (2'-OmeN) in a DNA template, but is not
limited thereto.
In another embodiment of the present invention, it is preferable,
but not limited, that the primer is from 5 mers or more to 50 mers
or less in length of the cleaved complementary tag fragment as
generated.
[0015] In one embodiment of the present invention, the primer is
one or more one selected from the group consisting of SEQ ID NOS:
1, 3, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 74, 76,
78, 80, 82, 86, 115, 117, 119, 121, 123, 125, 127, 129, 131, 151,
153, 155, 156, 159, 161, 164, 166, 168, 170, 204, 205, 207, 218,
220 and 222, but is not limited thereto.
[0016] Furthermore, the present invention provides a method for
forming a tag to be used in classifying and analyzing the kinds of
the target sequences amplified in the Polymerase Chain Reaction,
and identifying it, comprising: [0017] a) hybridizing a target
sequence with a primer of the present invention comprising a
template of a tag for generating the tag that is a cleaved
complementary tag fragment, [0018] b) generating the complementary
tag fragment cleaved from the primer by the activity of a
restriction enzyme when the amplification procedure is proceeded by
the hybridization of a) and introducing it into a reaction
solution, and [0019] c) identifying the generated cleaved
complementary tag fragment through an analyzer to confirm the
presence of the target sequence.
[0020] In one embodiment of the present invention, it is preferable
to analyze the mass of the cleaved complementary tag fragment to
identify the cleaved complementary tag fragment in the above
method, and the instrument used for the mass spectrometry is
preferably a matrix-assisted laser
desorption-ionization-time-of-flight mass spectrometer ((MALDI-TOF
MS), a Liquid Chromatography Mass Spectrometer, or a High
Performance Liquid Chromatography Mass Spectrometer, but is not
limited thereto.
[0021] In another embodiment of the present invention, the mass per
unit electric charge (m/z) of the cleaved tag fragment to be used
for mass spectrometry is preferably from greater than 0 to 10000 Da
or less, but is not limited thereto.
[0022] In another embodiment of the present invention, in order to
preserve the mass of a cleaved complementary tag fragment to be
used in mass analysis during the amplification process, it is
preferable to use DNA polymerase that the function of
adenine-addition elongation effect (A tailing) at the 3'end, which
is an intrinsic property of the nucleic acid polymerase, is
inhibited, but is not limited thereto.
[0023] In another embodiment of the present invention, it is
preferable to analyze the fluorescence signal using the
oligonucleotide that is tagged by fluorescence and Quencher and has
the complementary sequence of the cleaved complementary tag
fragment as the identification method of the cleaved complementary
tag fragment, but it is not limited thereto.
[0024] In another embodiment of the invention, it is preferable to
analyze the dissociation temperature and melting peak by varying
the inherent dissociation temperature at which the double strand of
the oligonucleotide and the cleaved complementary tag fragment are
dissociated into a single strand, and to identify the presence of
the target sequence by identifying the cleaved complementary tag
fragment in the method, but is not limited thereto.
[0025] In yet another embodiment of the present invention, the
oligonucleotide is preferably 5 or more in length, but is not
limited thereto.
[0026] In another embodiment of the present invention, it is
preferable to attach a quencher to the nucleotide at the 3'end of
the oligonucleotide in order to prevent elongation of the base
sequence from the oligonucleotide in the method, but is not limited
thereto.
[0027] In another embodiment of the present invention, it is
preferable to identify the complementary tag fragment cleaved by
analyzing the cycle threshold (C) value of the fluorescence signal
of the oligonucleotide, but not limited thereto.
[0028] In a preferred embodiment of the present invention, it is
preferable to identify causative organisms of a sexually
transmitted disease in the said method, and the sexually
transmitted disease causative organism is preferable one selected
from the group consisting of Chlamydia trachomatis, Neisseria.
Gonorrhea, Mycoplasma hominis, Mycoplasma genitalium, Trichononas
vaginalis, Ureaplasma urealyticum, Ureaplasma parvum, Candida
albicans, Gardnerella vaginalis, Herpes simplex virus 1, Herpes
simplex virus 2, Treponema pallidum, but is not limited
thereto.
[0029] The present invention also provides a composition for
diagnosing sexually-transmitted diseases, comprising the primer of
the present invention as an effective component.
[0030] In another embodiment of the present invention, it is
preferable to identify the causative organism of gastrointestinal
tract disease, wherein the causative organism of gastrointestinal
tract disease is selected from the group consisting of Rotavirus A,
Astrovirus, Adenovirus F40, Adenovirus F41, Norovirus GI and
Norovirus GII, but is not limited thereto.
[0031] The present invention also provides a composition for
diagnosing a gastrointestinal disease agent comprising the primer
of the present invention as an effective component.
[0032] In another preferred embodiment of the present invention, it
is preferable to identify a human papilloma virus in the method,
and the subpopulations of the human papilloma virus is preferably
selected from the group consisting of types 16, 18, 33, 35, 51, 53,
59, 68a, and 82, but is not limited thereto.
[0033] The present invention also provides a composition for
diagnosing HPV comprising the primer of the present invention as an
effective component.
[0034] In another preferred embodiment of the present invention, it
is preferable to identify a causative organism of the respiratory
disease in the method, and the causative organism of the
respiratory disease is one being selected from the group consisting
of Influenza A/H1N1, Influenza A/H3N2, influenza A/H1N1/2009pdm,
influenza B, Parainfluenza 1, Parainfluenza 3, Respiratory
syncytial virus A, Respiratory syncytial virus B, Human
metapneumovirus, Adenovirus, but is not limited thereto
[0035] The present invention also provides a composition for the
diagnosis of respiratory diseases, comprising the primer of the
present invention as an effective component.
[0036] In another preferred embodiment of the present invention,
the method is preferably a single nucleotide polymorphism (SNP),
wherein the single base mutation is preferably one selected from
the group consisting of r6265 of the Brain-derived neurotrophic
factor gene (BDNF gene), but is not limited thereto.
[0037] The present invention also provides a composition for
analyzing the BDNF gene rs6265 gene comprising the primer of the
present invention as an effective component.
[0038] Hereinafter, the present invention will be described.
[0039] The present inventors have tried our best to develop the
method that can perform a multiplex amplification reaction on a
large number of targets at one time by clearly distinguishing each
amplification product through an easier, faster and more efficient
method in preforming the amplification reaction and can analyze the
results.
[0040] As a result, so as to be able to generate a nucleic acid
sequence which can be used as a tag in an amplification reaction,
when a sequence serving as a template for a tag was inserted into a
primer, and only tag was cleaved by a restriction enzyme, we
confirmed that the generated tag can play a role as the tag for
detecting the target sequence and also identified that it can
identify the amplification efficiently and rapidly than other
existing methods in the multiplex amplification reaction analysis
by applying it to various analysis methods, and thus, has been
completed the present invention.
[0041] The present invention relates to a method of forming tags to
be used for sorting and analyzing kinds of amplified target
sequences during a PCR reaction.
[0042] In particular, the present invention is characterized in
comprising the steps of: (1) hybridizing a target sequence with a
primer comprising a template of a tag for generating the tag, (2)
generating the tag from the template of the tag using a restriction
enzyme during the PCR reaction, and (3) analyzing the generated tag
with various analysis equipment to identify the tag.
[0043] (1) As the step for hybridizing a target sequence with a
primer (CTPO-Cleavable Tag Primer Oligonucleotide, hereinafter
referred to as CTPO) comprising a template of a tag for generating
the tag (CCTF-Cleaved Complementary Tag Fragment, hereinafter
referred to as CCTF); wherein CTPO comprises a sequence
non-complementary to the target sequence (the template of CCTF),
followed by a restriction enzyme recognition sequence and a nucleic
acid sequence complementary to the target sequence, and the nucleic
acid sequence site complementary to the target sequence located at
the 3'end hybridizes with the target sequence, thereby playing a
role as a primer during the PCR reaction,
[0044] (2) as a step for generating and releasing CCTF from CTPO by
the activity of a restriction enzyme in the amplification process;
wherein the restriction enzyme recognition sequence is inserted
into the amplified product elongated from the above-described CTPO,
and CCTF is generated by the activity of the thermostable
restriction enzyme recognizing it and introduced into the reaction
solution,
[0045] (3) as the step for analyzing and identifying the generated
CCTF through various analysis equipment to confirm existence of a
target nucleic acid sequence; wherein the mass of the generated
CCTF is measured to identify the type of CCTF, and the amplified
product is sorted to confirm the presence of the target nucleic
acid sequence, or the fluorescence is emitted during the procedure
that the oligonucleotide composed of the sequence complementary to
the generated CCTF and tagged with the fluorescence and the
quencher (Signal Capture Oligonucleotide--SCO, hereinafter referred
to as SCO) and CCTF are hybridized to form a double strand and
dissociate again into a single strand, and such inherent
dissociation temperature is analyzed to identify the type of CCTF,
and to identify whether the amplification of the target nucleic
acid sequence is occurred or not.
[0046] Hereinafter, the present invention will be described in
detail.
[0047] In step (1), prior to hybridizing the CTPO and the target
sequence, the structure of CTPO is divided into a template portion
of the CCTF, a restriction enzyme recognition sequence, and a
sequence complementary to the target as shown in the following
Formula 1.
5'-A-B-C-3' Formula 1
[0048] The A site in the structural formula 1 is comprised of a
random sequence to be a template of the CCTF, and the complementary
sequence of the CCTF template, that is, the CCTF site, is elongated
by amplifying it after annealing with the target sequence and then
the CCTF site is released by the restriction enzyme during the
amplification. The released CCTF is characterized by being a random
sequence having 5 or more oligonucleotides in length so that it can
be specifically analyzed as a tag. Random sequences can be used in
any sequence that does not create a by-product during the PCR
reaction. The nucleotide sequence to be used as a template for CCTF
is free from any sequence that does not cause a hybridization
reaction during the amplification reaction
[0049] B is a restriction enzyme recognition sequence, which means
a specific recognition sequence of restriction enzymes and Nick
restriction enzymes having thermal stability that can be used
during amplification. For example, it includes Pho I, PspGI, BstNI,
TfiI, ApeKI, TspMI, BstBI, BstEII, BstNI, BstUI, BssKI, BstYI,
TaqI, MwoI, TseI, Tsp45I, Tsp509T, TspRI, Tth111I, Nb.BsmI,
Nb.BsrDI, Nt.BspQI, Nt.BstNBI, etc.
[0050] Most preferably, among them, PspGI can be used, and the
restriction enzyme used in Example of the present invention is
PspGI.
[0051] The modified dNTP is inserted into a site cleaved by the
restriction enzyme in the restriction enzyme recognition sequence
of CTPO so as not to exist and participate the cleaved by-products
other than CCTF in the reaction. Examples thereof include
phosphorothioated dNTPs, dNTPs containing 7-deazapurine, or
2'-O-methyl nucleotides (2'-OMeN) in DNA templates, etc. The prior
art, PNAS 89 (1992) 392-396 and Nucleic Acids Research 20 (1)
199155-61 can be applied to the present invention. Most preferably,
a phosphothiolated bond is inserted into the cleavage site among
the recognition sequence to prevent the cleavage of the template of
CCTF by a restriction enzyme, thereby securing a template capable
of generating CCTF and to prevent a by-product which can be
generated by releasing the template of CCTF into the reaction
solution, thereby increasing the efficiency of the reaction. It
represents the effects of the invention different from the prior
art, SDA (Strand Displacement Amplification) method (US Pat. No.
92-819,358) in view of that it generates CCTF and prevents the
template to inflow to the reaction solution.
[0052] The C site shown in the structural formula 1 means a part
after the restriction enzyme recognition sequence up to the 3'end,
and is composed of a target specific sequence so that it binds
specifically to the target during amplification so as to maintain
its role as a primer.
[0053] In step (2), when the amplification product is formed by
CTPO, and the amplified product present in the double strand is
cleaved to CCTF by the restriction enzyme and released into the
reaction solution, the appropriate concentration of the restriction
enzyme to be used can be varied depending on the purpose of use. In
addition, the results are different depending on the type of
polymerase to be used, which can be also varied depending on the
purpose of use. For example, when CCTF is formed for the purpose of
mass spectrometry, it is preferable that the weight of CCTF should
be kept constant regardless of the amplification process and should
not reflect the intrinsic property of the nucleic acid polymerase.
Therefore, a nucleic acid polymerase having no adenine addition
extension effect (A tailing) at the 3'end, which is an intrinsic
property of the nucleic acid polymerase, should be selected and
used. Among the nucleic acid polymerase enzymes that do not make A
tailing, Phusion polymerase, Vent polymerase, Deep Vent polymerase,
Bst polymerase, etc. are present.
[0054] However, when CCTF analysis method using other techniques
than mass analysis is applied, there is no variation in the results
due to the A tailing effect, and thus, any polymerase can be
used.
[0055] In order to increase the efficiency of the restriction
enzyme to generate CCTF and to maximize the effect by promoting the
influx into the reaction solution, a restriction enzyme reaction
time can be further added during the PCR process. Reaction time,
reaction temperature, etc. can be applied differently depending on
the kind of the specific restriction enzyme and the reaction
intention.
[0056] In step (3), as the step that the generated CCTF is analyzed
through various analysis equipment to identify the target nucleic
acid sequence, when the mass of the generated CCTF is directly
analyzed, the kinds of CCTF are diversified through recombination
of length and sequence, Mass spectrometry such as MALDI-TOF MS, LC
MS and HPLC MS can be used to observe the intrinsic mass of the
generated CCTF, and the amplified target sequence can be identified
and identified using the said mass. It is preferable to observe it
through MALDI-TOF MS, the range of mass of CCTF which is easy to
observe is 1200 Da or more. The amplification products can be
observed by forming various CCTFs in the mass range as above.
[0057] The amplified target sequence can be identified by observing
the fluorescence signal of CCTF, and this is the method which
comprises hybridizing CCTF with SCO which is tagged with the
fluorescence and the quencher so that the generated CCTF can
provide the fluorescence signal at the inherent dissociation
temperature, and is the sequence complementary to the CCTF having
the inherent dissociation temperature, analyzing the fluorescence
signal at the inherent dissociation temperature, and confirming the
generation of CCTF, thereby identifying the presence of the target
nucleic acid sequence.
[0058] For the release of CCTF, as described above, the use
concentration of the restriction enzyme is designated according to
the purpose of use, and the kind of the polymerase is not related
to the A tailing unlike the mass analysis. The CCTF released from
the amplification product and introduced into the reaction solution
reacts with the SCO present in the reaction solution, wherein the
component of the SCO is as follows.
[0059] The complementary sequence of CCTF exists to enable
hybridization with CCTF from the 5'end to the 3' end and the
sequence of SCO is determined by CCTF length, sequence
recombination depending on CCTF. In order to diversify the kinds of
tags in step (1), the combination of the length and the sequence
may be designed differently to give the inherent dissociation
temperature of CCTF and SCO, such as in the case using the method
such the length of CCTF and the method of sequence recombination,
etc. In this case, the SCO is composed of a complementary sequence
of CCTF, and the fluorescent substance is contained in the
sequence, and the position of the fluorescent substance is possible
in anywhere at least a certain length apart from the quencher. At
the 3'end of the SCO, a blocker is positioned so that SCO serves as
a primer during the reaction to prevent the nucleotide sequence
from elongation. Spacer C3, Phosphat, ddC, Inverted END and
Quencher, etc. may be used as the blocker, but not limited thereto.
In particular, when the quencher is located at the 3'end of SCO,
the SCO is served as a primer during the reaction to prevent the
nucleotide sequence from elongation, and simultaneously hybridizes
with CCTF to suppress the emission of the fluorescent material by
the FRET phenomenon, before it forms a double strand with CCTF. By
using a quencher in combination with a substance preventing
nucleotide sequence elongation, an unnecessary modification
reaction can be shortened in the production of SCO, thereby
increasing the yield of the production reaction and further
reducing the manufacturing cost. By using the hybridization of CCTF
generated during the reaction with SCO contained in the reaction,
it can be identified as to whether CCTF is generated by identifying
the dissociation of the double strand with the fluorescence and
analyzing it to confirm whether CCTF is generated due to the target
sequence, and then the target sequence can be identified. The range
of temperature that can be defined by the inherent dissociation
temperature of the SCO is .about.95.degree. C., and if there is no
interference of the dissociation temperature of each double strand,
there is no limitation in defining the inherent dissociation
temperature for each fluorescent substance.
[0060] The combination of SCO's fluorophore and quencher can be
exemplified as Alexa Fluor 350, Alexa Fluor 405. Alexa Fluor 430.
Alexa Fluor 488, Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 546,
Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 610,
Alexa Fluor 633, Alexa Fluor 635, Alexa Fluor 647, Alexa Fluor 660,
Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750, Alexa Fluor 790,
ATTO 390, ATTO 425, ATTO 465, ATTO 488, ATTO 495, ATTO 514, ATTO
520, ATTO 532, ATO Rho6G, ATTO 540Q, ATTO 550. ATTO 565, ATTO
Rho3B, ATTO Rho11, ATTO Rho12, ATO Thio12, ATO 580Q, ATTO Rho101,
ATO 590, ATTO Rho13, ATTO 594, ATTO 610, ATTO 612Q, ATTO 620, ATTO
Rho14, ATO 633, ATTO 647, ATTO 647N, ATTO 655, ATTO Oxa12, ATTO
665, ATTO 680, ATTO 700, ATO 725, ATTO 740, ATTO MB2, AMCA, AMCA-S,
BODIPY FL, BODIPY R6G, BODIPY 530/550, BODIPY TMR, BODIPY 558/568,
BODIPY 564/570, BODIPY 576/589, BODIPY 581/591, BODIPY TR, BODIPY
630/650, BODIPY 650/665, Biosearch Blue, CAL Fluor Gold 540, CAL
Fluor Orange 560, CAL Fluor Red 590, CAL Fluor Red 610, CAL Fluor
Red 635, Pulsar 650, Quasar 570, Quasar 670, Quasar 705. FAM,
Fluorescein, Fluorescein-C3, Calcein, Carboxyrhodamine 6G,
Carboxy-X-rhodamine (ROX), Cascade Blue, Cascade Yellow, Cy2, Cy3,
Cy5, Cy3.5, Cy5.5, Cy7, Dansyl, Dapoxyl, Dialkylaminocoumarin,
4',5'-Dichloro-2',7'-dimethoxy-fluorescein, DM-NERF, Eosin,
Erythrosin, HEX, Hydroxycoumarin, IRD40, IRD 700, IRD 800, JOE,
Lissamine rhodamine B, LC Red 610, LC Red 640, Marina Blue,
Methoxycoumarin, Naphthofluorescein, NED, Oregon Green 488, Oregon
Green 500, Oregon Green 514, Pacific Blue, PyMPO, Pyrene,
Phycoerythrin, Rhodamine 6G, Rhodamine Green, Rhodamine Red, Rhodol
Green, 2',4',5',7'-Tetra-bromosulfonefluorescein,
Tetramethyl-rhodamine (TMR), Carboxytetramethylrhodamine (TAMRA),
Texas Red, Texas Red-X. TET, VIC, Yakima Yellow, BMN-Q460, DDQ-1,
Dabcyl, BMN-Q530, BMN-Q535, Eclipse, Iowa Black FQ, BHQ-1, TQ2,
IQ4, QSY-7, BHQ-2, TQ3, DDQ-II, BBQ-650, Iowa Black RQ, QSY-21,
BHQ-3, etc., and may also include any fluorescent material and
quencher.
[0061] In addition, the reaction between SCO and CCTF occurs
simultaneously with the amplification reaction and the CCTF
formation reaction, and in this case, by utilizing the fact that
the double strand formation ratio of SCO represents a similar
efficiency to the amplification amount of the target sequence, the
Ct graph of the SCO having the inherent dissociation temperature
can be made, and by using this, it is possible to identify the
target sequence in a different manner from the inherent
dissociation temperature analysis method.
[0062] The above content made by the solving means of the present
invention will be described in more detail as the most preferable
embodiment through the Examples of the present invention.
Advantageous Effects
[0063] In the method of the present invention, since an arbitrary
tag (CCTF) is generated and cleaved by restriction enzymes during
the amplification reaction, the double strand of the restriction
enzyme recognition sequence is not formed before the amplification
reaction of the artificial sequence (CTPO) added to form the tag
and thus, there is no possibility that it is randomly cleaved;
since the tags are generated only by the reaction products
specifically generated to the target sequence during PCR, the
method of the present invention has the high accuracy for forming
CCTF, and can obtain more delicate analysis results than the
existing PCR result analysis depending on the length of the PCR
amplification product or the specificity of the specific sequence;
and the method of the present invention can distinguish and
interpret amplification products specifically even if various kinds
of amplification products are produced in the same length. In
addition, since the analysis of the resultant CCTF can be applied
to most of the analysis using base sequence, the device for
interpretation can be selected and applied ordinarily. In
particular, the method of the present invention can be used in the
fields of diagnosis, etc., which require rapid multiple analysis
using an amplification reaction.
DESCRIPTION OF DRAWINGS
[0064] FIG. 1 is a representative diagram illustrating the
formation process of CTPO and CCTF used in a PCR reaction, and an
example for the analysis of CCTF, as a schematic diagram of CCTF
formation.
[0065] FIG. 2 shows the results of the formation of CCTF and MALDI
analysis in dual target PCR. CTPO was designed to form different
CCTFs for each target sequence, amplified, and analyzed by MALDI,
and as a result, a peak corresponding to the masses of CCTF 1
obtained by amplifying Neisseria gonorrhoeae (NG) and cleaving it
and CCTF2 obtained by amplifying Mycoplasma hominis (MH) and
cleaving it, were observed.
[0066] FIG. 3 shows the results of Real-time PCR Melting Peak
analysis for causative organisms of sexually transmitted diseases.
As the results representing the multiple target dissociation
temperature measurements to each target of Chlamydia
trachomatis(CT), Neisseria gonorrhea (NG) Mycoplasma hominis(MH),
Mycoplasma genitalium(MG), Trichomonas vaginalis(TV), Ureaplasma
urealyticum(UU), Ureaplasma parvum(UP), Candida albicans(CA),
Gardnerella vaginalis(GV), Herpes simplex virus 1 (HSV 1), Herpes
simplex virus 2(HSV 2), Treponema pallidum(TP) and Internal Control
(IC), the peak was observed at the inherent dissociation
temperature that each SCO has (CT:FAM 80.degree. C., NG:HEX
76.5.degree. C., MH:HEX 68.degree. C., MG:CaRed610 67.5.degree. C.,
TV:Quasar670 71.5.degree. C., UU:CalRed610 77.degree. C., UP:FAM
77.degree. C., CA:FAM 65.degree. C., GV:Quasar670 78.5.degree. C.,
HSV 1: Quasar705 73.5.degree. C., HSV 2:Quasar705 79.degree. C.,
TP:Quasar705 66.degree. C., IC:Quasar670 63.5.degree. C.)
(a)(b)(c)(d)(e)(f), and no peak of SCO that visualized CCTF was
observed when the target sequence was not added in the same
composition (g).
[0067] FIG. 4 shows the results of Real-time PCR Melting Peak
analysis for the causative organism of the gastrointestinal
diseases. As the results representing the multiple inherent
dissociation temperature measurements to each target of Rotavirus
A(RVA), Astrovirus(AstV), Adenovirus F40(AdV 40), Adenovirus
F41(AdV 41), Norovirus GI(NoV GI), Norovirus GII(NoV GII) and
External Control, the peak was observed at the inherent
dissociation temperature that each SCO has (RVA:HEX 78.degree. C.,
AstV:CalRed610 78.degree. C., AdV 40:CaRed610 67.degree. C., AdV
41:CaRed610 67.degree. C., NoV GI:FAM 68.degree. C., NoV GII:FAM
84.degree. C., EC:HEX 69.degree. C.) (a)(b)(c)(d), and no peak of
SCO that visualizes CCTF was observed when the target sequence was
not added in the same composition (e).
[0068] FIG. 5 shows the results of Real-time PCR Melting Peak
analysis for Human Papilloma Virus (HPV) detection. As a result of
multiple inherent dissociation temperature measurements of each
target of type 16, type 18, type 33, type 35, type 51, type 53,
type 59, type 68a, type 82 and IC, the peak was observed at the
inherent dissociation temperature that each SCO has (type 16: HEX
76.5.degree. C., type 18: FAM 78.degree. C., type 33: Quasar670
71.degree. C., type 35: Quasar670 71.degree. C., type 51: Quasar670
71.degree. C., type 53: Quasar670 71.degree. C., type 59: Quasar670
71VC, type 68a: Quasar670 71.degree. C., type 82: Quasar670
71.degree. C., IC: Quasar670 67.5.degree. C.) (a)(b)(c)(d), and no
peak of SCO that visualizes CCTF was observed when the target
sequence was not added in the same composition (e).
[0069] FIG. 6 shows the result of Real-time PCR Melting Peak
analysis for detection of respiratory disease-induced virus. As a
result of multiple inherent dissociation temperature measurements
of each target of Influenza A/H1N1(H1), Influenza A/H3N2(H3),
Influenza A/H1N1/2009pdm (2009pdm), Influenza B (Flu B),
Parainfluenza 1 (PIV 1), Parainfluenza 3 (PIV 3), Respiratory
syncytial virus A (RSV A), Respiratory syncytial virus B (RSV B),
Human metapneumovirus (MPV), Adenovirus (AdV), External control
(EC), the peak was observed at the inherent dissociation
temperature that each SCO has (H1: FAM 67.5.degree. C., H3: FAM
76.5.degree. C., 2009pdm: FAM 86.5.degree. C., Flu B: CalRed610
83.5.degree. C., PIV 1: Quasar670 66.degree. C., PIV 3: Quasar670
74.degree. C., RSV A: HEX 63.5.degree. C., RSV B: CalRed610
72.degree. C., MPV: HEX 86.degree. C., ADV: Quasar670 85.degree.
C., EC: CalRed610 68.5t) (a)(b)(c)(d)(e), and no peak of SCO that
visualizes CCTF was observed when the target sequence was not added
in the same composition (f).
[0070] FIG. 7 shows the results of Real-time PCR Melting Peak
analysis to analyze the genotype of rs6265, a single nucleotide
polymorphism of BDNF gene. As a result representing the multiple
inherent dissociation temperature measurements of each target of
mutant A/A, wild type G/G and heterozygote A/G, the peak was
observed at the inherent dissociation temperature that each SCO has
(A/A: 76.5.degree. C., A/G: 76.5.degree. C. 75.degree. C., G/G
75.degree. C., IC: 66.degree. C.) (a)(b)(c)(d), and no peak of SCO
that visualizes CCTF was observed when the target sequence was not
added in the same composition (e).
[0071] FIG. 8 shows the results of real-time PCR Ct graph. As a
result representing fluorescent amplification curves and standard
curves of SCO under the experimental condition of a multi-real-time
polymerization chain reaction experiment of Neisseria gonorrhea
(NG), Mycoplasma. hominis (MH), Ureaplasma. parvum (UP) in which
genomic DNA of each of the above causative organism was diluted by
10-folds from 100 pg/ul concentration, (a) graph shows the results
of fluorescence amplification curves plotted when three target
sequences are present at each concentration simultaneously, (b)
graph shows a negative result plotted when all three target
sequences are not included. When the standard curve is represented
by a single fluorescence amplification curve of the graph
corresponding NG of (a) graphs, it can be represented as (c) and
(d), and the graph corresponding to MG graph can be represented as
(e) and (f), and the curve corresponding to UP can be represented
as (g) and (h).
MODE FOR INVENTION
[0072] Hereinafter, the present invention will be described in
detail with reference to Examples. These examples are for
illustrative purposes only and thus, are not interpreted to limit
the scope of the present invention.
Example 1. Formation of CCTF and MALDI Analysis in Dual Target
PCR
[0073] This experiment was conducted to prove that the CCTF formed
during the PCR reaction for the detection of multiple target
sequences can be detected in a target-specific manner by analyzing
the mass using MALDI-TOF MS. In Example 1, the causative organism
of sexually transmitted diseases, DNAs of Neisseria. gonorrhoeae
(NG) and Mycoplasma. Hominis (MH) were used as the targets.
[0074] 1. Target Template DNA, and Primers Manufactured by Sequence
Specific Manner
[0075] The forward primers of NG and MH targeting in this example
were manufactured based on the method described in the Detailed
Description of the Invention as CTPO. The 5'end of the forward
primer was an arbitrary nucleotide sequence consisting of a
sequence non-complementary to the DNA of NG and MH so that it could
be used as a template of CCTF, and a restriction enzyme recognition
sequence was consecutively located thereon. The sequence after the
restriction enzyme recognition sequence up to the 3'end is composed
of a sequence complementary to the target region of the DNA of NG
and MH, and plays a role as a primer. In addition, the 5'end of
forward primer is composed of a different number of nucleotides
with each other and has a different mass value for each CCTF
generated, in order to design that the amplification products can
be distinguished from each other as the mass when CCTF is formed.
The reverse primer is consisted of a sequence complementary to the
target site of the DNA of NG and MH.
[0076] Primer information and target sequence information being
amplified and generated are as follows.
TABLE-US-00001 Primer 1: (SEQ ID. NO: 1) 5'-TGAACTAT*
TCCGACGTTTCGGTTGTGTTGAAACACCGCCCGG-3' Primer 2: (SEQ ID. NO: 2)
5'-GCTCCTTATTCGGTTTGACCGG-3' Primer 3: (SEQ ID. NO: 3)
5'-ATCTATGATA* TTTAGCTCCTATTGCCAACGTATTGG-3' Primer 4: (SEQ ID. NO:
4) 5'-TGTGTGGAGCATCTTGTAATCTTTGGTC-3'
[0077] Amplified product 1: GenBank: CP012028.1/Position
(start-end): 251416-251506
TABLE-US-00002 (SEQ ID. NO: 5) 5'-TGAACTAT*
TCCGACGTTTCGGTTGTGTTGAAACACCGCC
CGGAACCCGATATAATCCGCCCTTCAACATCAGTGAAAATCTTTTTTTTA
ACCGGTCAAACCGAATAAGGAGC-3'
[0078] Amplified product 2: GenBank: AJ243692.1/Position
(start-end): 835-944
TABLE-US-00003 (SEQ ID NO: 6) 5' ATCTATGATA*
TTTAGCTCCTATTGCCAACGTATTGGAAA AAAACTTTGGTATTGAAAAAGGATT
TATGACAACAGTCCACTCATATACAGCAGACCAAAGATTACAAGATGCTC CACACA-3'
[0079] The bold and slanted font of the Primer sequence means the
restriction enzyme recognition sequence, and the underline is the
complementary sequence of the CCTF produced thereby. In the
examples of the present invention, the part represented by * is a
tag that modified dCTP was inserted into C in the recognition
sequence to block the site cleaved by the PspGI restriction
enzyme.
[0080] The sequence and mass of the CCTF produced in the amplified
product are as follows.
TABLE-US-00004 (SEQ ID. NO: 7) CCTF 1: 5'-CCAGGATAGTTCA-3'/4038.6
Da (SEQ ID NO: 8) CCTF 2: 5'-CCAGGTATCATAGAT-3'/4351.8 Da
[0081] 2. PCR Amplification
[0082] Primer 1 and Primer 3 as forward primers, and Primer 2 and
Primer 4 as reverse primers were subjected and PCR reaction was
performed simultaneously, and then, the formation of CCTF was
determined.
[0083] 20 Of the total reaction solution comprising each Primer 3
.mu.M, PspGI (NEB, USA) 2U, PCR buffer (1.times.), MgSO.sub.4 3 mM,
dNTP 400 .mu.M, Vent Polymerase (NEB, USA) 1 U and NG, MH template
DNA 100 pg/ul was subjected to PCR reaction using C1000 PCR
(Bio-Rad, USA) under the following conditions:
[0084] 94.degree. C. 10 mins,
[0085] 94.degree. C. 30 secs, 62.degree. C. 30 secs 72.degree. C.
30 secs (35 cycles),
[0086] 85.degree. C. 2.5 hours
[0087] 3. Purification and Desalting of the Cleaved Fragments
During the PCR Reaction
[0088] Oasis (Waters) C18 reverse phase column chromatography was
used to isolate the DNA fragments cleaved by treatment with a
restriction enzyme during the PCR reaction from the above solution.
To the solution treated with the restriction enzyme, 70 .mu.l of
0.15 M triethylammonium acetate (TEAA, pH 7.6) was added and
allowed to stand for 1 minute. Resin was activated by passing 1 ml
of 100% acetonitrile (ACN; Sigma, USA) and 0.1 M TEAA to the
column, and then, 100 s of a mixed solution of the solution treated
with the restriction enzyme and 0.15M TEAA, 2 ml of 0.1M TEAA and 1
ml of the third distilled water were passed through in this order.
The column was placed on a Collection Plate and 100 .mu.l of 70%
ACN was passed. When the eluate was collected on the collection
plate, the collection plate was dried at 120.degree. C. for 60
minutes.
[0089] 4. MALDI-TOF MS Analysis
[0090] 4 .mu. of MALDI matrix [22.8 mg ammonium citrate, 148.5 mg
hydroxypicolinic acid, 1.12 m acetonitrile, 7.8 m H.sub.2O] was
previously dotting on Anchor chip plate of MALDI-TOF mass
spectrometry (Biflex IV, Bruker), and then, was dried at 37.degree.
C. for 30 minutes. 10 .mu.l of the third distilled water was
dissolved in a sample of the collection plate after the
purification and desalting procedure, and 2 .mu.l of the solution
was dropped onto the dried MALDI Matrix, the Maldin Matrix was
dried again at 37.degree. C. for 30 minutes, and then was analyzed
by MALDI-TOF mass spectrometry. The analysis method follows the
manual of the MALDI-TOF mass spectrometry.
[0091] The result of analyzing the CCTF produced by the above
reaction using a mass spectrometer is as shown in FIG. 2. From the
result of FIG. 2, it can be confirmed the peaks of 4083 Da, the
mass of CCTF 1 which can be formed when performing PCT with the
combination of Primer 1 and Primer 2, and 4351 Da, the mass of CCTF
2 which can be formed when performing PCT with the combination of
Primer 3 and Primer 4 (a). These results demonstrated that the PCR
amplification product can be analyzed using CCTF formed by CTPO,
and that CCTF can be used to accurately amplify and differentiate
the target sequence in the reaction product comprising various
primers.
[0092] Therefore, it was demonstrated that the target nucleic acid
sequence can be detected more precisely than the conventional PCR
method by performing the PCR using the CCTF marking technique and
distinguishing the tag fragments of various lengths through mass
analysis using MALDI-TOF MS after performing PCR.
Example 2. Formation of CCTF and Analysis of Inherent Dissociation
Temperature Peak of CCTF in Multiple Target PCR
[0093] The CCTF generated during the PCR reaction is combined with
the SCO capable of generating a fluorescence signal at the inherent
dissociation temperature to form an intrinsic dissociation
temperature peak, which can be observed directly after the PCR
process using a real-time PCR instrument. During the PCR reaction,
CCTF is formed, and at the same time it is hybridized with the CCTF
complementary sequence region of SCO to form a double strand. By
measuring the inherent dissociation temperature of SCO seen when
the double strand is dissociated into a single strand, the kinds of
CCTF can be discriminated and analyzed simultaneously with PCR
through a real-time PCR instrument. The SCO used in this example
used different fluorescent reporters, respectively, and the
inherent dissociation temperature was adjusted to enable
discrimination of CCTF.
[0094] In this example, CCTF analysis was performed using a
real-time PCR instrument using 12 kinds of the causative organisms
of the sexually transmitted diseases, 5 types of the causative
organisms of gastrointestinal diseases, 9 types of HPV subtypes, 10
types of the causative organisms of the respiratory disease and
single base mutation rs6265 nucleic acid of BDNF gene,
respectively.
[0095] 1. Formation of CCTF in multi-target PCR of the causative
organisms of the sexually transmitted diseases and analysis of the
inherent dissociation temperature peak of CCTF
[0096] CCTF analysis for Chlamydia trachomatis(CT), Neisseria.
gonorrhea (NG) Mycoplasma hominis(MH), Mycoplasma genitalium(MG),
Trichomonas vaginalis(TV), Ureaplasma urealyticum(UU), Ureaplasma
parvum(UP), Candida albicans(CA), Gardnerella vaginalis(GV), Herpes
simplex virus 1 (HSV 1), Herpes simplex virus 2 (HSV 2), Treponema
pallidum (TP), the causatives agents of sexually transmitted
diseases and Internal control (IC) DNA was performed using
Real-time PCR instrumentation.
[0097] 1) Primer for Target Sequence Template DNA Constructed by
the Sequence-Specific Manner
[0098] The forward primer used in this example was CTPO and was
constructed on the same principle as in Example 1 above. The 5'end
of CTPO was composed of 19-20 mers of nucleotide sequences, and was
composed of a sequence non-complementary to DNA of the target
sequence to form CCTF. The restriction enzyme recognition sequence
was then located, and from this up to the 3' end, it was composed
of the sequence complementary to each target site was composed to
play a role as a primer. The reverse primer was composed of
sequence complementary to the target site to be amplified.
[0099] In addition, SCO, which forms a complementary bond with CCTF
to be a double-stranded template, was positioned by positioning
fluorescent offsetting molecules (BHQ-1 or BHQ-2), and the
fluorescent reporter molecular was positioned so as to have a
certain distance.
[0100] Primer information and target sequence information which is
amplified and generated are as follows
TABLE-US-00005 Primer 5: (SEQ ID. NO: 9) 5'-
CCACTCCAGCCGGCTGACA*CCAGGACTTGGTGTGACGCTATCAGCAT- 3' Primer 6: (SEQ
ID. NO: 10) 5'-GTTTTCAAAACACGGTCGAAAACAAAGTC-3' Primer 7: (SEQ ID.
NO: 11) 5'- CATCGCCACGAGCCGGTTAA*CCAGGTTGAAACACCGCCCGGAACCC-3'
Primer 8: (SEQ ID. NO: 12) 5'-GCTCCTTATTCGGTTTGACCGGT-3' Primer 9:
(SEQ ID. NO: 13) 5'-
ACTCACGCTAATGGAGCGCA*CCAGGTTTAGCTCCTATTGCCAACGTATT GG-3' Primer 10:
(SEQ ID. NO: 14) 5'-TGTGTGGAGCATCTTGTAATCTTTGGTC-3' Primer 11: (SEQ
ID. NO: 15) 5'- GCTACCCAGCCGGCTACAAG*CCAGGCTTTATGGTGCTTATATTGGTGGC
ATG-3' Primer 12: (SEQ ID. NO: 16) 5'-CTGTATAACGTTGTGCAGCAGGTC-3'
Primer 13: (SEQ ID. NO: 17) 5'-
TGCCGCGTGATTCGATCCCA*CCAGGTATGTCCGGCACAACATGCGCT- 3' Primer 14:
(SEQ ID. NO: 18) 5'-GAGGCTTACGAAGGTCGGAGTTGA-3' Primer 15: (SEQ ID.
NO: 19) 5'- TCTCATAGCTGGGCCGCTG*CCAGGAAGTAGCATATGATGAAGCACACAA
CA-3' Primer 16: (SEQ ID. NO: 20) 5'-TAATGCAACGTGCATTTGCTTCAAC-3'
Primer 17: (SEQ ID. NO: 21) 5'-
CAGATCGTTGGCACTCTGCGA*CCAGGTTAAAGTAGCATATGATCAAGCT CATTCA-3' Primer
18: (SEQ ID. NO: 22) 5'-TTGTAATGATACAACGAGCATCATCATTAAT-3' Primer
19: (SEQ ID. NO: 23) 5'-
GCTCGTATGCCGCTCCATATA*CCAGGCCAAATCTGGATCTTCCTCTGCA TC-3' Primer 20:
(SEQ ID. NO: 24) 5'-GAGCTTGAGCTGGACCCAGAG-3' Primer 21: (SEQ ID.
NO: 25) 5'- ACGTGCCGTGCATCGTTGCA*CCAGGCAACCGGCTCCATTTTGGTGGAG- 3'
Primer 22: (SEQ ID. NO: 26) 5'-CGTCACGTCCTTCATCGGTCC-3' Primer 23:
(SEQ ID. NO: 27) 5'-
TCGCAGTCCCGTCGAGGAA*CCAGGAGGCCTGGCTATCCGGAGAAAC-3' Primer 24: (SEQ
ID. NO: 28) 5'-CGTTGTGTTGGCCGCAGGTC-3' Primer 25: (SEQ ID. NO: 29)
5'- CTCATAGCTAGGCGCCTG*CCAGGGCTGCACGTGGGTCTGTTGTG-3' Primer 26:
(SEQ ID. NO: 30) 5'-GGAAACGCAGGCCACGAAACC-3' Primer 27: (SEQ ID.
NO: 31) 5'-GCTTCGCGTCTCAGGCCTGT*CCAGGGGGCATTACAGTTTTGCGTCA TGAC-3'
Primer 28: (SEQ ID. NO: 32) 5'-CAAGTCTGAGCACTTGCACCG-3' Primer 29:
(SEQ ID. NO: 33) 5'-
CTGTTAGCTCTGCGAGCT*CCAGGGGAGCGACACTTGTTGGTGTTGAC- 3' Primer 30:
(SEQ ID. NO: 34) 5'-TGATGAAATGAAGCCACCCGTGC-3' SCO 1: (SEQ ID. NO:
35) TCGGAGCCAGCGCGGCGTAAAC[T(FAM)]CCACTCCAGCCGGCTGACA [BHQ1] SCO 2:
(SEQ ID. NO: 36) TACAACAGCAGTACGGAGACGAC[T(HEX)]CATCGCCACGAGCCGGTTA
A[BHQ1] SCO 3: (SEQ ID. NO: 37)
ATTTATTCTTACTCGATGTTAAA[T(HEX)]ACTCACGCTAATGGAGCGC A[BHQ1] SCO 4:
(SEQ ID. NO: 38) TATATATATATATTATTATAAA[T(CalRed610)]GCTACCCAGCCGGC
TACAAG[BHQ2] SCO 5: (SEQ ID. NO: 39)
AAGAATAACTACTACAATCTACT[T(Quasar670)]TGCCGCGTGATTC GATCCCA[BHQ2]
SCO 6: (SEQ ID. NO: 40)
TTATTATTATTATTATTATATA[T(CalRed610)]TCTCATAGCTGGGC CGCTG[BHQ2] SCO
7: (SEQ ID. NO: 41)
AATCTTCAATGCTTACCGTA[T(FAM)]CAGATCGTTGGCACTCTGCGA [BHQ1] SCO 8:
(SEQ ID. NO: 42) AAAATAAATAATATAATATA[T(FAM)]GCTCGTATGCCGCTCCATATA
[BHQ1] SCO 9: (SEQ ID. NO: 43)
TCGGAGCCAGCGCGGCGTAACG[T(Quasar670)]ACGTGCCGTGCATC GTTGCA[BHQ2] SCO
10: (SEQ ID. NO: 44)
AAGAATAACTACTACAATCTAC[T(Quasar705)]TTCGCAGTCCCGTC GAGGAA[BHQ2] SCO
11: (SEQ ID. NO: 45)
TCGGAGCCAGCGCGGCGTAA[T(Quasar705)]CTCTCATAGCTAGGCG CCTG[BHQ2] SCO
12: (SEQ ID. NO: 46)
AAAATAAATAATATAATATAG[T(Quasar705)]CTTCGCGTCTCAGGC CTGT[BHQ2] SCO
13: (SEQ ID. NO: 47)
AAAATAAATAATATAATATA[T(Quasar670)]TCTGTTAGCTCTGCGA GCT[BHQ2]
Amplified product 3: GenBank: X52557.1/Position (start-end):
157-227
TABLE-US-00006 (SEQ ID. NO: 48) CCACTCCAGCCGGCTGACA*
ACTTGGTGTGACGCTATCAGCAT
GCGTATGGGTTACTATGGTGACTTTGTTTTCGACCGTGTTTTGAAAAC
Amplified product 4: GenBank: X52364.1/Position (start-end):
375-459
TABLE-US-00007 (SEQ ID. NO: 49) CGCCCACCGCATCCCGCGCCCCTCCCTCAGCA*
TTGAAACACC GCCCGGAACCCGATATAATCCGCCCTTCAACATCAGTGAAAATCTTTTTT
TAACCGGTCAAACCGAATAAGGAGC
Amplified product 5: GenBank: AJ243692.1/Position (start-end):
835-944
TABLE-US-00008 (SEQ ID. NO: 50) ACTCACGCTAATGGAGCGCA*
TTTAGCTCCTATTGCCAACGTA
TTGGAAAAAAACTTTGGTATTGAAAAAGGATTTATGACAACAGTCCACTC
ATATACAGCAGACCAAAGATTACAAGATGCTCCACACA
Amplified product 6: GenBank: U09251.1/Position (start-end):
3462-3687
TABLE-US-00009 (SEQ ID. NO: 51) GCTACCCAGCCGGCTACAAG*
CTTTATGGTGCTTATATTGGTG
GCATGCACCATGATCGTCCTTTTAAAAAGTCTGCGAGGATTGTTGGTGAT
GTAATGAGTAAATTCCACCCTCATGGTGATATGGCAATATATGACACCAT
GTCAAGAATGGCTCAAGACTTTTCATTAAGATACCTTTTAATTGATGGTC
ATGGTAATTTTGGTTCTATAGATGGTGATAGACCTGCTGCACAACGTTAT ACAG
Amplified product 7: GenBank: XM_001582993.1/Position (start-end):
705-768
TABLE-US-00010 (SEQ ID. NO: 52) TGCCGCGTGATTCGATCCCA*
TATGTCCGGCACAACATGCGCT
TATGTCCGGCACAACATGCGCTCTCCGCTTCCCAGGTCAGCTCAACTCCG
ACCTTCGTAAGCTC
Amplified product 8: GenBank: AF085700.2/Position (start-end):
4673-4873
TABLE-US-00011 (SEQ ID. NO: 53) TCTCATAGCTGGGCCGCTG*
AAGTAGCATATGATGAAGCACAC
AACAAAATGGCGCATACTGTGTATTTCACTAATTTCTATCGTTCATCAAA
ACCACTATTTTTAGATGAAGAAGACCCAATTAATCCCTGTTTTCAAACTA
TTAGTATGGGTGGGGGTTATGTATCTGGTGAAGTGTATCGTTCTGATTTT
GAAGTTGAAGCAAATGCACGTTGCATTA
Amplified product 9: GenBank: AF085733.2/Position (start-end):
4677-4886
TABLE-US-00012 (SEQ ID. NO: 54) CAGATCGTTGGCACTCTGCGA*
TTAAAGTAGCATATGATCAAG
CTCATTCAAAAATGGCACATACTGTCTATTTTACGAATTTTTATCGTTCA
TCTAAACCTTTATTTTTAGATGAAGAAGATCCAATCAACCCCTGTTTTCA
AACAATTAGTATGGGTGGTGGATATGTTTCAGGTGAAATTTATCGTTCTG
ATTTTGAAATTAATGATGATGCTCGTTGTATCATTACAA
Amplified product 10: GenBank: M90812.1/Position start-end):
1736-1811
TABLE-US-00013 (SEQ ID. NO: 55) GCTCGTATGCCGCTCCATATA*
CCAAATCTGGATCTTCCTCTG
CATCTGCTTCTGGATCATCAAGCAGCAGCACCAGCTCTGGGTCCAGCTCA AGCTC
Amplified product 11: GenBank: L08167.1/Position (start-end):
273-434
TABLE-US-00014 (SEQ ID. NO: 56) ACGTGCCGTGCATCGTTGCA*
CAACCGGCTCCATTTTGGTGGA
GTCGCTTGATCGTTTTGTGATCGTTTAGTGTGATGATTTATTATGTCTAG
AGAGTTAAGCGATAGGCTTTTACTGGTGTATCACTGTAAGGGCGTATTGG
TTGGATGCCTTGGTAGACAGGACCGATGAAGGACGTGACG
Amplified product 12:DQ889502.1/Position (start-end):
123860-124007
TABLE-US-00015 (SEQ ID. NO: 57) TCGCAGTCCCGTCGAGGAA*
AGGCCTGGCTATCCGGAGAAACA
GCACACGACTTGGCGTTCTGTGTGTCGCGATGTCTCTGCGCGCAGTCTGG
CATCTGGGGCTTTTGGGAAGCCTCGTGGGGGCTGTTCTTGCCGCCACCCA
TCGGGGACCTGCGGCCAACACAACG
Amplified product 13: GenBank: EU018100.1/Position (start-end):
561-746
TABLE-US-00016 (SEQ ID. NO: 58) CTCATAGCTAGGCGCCTG*
GCTGCACGTGGGTCTGTTGTGGGT
AGAGGTGGGCGGGGAGGGCCCCGGCCCCACCGCCCCCCCCACAGGCGGCG
CGTGCGGAGGGCGGCCCGTGCGTCCCCCCGGTCCCCGCGGGCCGCCCGTG
GCGCTCGGTGCCCCCGGTATGGTATTCCGCCCCCAACCCCGGGTTTCGTG GCCTGCGTTTCC
Amplified product 14: GenBank: U57757.1/Position (start-end):
910-1067
TABLE-US-00017 (SEQ ID. NO: 59) GCTTCGCGTCTCAGGCCTGT*
GGGCATTACAGTTTTGCGTCAT
GACGGCTTTGAAGCTGACGACCTCATTGCAACCCTAGCAAAACGAGTTGC
GGCTGAGCACTGTCATGTTGTGATTATCTCCTCAGATAAAGATGTACTTC
AGCTTGTGTGTGATACGGTGCAAGTGCTCAGACTTG
Amplified product 15: GenBank: NM 001035551.2/Position (start-end):
214-369
TABLE-US-00018 (SEQ ID. NO: 60) CTGTTAGCTCTGCGAGCT*
GGAGCGACACTTGTTGGTGTTGACA
AGTTCGGTAACAAATACTACCAGAAGCTAGGCGATACTCAATACGGTATG
CACAGATGGGTAGAGTATGCTTCAAAGGATCGTTACAACGCATCTCAAGT
ACCAGCTGAATGGCACGGGTGGCTTCATTTCATCA
[0101] The bold and slanted font of the Primer sequences means the
restriction enzyme recognition sequence, and the underline is the
complementary sequence of the CCTF produced thereby. the part
represented by * is a tag that modified dCTP was inserted into C in
the recognition sequence to block the site cleaved by the PspGI
restriction enzyme. In SCO, the parentheses mean the position of
the nucleotide sequence in which the fluorescent offsetting
molecule and the fluorescent reporter are located. The sequence of
the CCTF produced from the amplified product is as follows.
TABLE-US-00019 CCTF 3: (SEQ ID. NO: 61) 5'-
CCTGGTGTCAGCCGGCTGGAGTGG -3' CCTF 4: (SEQ ID. NO: 62) 5'-
CCTGGTTAACCGGCTCGTGGCGATG -3' CCTF 5: (SEQ ID. NO: 63) 5'-
CCTGGTGCGCTCCATTAGCGTGAGT -3' CCTF 6: (SEQ ID. NO: 64) 5'-
CCTGGCTTGTAGCCGGCTGGGTAGC -3' CCTF 7: (SEQ ID. NO: 65) 5'-
CCTGGTGGGATCGAATCACGCGGCA -3' CCTF 8: (SEQ ID. NO: 66) 5'-
CCTGGCAGCGGCCCAGCTATGAGA -3' CCTF 9: (SEQ ID. NO: 67) 5'-
CCTGGTCGCAGAGTGCCAACGATCTG -3' CCTF 10: (SEQ ID. NO: 68) 5'-
CCTGGTATATGGAGCGGCATACGAGC -3' CCTF 11: (SEQ ID. NO: 69) 5'-
CCTGGTGCAACGATGCACGGCACGT -3' CCTF 12: (SEQ ID. NO: 70) 5'-
CCTGGTTCCTCGACGGGACTGCGA -3' CCTF 13: (SEQ ID. NO: 71) 5'-
CCTGGCAGGCGCCTAGCTATGAG -3' CCTF 14: (SEQ ID. NO: 72) 5'-
CCTGGACAGGCCTGAGACGCGAAGC -3' CCTF 15: (SEQ ID. NO: 73) 5'-
CCTGGAGCTCGCAGAGCTAACAG -3'
[0102] 2) PCR Amplification and Determination of SCO Inherent
Dissociation Temperature [0103] PCR reaction was performed using
the following CFX96 Real-time PCR (Bio-Rad, USA) with 20 .mu. of
total reaction solution of each of Primer 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,
29, 30 and SCO 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 prepared
by adding 0.15 .mu.M, PspGI (NEB, USA) 51, PCR buffer (1.times.),
MgCl.sub.2 2.5 mM, dNTP 200 .mu.M, h-Taq DNA polymerase (Solgent,
Korea) 1.6 U, template DNA of genomic DNA of CT, NG, MH, MG, TV,
UU, UP, CA, GV, HSV1, HSV2, TP and IC 100 pg/rxn, respectively.
[0104] 95.degree. C. 15 mins,
[0105] 95.degree. C. 30 secs, 63.degree. C. 1 min (50 cycles).
[0106] A reaction was performed using a cycle at the denaturation
temperature of 95.degree. C. for 15 minutes once, and with a cycle
at the denaturation temperature of 95.degree. C. for 30 seconds,
and an annealing temperature of 63.degree. C. for 1 minute 50
times. After the reaction, the reaction mixture was cooled to
50.degree. C. in the same apparatus, held at 50.degree. C. for 30
seconds, and then slowly heated from 50.degree. C. to 95.degree. C.
to obtain an inherent dissociation temperature analysis curve. Data
analysis was performed with Bio-Rad CFX Manager 1.6.
[0107] FIG. 3, (a) shows the results of multiple inherent
dissociation temperature measurements for causative organisms of
CT, NG, MH, MG, TV, UU, UP, CA, GV, HSV1, HSV2, TP, IC. The peak
was observed at the inherent dissociation temperature that each SCO
has (CT: FAM 80.degree. C., NG: HEX 76.5.degree. C., MH: HEX
68.degree. C., MG: CaRed610 67.5.degree. C., TV: Quasar670
71.5.degree. C., UU: CaRed610 77.degree. C., UP: FAM 77.degree. C.,
CA: FAM 65.degree. C., GV: Quasar670 78.5.degree. C., HSV 1:
Quasar705 73.5.degree. C., HSV 2: Quasar705 79.degree. C., TP:
Quasar705 66.degree. C., IC: Quasar670 63.5.degree. C.)
(a)(b)(c)(d)(e)(f), and no peak of SCO visualizing CCTF was
observed when the target sequence was not added in the same
composition (g).
[0108] Therefore, it was proved that the target nucleic acid
sequence can be detected more quickly and simply than the
conventional PCR method by analyzing the fluorescence of the SCO
using the real-time PCR instrument, simultaneously with the PCR
using the marking technique of CCTF.
[0109] 2. Formation of CCTF in Multi-Target PCR of the Causative
Organism for the Gastrointestinal Diseases and Analysis for the
Inherent Dissociation Temperature Peak of CCTF
[0110] CCTF analysis was performed with Real-time PCR instrument
for DNA of the causative organisms of the gastrointestinal
diseases, Rotavirus A(RVA), Astrovirus(AstV), Adenovirus F40(AdV
40), Adenovirus F41(AdV 41), Norovirus GI(NoV GI), Norovirus
GII(NoV GII), and External control (EC).
[0111] 1) Primer for the Target Sequence of Template DNA
Constructed in the Sequence-Specific Manner
[0112] The forward primer used in this example was CTPO and was
constructed on the same principle as in Example 1 above. The 5'end
of CTP was composed of 19-20 mers of nucleotide sequences, and was
composed of a sequence non-complementary to DNA of the target
sequence so as to form CCTF. The restriction enzyme recognition
sequence was consecutively located, and after this up to the 3'
end, it was composed of the sequence complementary to each target
site to play a role as a primer. The reverse primer was composed of
sequence complementary to the target site to be amplified.
[0113] In addition, SCO, which forms a complementary bond with CCTF
to be a double-stranded template, was positioned by positioning
fluorescent offsetting molecules (BHQ-1 or BHQ-2), and the
fluorescent reporter molecular was positioned so as to have a
certain distance.
[0114] Primer information and target sequence information which is
amplified and generated are as follows.
TABLE-US-00020 Primer 31: (SEQ ID. NO: 74)
5'-GCAGGAGCCTCTCATCTCG*CCAGGCTCATTTATAGACARCTTCTCA CTAATTC-3'
Primer 32: (SEQ ID. NO: 75) 5'-AGTTTTTTCTGATCCAATYTGYTCTATTTC-3
Primer 33: (SEQ ID. NO: 76)
5'-TCAGACGGTTCGAGGCTCC*CCAGGARGATYAAGCGTGGAGTATAYA TGG-3' Primer
34: (SEQ ID. NO: 77) 5'-TTTGCGTGCYTCTTCACACGC-3' Primer 35: (SEQ
ID. NO: 78) 5'-AACGCGAATCGACCGGAT*CCAGGCGCGATGTGTTTGCCGATAAAA C-3'
Primer 37: (SEQ ID. NO: 79) 5'-CATTGCGTCTGCCECACTTG-3' Primer 38:
(SEQ ID. NO: 80) 5'-AACGCGAATCGACCGGAT*CCAGGAAACAAGAACACCTATGCCTACA
TGAAC-3' Primer 39: (SEQ. ID. NO: 81)
5'-ATGTTAACGTCCTTCCTGAAGTTCCAC-3 Primer 40: (SEQ ID. NO: 82)
5'-TAGATCGGACTGCGAATCG*CCAGGGAGATCGCRATCTYCTGCCCGA -3 Primer 41:
(SEQ ID. NO: 83) 5'-RGCGTCCTTAGACGCCATCATC-3 Primer 42: (SEQ ID.
NO: 84) 5'-ATCTACAGCGTCGCATCACG*CCAGGCGCAATCTGGCTCCCARTTTT GTG-3
Primer 43: (SEQ ID. NO: 85) 5'-GCGTCAYTCGACGCCATCYTCA-3 Primer 44:
(SEQ ID. NO: 86) 5'-CATAGGTCGAGGTCCTCAC*CCAGGGCAAACTCCGGCATCTACTAAT
AGACG-3 Primer 45: (SEQ ID. NO: 87) 5'-AAGCGGTGATCCGCACAGTG-3 SCO
14: (SEQ ID. NO: 88)
TCGGCCGATCGTCCATAGAGTCAAGC[T(HEX)]CGCAGGAGCCTCTCAT CTCG[BHQ1] SCO
15: (SEQ ID. NO: 89)
TCACGATGAGCGAGTTGAGCTACG[T(Calred610]ATCAGACGGTTCG AGGCTCC[BHQ2]
SCO 16: (SEQ ID. NO: 90)
TGTTCAATATATAATGATAATATG[T(Calred610)]AACGCGAATCGA CCGGAT[BHQ2] SCO
17: (SEQ ID. NO: 91)
TGTTCAATATATAATGATAATATG[T(Calred610)]AACGCGAATCGA CCGGAT[BHQ2] SCO
18: (SEQ ID. NO: 92)
ACATTTATAATACAGTATTTTA[T(FAM)]TAGATCGGACTGCGAATCG [BHQ1] SCO 19:
(SEQ ID. NO: 93) AGCTCCTGCCAGTACTGCCATCCA[T(FAM)]ATCTACAGCGTCGCATCA
CG[BHQ1] SCO 20: (SEQ ID. NO: 94)
TAGTTATAATGAATAACTATTAT[T(HEX)]CATAGGTCGAGGTCCTCA C[BHQ1]
Amplified product 16: GenBank: KT694942.1/Position (start-end):
19-99
TABLE-US-00021 (SEQ ID NO: 95) GCAGGAGCCTCTCATCTCG *
CTCATTTATAGACARCTTCTCACT
AATTCATATTCAGTAGATTTACATGATGAAATAGARCARATTGGATCAGA AAAAACT
Amplified product 17: GenBank: AB000287.1/Position (start-end):
2232-2321
TABLE-US-00022 (SEQ ID NO: 96) TCAGACGGTTCGAGGCTCC *
ARGATYAAGCGTGGAGTATAYATG
GACCTGCTTGTCTCGGGGGCAAGCCCAGGCAATGCATGGTCCCATGCGTG
TGAAGARGCACGCAAA
Amplified product 18: GenBank: KM274923.1/Position (start-end):
121-179
TABLE-US-00023 (SEQ. ID NO: 97) AACGCGAATCGACCGGAT*
CGCGATGTGTTTGCCGATAAAACGT CACAACCGGAGCCCCAAGTGGGGCAGACGCAATG
Amplified product 19: GenBank: AB330122.1/Position (start-end):
1407-1691
TABLE-US-00024 (SEQ ID. NO: 98) AACGCGAATCGACCGGAT *
AAACAAGAACACCTATGCCTACATG
AACGGTCGGGTGGCGGTTCCTAGCGCCCTCGATACCTACGTAAACATCGG
GGCACGGTGGTCTCCAGATCCCATGGACAATGTTAACCCCTTCAATCACC
ACCGTAACGCCGGTCTGCGCTATCGATCCATGCTCTTUGGCAACGGGCGT
TACGTACCCTTCCACATTCAAGTCCCCCAGAAGTTTTTTGCCATTAAAAA
TCTCCTCCTCTTACCGGGTTCCTACACCTACGAGTGGAACTTCAGGAAGG ACGTTAACAT
Amplified product 20: GenBank: LN854564.1/Position (start-end):
5325-5378
TABLE-US-00025 (SEQ ID NO: 99) TAGATCGGACTGCGAATCG *
GAGATCGCRATCTYCTGCCCGAAT TCGTAAATGATGATGGCGTCTAAGGACGCY
Amplified product 21: GenBank: KT202798.1/Position (start-end):
5060-5107
TABLE-US-00026 (SEQ ID. NO: 100) ATCTACAGCGTCGCATCACG *
CGCAATCTGGCTCCCARTTTTGT GAATGARGATGGCGTCGARTGACGC
Amplified product 22: GenBank: EF204940.1/Position (start-end):
1707-1878
TABLE-US-00027 (SEQ ID. NO: 101) CATAGGTCGAGGTCCTCAC*
GCAAACTCCGGCATCTACTAATAG
ACGCCGGCCATTCAAACATGAGGATTACCCATGTCGAAGACAACAAAGAA
GTTCAACTCTTTATGTATTGATCTTCCTCGCGATCTTTCTCTCGAAATTT
ACCAATCAATTGCTTCTGTCGCTACTGGAAGCGGTGATCCGCACAGTG
[0115] The bold and slanted font of the Primer sequence means the
restriction enzyme recognition sequence, and the underline is the
complementary sequence of the CCTF produced thereby. the part
represented by * is a tag that modified dCTP was inserted into C in
the recognition sequence to block the site cleaved by the PspGI
restriction enzyme. In SCO, the parentheses mean the position of
the nucleotide sequence in which the fluorescent offsetting
molecule and the fluorescent reporter are located. The sequence of
the CCTF produced from the amplified product is as follows.
TABLE-US-00028 CCTF 16: (SEQ ID. NO: 102) 5'-
CCTGGTGTCAGCCGGCTGGAGTGG3' CCTF 17: (SEQ ID. NO: 103) 5'-
CCTGGTTAACCGGCTCGTGGCGATG3' CCTF 18: (SEQ ID. NO: 104) 5'-
CCTGGTGCGCTCCATTAGCGTGAGT3' CCTF 19: (SEQ ID. NO: 105) 5'-
CCTGGCTTGTAGCCGGCTGGGTAGC -3' CCTF 20: (SEQ ID. NO: 106) 5'-
CCTGGTGGGATCGAATCACGCGGCA -3' CCTF 21: (SEQ ID. NO: 107) 5'-
CCTGGCAGCGGCCCAGCTATGAGA -3' CCTF 22: (SEQ ID. NO: 108) 5'-
CCTGGTCGCAGAGTGCCAACGATCTG -3' CCTF 23: (SEQ ID. NO: 109) 5'-
CCTGGTATATGGAGCGGCATACGAGC -3' CCTF 24: (SEQ ID. NO: 110) 5'-
CCTGGTGCAACGATGCACGGCACGT -3' CCTF 25: (SEQ ID. NO: 111) 5'-
CCTGGTTCCTCGACGGGACTGCGA -3' CCTF 26: (SEQ ID. NO: 112) 5'-
CCTGGCAGGCGCCTAGCTATGAG -3' CCTF 27: (SEQ ID. NO: 113) 5'-
CCTGGACAGGCCTGAGACGCGAAGC -3' CCTF 28: (SEQ ID. NO: 114) 5'-
CCTGGAGCTCGCAGAGCTAACAG -3'
[0116] 2) PCR Amplification and Determination of the Inherent
Dissociation Temperature of SCO
[0117] The following PCR reaction was performed using CFX96
Real-time PCR (Bio-Rad, USA) with 20 .mu. of total reaction
solution of each of Primer 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,
41, 42, 43, 44, 45 and SCO 14, 15, 16, 17, 18, 19, 20 prepared by
adding 0.15 .mu.M, PspG (NEB, USA) 1U, PCR buffer (1.times.),
MgCl.sub.2 2.5 mM, dNTP 200 .mu.M, DTT 0.1 mM, RNase Inhibitor IU,
SuperiorScript II (Enzynomics, Korea) 1U and the nucleic acid of
the genomic RNA of RVA, AstV, AdV 40, AdV41, NoV GI, NoV GII and
EC(MS2 phage) 1.times.10{circumflex over ( )}.sup.4 pg/rxn,
respectively.
[0118] 55.degree. C. 20 mins, 95.degree. C. 10 mins
[0119] 95.degree. C. 30 secs, 63.degree. C. 1 mins (50 cycles).
[0120] A reverse transcription reaction was performed using a cycle
at the denaturation temperature of 55.degree. C. for 20 minutes
once, and with a cycle at the denaturation temperature of
95.degree. C. for 10 minutes 1 time, and with a cycle at an
annealing temperature of 63.degree. C. for 1 minute 50 times
repeatedly. After the reaction, the reaction mixture was cooled to
50.degree. C. in the same apparatus, held at 50.degree. C. for 30
seconds, and then slowly heated from 50.degree. C. to 95.degree. C.
to obtain an inherent dissociation temperature analysis curve. Data
analysis was performed with Bio-Rad CFX Manager 1.6.
[0121] FIG. 4 shows the results of multiple inherent dissociation
temperature measurements for causative organisms of RVA, AstV, AdV
40, AdV 41, NoV GI, NoV GII. It was identified that the peak was
observed at the inherent dissociation temperature that each SCO
has: RVA: HEX 78.degree. C., AstV: CalRed60 78.degree. C., AdV 40:
CalRed610 67.degree. C., AdV 41: CalRed610 67.degree. C., NoV GI:
FAM 68.degree. C., NoV GII: FAM 84.degree. C., EC: HEX 69.degree.
C. (a)(b)(c)(d), and no peak of SCO visualizing CCTF was observed
when the target sequence was not added in the same composition
(e).
[0122] Therefore, it was proved that the target nucleic acid
sequence can be detected more quickly and simply than the
conventional PCR method by analyzing the fluorescence of the SCO
using the real-time PCR instrument, simultaneously with the PCR
using the marking technique of CCTF.
[0123] 3. Formation of CCTF and Analysis for the Inherent
Dissociation Temperature Peak of CCTF in Multi-Target PCR for
Detecting the Human Papillomavirus
[0124] CCTF analysis was performed with Real-time PCR instrument
for DNA of subtypes of Human Papillomavirus (HPV), 16 type, 18
type, 33 type, 35 type, 51 type, 53 type, 59 type, 68a type, 82
type and Internal control (IC).
[0125] 1) Primer of the Target Sequence Template DNA, Constructed
in the Sequence-Specific Manner
[0126] The forward primer used in this example was CTPO and was
constructed on the same principle as in Example 1 above. The 5'end
of CTPO was composed of 19-20 mers of nucleotide sequences, and was
composed of a sequence non-complementary to DNA of the target
sequence to form CCTF. The restriction enzyme recognition sequence
was consecutively located, and after this up to the 3' end, a
sequence complementary to each target site was composed to play a
role as a primer. The reverse primer was composed of sequence
complementary to the target site to be amplified.
[0127] In addition, SCO, which forms a complementary bond with CCTF
to be a double-stranded template, was positioned by positioning
fluorescent offsetting molecules (BHQ-1 or BHQ-2), and the
fluorescent reporter molecular was positioned so as to have a
certain distance.
[0128] Primer information and target sequence information which is
amplified and generated are as follows.
TABLE-US-00029 Primer 46: (SEG ID. NO: 115)
5'-CTCTGATAGCGACTGCTCGCA*CCAGGATAATATAAGGGGTCGGTGG ACCGG-3' Primer
47: (SEQ ID. NO: 116) 5'-CTCCATGCATGATFACAGCTGGGTT-3' Primer 48:
(SEQ ID. NO: 117)
5'-ATCGGTCTCCTGAAAGCTGCG*CCAGGCAGAAGGTACAGACGGGGAG GGC-3' Primer
49: (SEQ ID. NO: 118) 5'-CACCTCCAGCCGCTCCCCTAAT-3' Primer 50: (SEQ
ID. NO: 119) 5'-CTGGCGTAGAGCACTTACGCT*CCAGGCAACGATAACCGACCACCAC
AAGCA-3' Primer 51: (SEQ ID. NO: 120) 5'-CGGGGTCTGCACAGAACAGCTTT-3'
Primer 52: (SEQ ID. NO: 121)
5'-CTGGCGTAGAGCACTTACGCT*CCAGGAGGACCCAGCTGAACGACCT TACAA-3' Primer
53: (SEQ ID. NO: 122) 5'-CTGTCCACCGTCCACCGATGTTATG-3' Primer 54:
(SEQ ID. NO: 123)
5'-CTGGCGTAGAGCACTTACGCT*CCAGGGCTGGCAACGTACACGACAA CG-3' Primer 55:
(SEQ ID. NO: 124) 5'-GCTGTACAACGCGAAGGGTGTC-3' Primer 56: (SEQ ID.
NO: 125) 5'-CTGGCGTAGAGCACTTACGCT*CCAGGTCCACCTATGCACCGAAACC
TCCAA-3' Primer 57: (SEQ ID. NO: 126)
5'-TGCAGTGACGAGTCCCCGTGTAGTA-3' Primer 58: (SEQ ID. NO: 127)
5'-CTGGCGTAGAGCACTTACGCT*CCAGGGACTGTACACCGTATGCAGC GTG-3' Primer
59: (SEQ ID. NO: 128) 5'-GCGTATCAGCAGCTCATGTAA-3' Primer 60: (SEQ
ID. NO: 129) 5'-CTGGCGTAGAGCACTTACGCT*CCAGGACAAACTCGACGTCGTCTCG
GAA-3' Primer 61: (SEQ ID. NO: 130) 5'-CAGGTCACCACAACAAAGGCTCCGT-3'
Primer 62: (SEQ ID. NO: 131)
5'-ATCAGGACGCAGCCGGTTCT*CCAGGCCAAGGACAGGTACGGCTGTC ATC-3' Primer
63: (SEQ ID. NO: 132) 5'-GGTGCCCTTGAGGTTGTCCAGGTG-3' SCO 21: (SEQ
ID. NO: 133) GAGACGTTTAAGTCCGCGACCGCTC[T(HEX)]CTGATAGCGACTGCTCG
CA[BHQ 1] SCO 22: (SEQ ID. NO: 134)
CAGGCGACGTCCATATGGTGCGCTA[T(FAM)]CGGTCTCCTGAAAGCTG CG[BHQ 2] SCO
23: (SEQ ID. NO: 135) CCCTTAGGTAACGTCTGGC[T(Qusar
670)]GGCGTAGAGCACTTACG CT[BHQ 2] SCO 24: (SEQ ID. NO: 136)
AAACTTTAATTATTGTATA[T(FAM)]CAGGACGCAGCCGGTTCT [BHQ 1]
Amplified product 23: GenBank: LC193821.1/Position (start-end):
480-571
TABLE-US-00030 (SEQ ID. NO: 137) CTCTGATAGCGACTGCTCGCA *
ATAATATAAGGGGTCGGTGGAC
CGGTCGATGTATGTCTTGTTGCAGATCATCAAGAACACGTAGAGAAACCC
AGCTGTAATCATGCATGGAG
Amplified product 24: GenBank: KC470209.1/Position (start-end):
538-747
TABLE-US-00031 (SEQ ID NO: 138) ATCGGTCTCCTGAAAGCTGCG *
CACGACAGGAACGACTCCAACG
ACGCAGAGAAACACAAGTATAATATTAAGTATGCATGGACCTAAGGCAAC
ATTGCAAGACATTGTATTGCATTTAGAGCCCCAAAATGAAATTCCGGTTG
ACCTTCTATGTCACGAGCAATTAAGCGACTCAGAGGAAGAAAACGATGAA
ATAGATGGAGTTAATCATCAACATTTACCAGCCCGACG
Amplified product 25: GenBank: KU298894.1/Position (start-end):
535-860
TABLE-US-00032 (SEQ ID. NO: 139) CTGGCGTAGAGCACTTACGCT *
ACGCCATGAGAGGACACAAGCC
AACGTTAAAGGAATATGTTTTAGATTTATATCCTGAACCAACTGACCTAT
ACTGCTATGAGCAATTAAGTGACAGCTCAGATGAGGATGAAGGCTTGGAC
CGGCCAGATGGACAAGCACAACCAGCCACAGCTGATTACTACATTGTAAC
CTGTTGTCACACTTGTAACACCACAGTTCGTTTATGTGTCAACAGTACAG
CAAGTGACCTACGAACCATACAGCAACTACTTATGGGCACAGTGAATATT
GTGTGCCCTACCTGTGCACAACAATAAACATCATCTACAATGGCCGATCC TGAA
Amplified product 26: GenBank: M74117.1/Position (start-end):
117-509
TABLE-US-00033 (SEQ ID. NO: 140) CTGGCGTAGAGCACTTACGCT*
AGGACCCAGCTGAACG ACCTTACAAACTGCATGATTTGTGCAACGAGGTAGAAGAAAGC
ATCCATGAAATTTGTTTGAATTGTGTATACTGCAAACAAGAAT
TACAGCGGAGTGAGGTATATGACTTTGCATGCTATGATTTGTG
TATAGTATATAGAGAAGGCCAGCCATATGGAGTATGCATGAAA
TGTTTAAAATTTTATTCAAAAATAAGTGAATATAGATGGTATA
GATATAGTGTGTATGGAGAAACGTTAGAAAAACAATGCAACAA
ACAGTTATGTCATTTATTAATTACGTGTATTACATGTCAAAAA
CCGCTGTCTCCAGTTGAAAAGCAAAGACATTTAGAAGAAAAAA
AACGATTCCATAACATCGGTGGACGGTGGACAG
Amplified product 27: GenBank: KU298905.1/Position (start-end):
512-812
TABLE-US-00034 (SEQ ID. NO: 141) CTGGCGTAGAGCACTTACGCT*
GCTGGCAACGTACAC GACAACGTAACGAAACCCAAGTGTAATAAAGCCATGCGTGGTAA
TGTACCACAATTAAAAGATGTAGTATTGCATTTAACACCACAGA
CTGAAATTGACTTGCAATGCTACGAGCAATTTGACAGCTCAGAG
GAGGAGGATGAAGTAGATAATATGCGTGACCAGCTACCAGAAAG
ACGGGCTGGACAGGCTACGTGTTACAGAATTGAAGCTCCGTGTT
GCAGGTGTTCAAGTGTAGTACAACTGGCAGTGGAAAGCAGTGGA
GACACCCTTCGCGTTGTACAGC
Amplified product 28: GenBank: KU298906.1/Position (start-end):
3374-3558
TABLE-US-00035 (SEQ ID. NO: 142) CTGGCGTAGAGCACTTACGCT*
TCCACCTATGCACCGA AACCTCCAAGACCTCCGCATTGTCCGTGGGTGCCAAAGACACAC
ACCTACAACCACCACAGAAACGACGACGACCAGACGTCACAGAC
TCCAGAAACACCAAGTACCCCAACAACCTTTTGCGGGGACAACA
ATCCGTGGACAGTACTACACGGGGACTCGTCACTGCA
Amplified product 29: GenBank: KU298922.1/Position (start-end):
226-366
TABLE-US-00036 (SEQ ID. NO: 143) CTGGCGTAGAGCACTTACGCT*
GTTAAGACCGAAAACG GTGCATATAAAGGTAGTTAGAAAGAAAAGGGCAACGGCATGGCA
CGCTTTGAGGATCCTACACAACGACCATACAAACTGCCTGACTT
GAGCACAACATTGAATATTCCTCTGCATGATATTCGC
Amplified product 30: GenBank: KC470271.1/Position (start-end):
3389-3541
TABLE-US-00037 (SEQ ID. NO: 144) CTGGCGTAGAGCACTTACGCT*
ATGGCGCTATTTCAC AACCCTGAGGAACGGCCATACAAATTGCCAGACCTGTGCAGGA
CATTGGACACTACATTGCATGACGTTACAATAGAGTGTGTCTA
TTGCAGAAGGCAACTACAACGGACAGAGGTATATGAATTTGCC TTTAGTGAC
Amplified product 31: GenBank: EF450778.1/Position (start-end):
431-681
TABLE-US-00038 (SEQ ID. NO: 145) GCTCATATGCGGCGCCATTTA*
GCAGGTTGCTATCAAG GTTACAAGACAGGTTTAAGGAGACCAATAGAAACTGGGCATGTG
GAGACAGAGAAGACTCTTGGGTTTCTGATAGGCACTGACTCTCT
CTGCCTATTGGTCTATTTTCCCACCCTTAGGCTGCTGGTGGTCT
ACCCTTGGACCCAGAGGTTCTTTGAGTCCTTTGGGGATCTGTCC
ACTCCTGATGCTGTTATGGGCAACCCTAAGGTGAAGGCTCATGG CAAGAAAGTGCTCGG
[0129] The bold and slanted font of the Primer sequence means the
restriction enzyme recognition sequence, and the underline is the
complementary sequence of the CCTF produced thereby. the part
represented by * is a tag that modified dCTP was inserted into C in
the recognition sequence to block the site cleaved by the PspGI
restriction enzyme. In SCO, the parentheses mean the position of
the nucleotide sequence in which the fluorescent offsetting
molecule and the fluorescent reporter are located. The sequence of
the CCTF produced from the amplified product is as follows.
TABLE-US-00039 CCTF 29: (SEQ ID. NO: 146)
5'-TGCGAGCAGTCGCTATCAGAG-3' CCTF 30: (SEQ ID. NO: 147)
5'-CGCAGCTTTCAGGAGACCGAT-3' CCTF 31: (SEQ ID. NO: 148)
5'-AGCGTAAGTGCTCTACGCCAG-3' CCTF 32: (SEQ ID. NO: 149)
5'-AGAACCGGCTGCGTCCTGAT-3'
[0130] 2) PCR Amplification and Determination of the Inherent
Dissociation Temperature of SCO
[0131] The following PCR reaction was performed using CFX96
Real-time PCR (Bio-Rad, USA) with 20 .mu. of total reaction
solution of each of Primer 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,
56, 57, 58, 59, 60, 61, 62, 63 and SCO 21, 22, 23, 24 prepared by
adding 0.15 .mu.M, PspGI (NEB, USA) 5U, PCR buffer (1.times.),
MgCl.sub.2 2.5 mM, dNTP 2 .mu.M, h-Taq DNA polymerase (Solgent,
Korea) 1.6 U and HPV type 16, type 18, type 33, type 35, type 51,
type 53, type 59, type 68a, type 82 and template DNA of genomic DNA
of IC 100 .mu.g/rxn, respectively.
[0132] 95.degree. C. 15 mins,
[0133] 95.degree. C. 30 secs, 63.degree. C. 1 mins (50 cycles).
[0134] A reaction was performed using a cycle at the denaturation
temperature of 95.degree. C. for 15 minutes once, and with a cycle
at the denaturation temperature of 95.degree. C. for 30 second and
at an annealing temperature of 63.degree. C. for 1 minute 50 times
repeatedly. After the reaction, the reaction mixture was cooled to
50.degree. C. in the same apparatus, held at 50.degree. C. for 30
seconds, and then slowly heated from 50.degree. C. to 95.degree. C.
to obtain an inherent dissociation temperature analysis curve. Data
analysis was performed with Bio-Rad CFX Manager 1.6.
[0135] FIG. 5 shows the results of multiple inherent dissociation
temperature measurements for each target of type 16, type 18, type
33, type 35, type 51, type 53, type 59, type 68a, type 82, IC. It
was identified that the peak was observed at the inherent
dissociation temperature that each SCO has (type 16: HEX
76.5.degree. C., type 18: FAM 78.degree. C., type 33: Quasar670
71.degree. C., type 35: Quasar670 71C, type 51: Quasar670
71.degree. C. type 53: Quasar670 71.degree. C., type 59: Quasar670
71PC, type 68 a: Quasar670 71.degree. C., type 82: Quasar670
71.degree. C., IC: Quasar670 67.5.degree. C.) (a)(b)(c)(d), and no
peak of SCO visualizing CCTF was observed when the target sequence
was not added in the same composition (e).
[0136] Therefore, it was proved that the target nucleic acid
sequence can be detected more quickly and simply than the
conventional PCR method by analyzing the fluorescence of the SCO
using the real-time PCR instrument, simultaneously with the PCR
using the marking technique of CCTF.
[0137] 4. Formation of CCTF and Analysis for the Inherent
Dissociation Temperature Peak of CCTF in Multi-Target PCR for
Detecting the Causative Organism of the Respiratory Diseases
[0138] CCTF analysis was performed using Real-time PCR instrument
of nucleic acids of the causative organisms of the respiratory
diseases, Influenza A/H1N1(Flu A/H1N1), Influenza A/H3N2(Flu
A/H3N2), Influenza A/H1N1/2009pdm(Flu A/H1N1/2009pdm), Influenza
B(Flu B), Parainfluenza 1(PIV1), Parainfluenza 3(PIV3), Respiratory
syncytial virus A(RSV A), Respiratory syncytial virus B(RSV B),
Human metapneumovirus(MPV), Adenovirus(AdV) and External control
(EC).
[0139] 1) Primer for the Target Sequence of Template DNA,
Constructed in the Sequence-Specific Manner
[0140] The forward primer used in this example was CTPO and was
constructed on the same principle as in Example 1 above. The 5'end
of CTPO was composed of 19.about.20 mers of nucleotide sequences,
and was composed of a sequence non-complementary to DNA of the
target sequence so as to form CCTF. The restriction enzyme
recognition sequence was then consecutively located, and after this
up to the 3' end, a sequence complementary to each target site was
composed to play a role as a primer. The reverse primer was
composed of sequence complementary to the target site to be
amplified.
[0141] In addition, SCO, which forms a complementary bond with CCTF
to be a double-stranded template, was positioned by positioning
fluorescent offsetting molecules (BHQ-1 or BHQ-2), and the
fluorescent reporter molecular was positioned so as to have a
certain distance.
[0142] Primer information and target sequence information which is
amplified and generated are as follows.
TABLE-US-00040 Primer 64: (SEQ ID. NO: 150)
5'-TTGCTATGGCTGACGGGGAAGAATGG-3' Primer 65: (SEQ ID. NO: 151)
5'-GCCCCGTTGAGAGCACGAAT*CCAGGG GGGTGAATCTTCTGCTTAATGTGAAGACAC-3'
Primer 66: (SEQ ID. NO: 152) 5'-GGGCACCATGCAGTACCAAACGGAAC-3'
Primer 67: (SEQ ID. NO: 153) 5'-CCGTGGCGCGAACTTATCGA*CCAGGATC
ACACTGAGGGTCTCCCAATAGAGC-3' Primer 68: (SEQ ID. NO: 154)
5'-TCAAAGACTAAGTGGTGCCATGGATGAAC-3' Primer 69: (SEQ ID. NO: 155)
5'-AAGTGACCTGCCATTGCGCG*CCAGGTATGTC TACAGCAGAGGGACCCAGC-3' Primer
70: (SEQ ID. NO: 156) 5'-GGCTTAGAGCACCGCGTCATT*CCAGGTGTCG
CTACTGGAAGCGGTGATC-3' Primer 71: (SEQ ID. NO: 157)
5'-GCGATAGCTAAGGTACGACGGGTC-3' Primer 72: (SEQ ID. NO: 158)
5'-GTAGATTCGATCCATGCTCCTCTACTACC-3' Primer 73: (SEQ ID. NO: 159)
5'-CGTCTTACATGCGCAAGCGG*CCAGGTGATATT GAGTTCGGTAATGCAAGATCTGC-3'
Primer 74: (SEQ ID. NO: 160) 5'-CCATAGAGATGGCAATAGATGAAGAGC-3'
Primer 75: (SEQ ID. NO: 161) 5'-AGGCGTTCCGCTTCAACGAG*CCAGGTTGTCAGA
TTCTGTAGCTTGCTCAGTC-3' Primer 76: (SEQ ID. NO: 162)
5'-GGTGGTGATCCCAACTTGTTATATCGAAG-3' Primer 77: (SEQ ID. NO: 163)
5'-TCCGTCTGCGAAGATCTGAGC*CCAGGTTCAATCT ATCRTCTGACAGATCTTGAAGT-3'
Primer 78: (SEQ ID. NO: 164) 5'-GTGTCACGACGCGCGAATCT*CCAGGAGATCGTGA
CCAGTATAATAGCTCAACAC-3' Primer 79: (SEQ ID. NO: 165)
5'GTTCAGACAATGCAGGGATAACACCAGC-3' Primer 80: (SEQ ID. NO: 366)
5'-CCCAGAACGATTTGCGGCGT*CCAGGCTTGGTC CTCTCTTAGGAGGCAAGC-3' Primer
81: (SEQ ID. NO: 167) 5'-AGGATGCTTCGGACTACCTGAG-3' Primer 82: (SEQ
ID. NO: 168) 5'-TGCATTGCCGTCGCAGAGAC*CCAGGCAACGGG CACGAAGCGCATC-3'
Primer 83: (SEQ ID. NO: 369) GCCCTAATGATAAGACAGGCAGTTGTGG Primer
84: (SEQ ID. NO: 170) 5'-ATGCGCTTGGATTGCCGATG*CCAGGAGCCCTGT
TAGTTCTGGATGCTGAACA-3' SCO 33: (SEQ ID. NO: 171)
CTTATAGATTATA[T(FAM)]TGCCCCGTTGAGAGC ACGAAT[BHQ1] SCO 34: (SEQ ID.
NO: 172) CTAAGTAAGCCTATATCGAAT[T(FAM)]CCGTGGC GCGAAGTTATCCA[BHQ1]
SCO 35: (SEQ ID. NO: 173) CGTACTGCACTCGCCTACGAC [T(Cal Fluor Red
610) AAGTGACCTGCCATTGCGCG[BHQ2] SCO 36: (SEQ ID. NO: 174)
CTTATAAGTTACA[T(Cal Fluor Red 610)]GGC TTAGAGCACCGCGTCATT[BHQ2] SCO
37: (SEQ ID. NO: 175) CTAATTGTAATAC[T(Quasar 670)]CGTCTTACA
TGCGCAAGCGG[BHQ2] SCO 38: (SEQ ID. NO: 176)
CTAATCGTATGAGATCTATGA[T(Quasar 670)] AGGCGTTCCGCTTCAACGAG[BHQ2] SCO
39: (SEQ ID. NO: 177) TCATAGACATTTA[T(Cal Fluor Gold 540)
TCCGTCTGCGAAGATCTGAGC[BHQ1] SCO 40: (SEQ ID. NO: 178)
TACGAATCTGACCTAGTAAGA [TYCal Fluor Gold
540)]GTGTCACGACGCGCGAATCT[BHQ1] SCO 41: (SEQ ID. NO: 179)
TGCCACTAACAGGCCGCTAGA[T(Cal Fluor Gold
540)]CCCAGAACGATTTGCGGCGT[BHQ1] SCO 42: (SEQ ID. NO: 180)
TCGAGCGTGCGCCAGATCCA[T(Quasar 670) TGCATFGCCGTCGCAGAGAC[BHQ2] SCO
43: (SEQ ID. NO: 181) TCGACTGTGCCTGCGTCCGTA[T(FAM)]ATGCGCTTG
GATTGCCGATG[BHQ1]
Amplified product 32: GenBank: KU558787.1/Position (start-end):
428-621
TABLE-US-00041 (SEQ ID. NO: 182)
TTGCTATGGCTGACGGGGAAGAATGGTTTGTACCCAAACCTGAGC
ATGTCCTATGTAAACAACAAAGAGAAAGAAGTCCTTGTGCTATGG
GGTGTTCATCACCCACCTAACATAGGGAACCAAAGGGCCCTCTAC
CATACAGAAAATGCTTATGTCTCTGTAGTGTCTTCACATTATAG CAGAAGATTCACCCC*
ATTCGTGCTCTCAACGGGGC
Amplified product 33: GenBank: CY221934.1/Position (start-end):
111-296
TABLE-US-00042 (SEQ ID. NO: 183)
GGGCACCATGCAGTACCAAACGGAACGATAGTGAAAACAATCACAA
ATGACCAAATTGAAGTTACTAATGCTACTGAGTTGGTTCAGAATTC
CTCAATAGGTGAAATATGCGACAGTCCTCATCAGATCCTTGATGGA
GAGAACTGCACACTAATAGATGCTCTATTGGGAGACCCTCA GTGTGAT*
TCGATAAGTTCGCGCCACGG
Amplified product 34: GenBank: CY221750.1/Position (start-end):
1291-1501
TABLE-US-00043 (SEQ ID. NO: 184)
TCAAAGACTAAGTGGTGCCATGGATGAACTCCACAACGAAATACT
CGAGCTGGATGAAAAAGTGGATGACCTCAGAGCTGACACTATAAG
CTCACAAATAGAACTTGCAGTCTTGCTTTCCAACGAAGGAATAAT
AAACAGTGAAGATGAGCATCTATTGGCACTTGAGAGAAAACTAAA
GAAAATGCTGGGTCCCTCTGCTCTAGACATA* CGCGCA ATGGCAGGTCACTT
Amplified product 35: GenBank: JF719743.1/Position (start-end):
1816-1950
TABLE-US-00044 (SEQ ID. NO: 185) GGCTTAGAGCACCGCGTCATT*
TGTCGCTACTGGAAG CGGTGATCCGCACAGTGACGACTTTACAGCAATTGCTTACTTA
AGGGACGAATTGCTCGCAAAGCATCCGACCTTAGGTTCTGGTA
ATGACGAGGCGACCCGTCGTACCTTAGCTATCGC
Amplified product 36: GenBank: KX639498.1 z/Position (start-end):
4035-4253
TABLE-US-00045 (SEQ ID. NO: 186)
GTAGATTCGATCCATGCTCCTCTACTACCATGGTCCAGCCGACTG
AGACAAGGGATGATATATAATGCCAATAAAGTAGCTCTGGCACCC
CAATGTCTCCCAGTCGACAAAGATATCAGATTCAGAGTrGTATTT
GTCAACGGAACATCACTGGGTAGAATCACAATTGCCAAGGTCGCA
AAAACTCTTGCAGATCTTGCATTACCGAACTCAATATCA* CCGCTTGCGCATGTAAGACG
Amplified product 37: GenBank: KY369876.1/Position (start-end):
1310-1463
TABLE-US-00046 (SEQ ID. NO: 187)
CCCATAGAGATGGCAATAGATGAAGAGCCAGAACAATTCGAACA
TAGAGCAGACCAAGAACAAGATGGGGAACCTCAATCATCTATAA
TCCAATATGCTTGGGCAGAAGGAAACAGAAGCGATGAGCGGACT
GAGCAAGGTAGAGAATCTGACAA* CTCGTTTGAAGC GGAACGCCT
Amplified product 38: GenBank: KX894800.1/Position (start-end):
11378-11529
TABLE-US-00047 (SEQ ID. NO: 188)
GGTGGTGATCCCAACTTGTTATATCGAAGTTTCTATAGAAGAAC
TCCTGATTTCCTCACAGAGGCTATAGTTCACTCTGTGTTCATAC
TTAGTTATTATACAAACCATGATTTAAAGGATAAACTTCAAGAT CTGTCAGAYGATAGATTGAA*
GCTCAGATCTTCGCAG ACGGA
Amplified product 39: GenBank: KY249683.1/Position (start-end):
11465-11577
TABLE-US-00048 (SEQ ID. NO: 189) GGTGGTGATCCTAATTTGTTATATCGAAGC
TTTTATAGGAGAACTCCAGACTTCCTTACA GAAGCTATAGTACATTCAGTGTTCGTGTTG
AGCTATTATACTGGTCACGATCT* AGATTCGCGCGTCGTGACAC
Amplified product 40: GenBank: KJ627391.1/Position (start-end):
3631-3933
TABLE-US-00049 (SEQ ID. NO: 190) TTTCAGACAATGCAGGGATAACACCAGCA
ATATCATTGGACCTAATGACTGATGCTGA ACTGGCCAGAGCTGTATCATACATGCCAA
CATCTGCAGGGCAGATAAAGCTGATGTTG GAGAACCGCGCAATGGTAAGGAGAAAAGG
ATTTGGAATCCTAATAGGGGTCTACGGAA GCTCTGTGATTTACATGGTTCAATTGCCG
ATCTTTGGTGTCATAGATACACTTGTTGG ATAATCAAGGCAGCTCCCTCTTGCTCAGA
AAAAAACGGGAATTATGCTTGCCTCCTAA GAGAGGACCAAG* ACGCCGC
AAATCGTTCTGGG
Amplified product 41: GenBank: KT963081.1/Position (start-end):
18437-18598
TABLE-US-00050 (SEQ ID. NO: 191)
AGGATGCTTCGGAGTACCTGAGTCCGGGTCTGGTGCAGT
TCGCCCGTGCAACAGACACCTACTTCAGTATGGGGAACA
AGTTTAGAAACCCCACAGTGGCGCCCACCCACGATGTGA
CCACCGACCGTAGCCAGCGACTGATGCTGCGCTTCGTGC CCGTTG*
GTCTCTGCGACGGCAATGCA
Amplified product 42: GenBank: CY221624.1/Position (start-end):
988-1252
TABLE-US-00051 (SEQ ID. NO: 192)
GCCCTAATGATAAGACAGGCAGTTGTGGTCCAGTATCGTC
TAATGGAGCAAATGGAGTAAAAGGATTTTCATTCAAATAC
GGCAATGGTGTTTGGATAGGGAGAACTAAAAGCATTAGTT
CAAGAAAAGGTTTTGAGATGATTTGGGATCCGAATGGATG
GACTGGGACTGACAATAAATTCTCAATAAAGCAAGATATC
GTAGGAATAAATGAGTGGTCAGGGTATAGCGGGAGTTTTG TTCAGCATCCAGAACTAACAGGGCT*
CATCGGCAATCCAAGCGCAT
[0143] The bold and slanted font of the Primer sequence means the
restriction enzyme recognition sequence, and the underline is the
complementary sequence of the CCTF produced thereby. the part
represented by * is a tag that modified dCTP was inserted into C in
the recognition sequence to block the site cleaved by the PspGI
restriction enzyme. In SCO, the parentheses mean the position of
the nucleotide sequence in which the fluorescent offsetting
molecule and the fluorescent reporter are located. The sequence of
the CCTF produced from the amplified product is as follows.
TABLE-US-00052 CCTF 33: (SEQ ID. NO: 193)
5'-ATTCGTGCTCTCAACGGGGC-3' CCTF 34: (SEQ ID. NO: 194)
5-TCGATAAGTTCGCGCCACGG-3' CCTF 35: (SEQ ID. NO: 195)
5'-CGCGCAATGGCAGGTCACTT-3' CCTF 36: (SEQ ID. NO: 196)
5'-AATGACGCGGTGCTCTAAGCC-3' CCTF 37: (SEQ ID. NO: 197)
5'-CCGCTTGCGCATGTAAGACG-3' CCTF 38: (SEQ ID. NO: 198)
5'-CTCGTTGAAGCGGAACGCCT-3' CCTF 39: (SEQ ID. NO: 199)
5-GCTCAGATCTTCGCAGACGGA-3' CCTF 40: (SEQ ID. NO: 200)
5'-AGATTCGCGCGTCGTGACAC-3' CCTF 41: (SEQ ID. NO: 201)
5'-ACGCCGCAAATCGTTCTGGG-3' CCTF 42: (SEQ ID. NO: 202)
5-GTCFCTGCGACGGCAATGCA-3' CCTF 43: (SEQ ID. NO: 203)
5'-CATCGGCAATCCAAGCGCAT-3'
[0144] 2) PCR Amplification and Determination of SCO Inherent
Dissociation Temperature
[0145] The following PCR reaction was performed using CFX96
Real-time PCR (Bio-Rad, USA) with 20 .mu. of total reaction
solution of each of 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,
76, 77, 78, 79, 80, 81, 82, 83, 84 and SCO 33, 34, 35, 36, 37, 38,
39, 40, 41, 42, 43 prepared by adding 0.15 .mu.M, PspGI (NEB, USA)
1U, PCR buffer (1.times.), MgCl.sub.2 2.5 mM, dNTP 200 IM, DTT 0.1
mM, RNase Inhibitor 1U, SuperiorScript III (Enzynomics, Korea) 1U
Flu A/H1N1, Flu A/H3N2, Flu A/H1N1/2009pdm, the template nucleic
acid of the genomic RNA of Flu B, PIV1, PIV3, RSV A, RSV B, hMPV,
ADV and MS2 phage 1.times.10{circumflex over ( )}.sup.4 copies/rx,
respectively.
[0146] 55.degree. C. 20 mins, 95.degree. C. 10 mins
[0147] 95.degree. C. 30 secs, 63.degree. C. 1 min (50 cycles).
[0148] A reaction was repeatedly performed with a cycle at the
reverse transcription reaction temperature of 55.degree. C. for 20
minutes once, and with a cycle at the denaturation temperature of
95.degree. C. for 30 seconds, and an annealing temperature of
63.degree. C. for 1 minute 50 times repeatedly. After the reaction,
the reaction mixture was cooled to 50.degree. C. in the same
apparatus, held at 50.degree. C. for 30 seconds, and then slowly
heated from 50.degree. C. to 95.degree. C. to obtain an inherent
dissociation temperature analysis curve. Data analysis was
performed with Bio-Rad CFX Manager 1.6.
[0149] FIG. 6 shows the results of multiple inherent dissociation
temperature measurements for causative organisms of Flu A/H1N1, Flu
A/H3N2, Flu A/H1N1/2009pdm, Flu B, PIV1, PIV3, RSV A, RSV B, hMPV,
ADV, EC(Ms2 phage). It was confirmed that the peak was observed at
the inherent dissociation temperature that each SCO has (Flu
A/H1N1: 67.5.degree. C., Flu A/H3N2: 76.5.degree. C., Flu
A/H1N1/2009pdm: 86.5.degree. C., Flu B: 83.5.degree. C., PIV1:
66.degree. C., PIV3: 74.degree. C., RSV A: 63.5.degree. C., RSV B:
72.degree. C., hMPV: 86.degree. C., ADV: 85.degree. C.)
(a)(b)(c)(d)(e), and no peak of SCO visualizing CCTF was observed
when the target sequence was not added in the same composition
(f).
[0150] Therefore, it was proved that the target nucleic acid
sequence can be detected more quickly and simply than the
conventional PCR method by analyzing the fluorescence of the SCO
using the real-time PCR instrument, simultaneously with the PCR
using the marking technique of CCTF.
[0151] 5. Formation of CCTF and Analysis for the Inherent
Dissociation Temperature Peak of CCTF in Multi-Target PCR for
Analyzing the Single Nucleotide Polymorphism Genotype of BDNF
Gene
[0152] CCTF analysis was performed with Real-time PCR instrument
for analyzing the genotype of rs6265, single nucleotide
polymorphism of BDNF gene.
[0153] 1) Primer for the Target Sequence of Template DNA,
Constructed in the Sequence-Specific Manner
[0154] The forward primer used in this example was CTPO and was
constructed on the same principle as in Example 1 above. The 5'end
of CTPO was composed of 19-20 mers of nucleotide sequences, and was
composed of a sequence non-complementary to DNA of the target
sequence so as to form CCTF. The restriction enzyme recognition
sequence was then located, and from this up to the 3' end, a
sequence complementary to each target site was composed to play a
role as a primer. The reverse primer was composed of sequence
complementary to the target site to be amplified.
[0155] In addition, SCO, which forms a complementary bond with CCTF
to be a double-stranded template, was positioned by positioning
fluorescent offsetting molecules (BHQ-1 or BHQ-2), and the
fluorescent reporter molecular was positioned so as to have a
certain distance.
[0156] Primer information and target sequence information which is
amplified and generated are as follows.
TABLE-US-00053 Primer 85: (SEQ ID. NO: 204)
5'-ACGAGGCCTGTCCGCTTACTAG*CCAGGCTG GTCCTCATCCAACAGCTCTTCTATCGC-3'
Primer86: (SEQ ID: NO: 205) 5'-CCGGGTACGCTAAGTCCGCTAT*CCAGGTTCT
GGTCCTCATCCAACAGCTCTTCTATCGT-3' Primer 87: (SEQ. ID. NO: 206)
5'-GACCCATGGGACTCTGGAGAGCGTGAA-3' Primer 88: (SEQ ID. NO: 207)
5'-GCTCATATGCGGCGCCATTTA*CCAGGGCAG GTTGCTATCAAGGTTACAAGACAG-3'
Primer 89: (SEQ ID. NO: 208) 5-CCGAGCACTTTCTTGCCATGAGCC-3' SCO 44:
(SEQ ID. NO: 209) GTAGCACGCTTCGAATGGC[T(HEX)]ATACGAG
GCCTGTCCGCTTACTAG[BHQ1] SCO 45: (SEQ ID. NO: 210)
GATACGGAGGTCCGAAGGCAG[T(FAM)]GTTGGT TACCCTAACGCGCCGGA[BHQ1] SCO 46:
(SEQ ID. NO: 211) ATTAGTTTAACTATTATATT[T(FAM)]TATGCT
CATATGCGGCGCCATTTA[BHQ1]
Amplified product 43: GenBank: NT_009237.19/Position (start-end):
27598340-27598451
TABLE-US-00054 (SEQ ID. NO: 212) ACGAGGCCTGTCCGCTTACTAG* CTGGTCCTCA
TCCAACAGCTCTTCTATCACGTGTTCGAAAGTGTCAGCCA
ATGATGTCAAGCCTCTTGAACCTGCCTTGGGCCCATTCAC GCTCTCCAGAGTCCCATGGGTC
Amplified product 44: GenBank: NT_009237.19/Position (start-end):
17598338-7598451
TABLE-US-00055 (SEQ ID. NO: 213) CCGGGTACGCTAAGTCCGCTAT*
TTCTGGTCCTCAT CCAACAGCTCTTCTATCACGTGTTCGAAAGTGTCAGCCAATG
ATGTCAAGCCTCTTGAACCTGCCTTGGGCCCATTCACGCTCT CCAGAGTCCCATGGGTC
Amplified product 45: GenBank: EF450778.1/Position (start-end):
431-681
TABLE-US-00056 (SEQ ID. NO: 214) GCTCATATGCGGCGCCATTTA* GCAGGTTGCTA
TCAAGGTTACAAGACAGGTTTAAGGAGACCAATAGAAACT
GGGCATGTGGAGACAGAGAAGACTCTTGGGTTTCTGATAG
GCACTGACTCTCTCTGCCTATTGGTCTATTTTCCCACCCT
TAGGCTGCTGGTGGTCTACCCTTGGACCCAGAGGTTCTTT
GAGTCCTTTGGGGATCTGTCCACTCCTGATGCTGTTATGG
GCAACCCTAAGGTGAAGGCTCATGGCAAGAAAGTGCTCGG
[0157] The bold and slanted font of the Primer sequence means the
restriction enzyme recognition sequence, and the underline is the
complementary sequence of the CCTF produced thereby. the part
represented by * is a tag that modified dCTP was inserted into C in
the recognition sequence to block the site cleaved by the PspGI
restriction enzyme. In SCO, the parentheses mean the position of
the nucleotide sequence in which the fluorescent offsetting
molecule and the fluorescent reporter are located. The sequence of
the CCTF produced from the amplified product is as follows.
TABLE-US-00057 CCTF 44: (SEQ ID. NO: 215)
5'-CTAGTAAGCGGACAGGCCTCGT-3' CCTF45: (SEQ ID. NO: 216)
5'-ATAGCGGACTTAGCGTACCCGG-3' CCTF 46: (SEQ ID. NO: 217)
5'-TAAATGGCGCCGCATATGAG-3'
[0158] 2) PCR Amplification and Determination of SCO Inherent
Dissociation
[0159] PCR reaction was performed using the following CFX96
Real-time PCR (Bio-Rad, USA) with 20 .mu. of total reaction
solution of each of Primer 85, 86, 87, 88, 89 and SCO 44, 45, 46
prepared by adding 0.15 .mu.iM, PspGI (NEB, USA) 5U, PCR buffer
(1.times.), MgCl.sub.2 2.5 mM, dNTP 200 .mu.iM, h-Taq DNA
polymerase (Solgent, Korea) 1.6 U and Flu A/H1N1, Flu A/H3N2, Flu
A/H1N1/2009pdm, the template nucleic acids of the genomic RNA of
Flu B, PIV1, PIV3, RSV A RSV B, hMPV, ADV and MS2 phage
1.times.10{circumflex over ( )}.sup.4 copies/rxn, respectively.
[0160] 95.degree. C. 15 mins,
[0161] 95.degree. C. 30 secs, 63.degree. C. 1 min (50 cycles).
[0162] A reaction was performed using a cycle at the denaturation
temperature of 95.degree. C. for 15 minutes once, and with a cycle
at the denaturation temperature of 95.degree. C. for 30 seconds,
and an annealing temperature of 63.degree. C. for 1 minute 50 times
repeatedly. After the reaction, the reaction mixture was cooled to
50.degree. C. in the same apparatus, held at 50.degree. C. for 30
seconds, and then slowly heated from 50.degree. C. to 95.degree. C.
to obtain an inherent dissociation temperature analysis curve. Data
analysis was performed with Bio-Rad CFX Manager 1.6.
[0163] FIG. 7, (a) shows the results of multiple inherent
dissociation temperature measurements for the genotype of mutant
type A/A, wild type G/G and heterozygote A/G of rs6265 and IC. It
was identified that the peak was observed at the inherent
dissociation temperature that each SCO has (A/A: 76.5.degree. C.,
A/G: 76.5.degree. C. 75.degree. C., G/G 75.degree. C., IC:
66.degree. C.) (a)(b)(c)(d), and no peak of SCO visualizing CCTF
was observed when the target sequence was not added in the same
composition (e).
[0164] Therefore, it was proved that the target nucleic acid
sequence can be detected more quickly and simply than the
conventional PCR method by analyzing the fluorescence of the SCO
using the real-time PCR instrument, simultaneously with the PCR
using the marking technique of CCTF.
Example 3. Formation of CCTF and Analysis for Ct Graph of CCTF in
Multiple Target PCR
[0165] It has been proved in Example 2 that SCO can be used to
confirm whether CCTF is generated with a real-time PCR device. The
SCO used in the above method is simultaneously formed during the
reaction in which the target sequence is generated during the PCR
amplification process, and it is possible to identify CCTF
generated by real-time fluorescence analysis. Based on this, the
present example demonstrated that a standard curve formation is
possible when analyzing the formation of CCTF using SCO in the case
of PCR with multiple target sequences.
[0166] In order to perform this experiment, the causative organisms
of sexually transmitted infections (STI), Neisseria. gonorrhea
(NG), Mycoplasma. hominis (MH), Ureaplasma. parvum (UP) were
selected.
[0167] 1. Construction of Specific Primer of Target Template
DNA
[0168] The forward primer used in this example was constructed
based on the method described in the detailed description of the
invention above as CPTO. The 5'end of the forward primer was
composed of a 19-mer or 21-mer nucleotide sequence, and was
composed of non-complementary sequences to DNA of each causative
organism so as to form CCTF. The restriction enzyme recognition
sequence was then consecutively located. After this up to the 3'
end, a sequence complementary to DNA of each causative organism was
composed to play a role as a primer. The reverse primer was
composed of sequence complementary to the target site of DNA by
each causative organism.
[0169] In addition, SCO, which forms a dimer with CCTF, was
designed to have a double tag, and was separately designed for each
causative organism. SCO was designed by positioning quencher (BHQ-1
or BHQ-2) at 3' end, with reporter molecular (each FAM, HEX, CAL
Fluor Red 610) positioned at a certain distance, and its sequence
was complementary to CCTF sequence to be analyzed.
[0170] Primer information and target sequence information which is
amplified and generated are as follows.
TABLE-US-00058 Primer 90: (SEQ ID. NO: 218)
5'-CTCATCGCCACGAGCCGGTTAA* TTGAAACACCGCCCGGAACCC-3' Primer 91: (SEQ
ID. NO: 219) 5'-GCTCCTTATTCGGTTTGACCGGT-3' Primer 92: (SEQ ID. NO:
220) 5'-GCTCGCAGGTACGGCACCATTCA* CAGAAGGTA TGATAACAACGGTAGAGC-3'
Primer 93: (SEQ ID. NO: 221) 5'-CCCCTTTGCACCGTTGAGGGG-3' Primer 94:
(SEQ ID. NO: 222) 5'-AGTCGATTATGTCTGAGGCCGCG* TTAAAGT
AGCATATGATCAAGCTCATTCA-3' Primer 95: (SEQ ID. NO: 223)
5'-GATCCTGACATATAATCATTATCTCCTTTTATAAA-3' SCO 47: (SEQ ID. NO: 224)
TC[T(HEX)]CATCGCCACGAGCCGGTTAA[BHQ] SCO 48: (SEQ ID. NO: 225)
TG[TTCAL Fluor Red 610)]CGCAGGTACGGCACC ATTCA[BHQ2] SCO 49: (SEQ
ID. NO: 226) TAG[T(FAM)]CGATTATGTCTGAGGCCGCG[BHQ]
Amplified product 46: GenBank: X52364.1/Position (start-end):
375-459
TABLE-US-00059 (SEQ ID. NO: 227) CTCATCGCCACGAGCCGGTTAA TTGAAACACCG
CCCGGAACCCGATATAATCCGCCCITCAACATCAGTGAAA
ATCTTTTTTTAACCGGTCAAACCGAATAAGGAGC
Amplified product 47: GenBank: M31431.1/Position (start-end):
1455-1535
TABLE-US-00060 (SEQ ID. NO: 228) GCTCGCAGGTACGGCACCATTCA* CAGAAGG
TATGATAACAACGGTAGAGCTTTATATGATATTAACTT
AGCAAAAATGGAAAACCCCTCAACGGTGCAAAGGGG
Amplified product 48: GenBank: AF085733.2/Position (start-end):
416-502
TABLE-US-00061 (SEQ ID. NO: 229) AGTCGATTATGTCTGAGGCCGCG*
GTTTCTGTAC ACGATCCAATT[T/c]ACAAATAACATTTACAATTCGTAAA
ATTTTTTTATAAAAGGAGATAATGATTATATGTCAGGATC
[0171] The bold and slanted font of the Primer sequence means the
restriction enzyme recognition sequence, and the underline is the
complementary sequence of the CCTF produced thereby. the part
represented by * is a tag that modified dCTP was inserted into C in
the recognition sequence to block the site cleaved by the PspGI
restriction enzyme. In SCO, the parentheses mean the position of
the nucleotide sequence in which the fluorescent offsetting
molecule and the fluorescent reporter are located. Primer and
primer corresponding to NG in SCO is the same as those used in
Example 2. The sequence of the CCTF produced from the amplified
product is as follows.
TABLE-US-00062 CCTF 47: (SEQ ID. NO: 230)
5'-CCTGGTTAACCGGCTCGTGGCGATGAG-3' CGTF48: (SEQ ID. NO: 231)
5'-CCTGGTGAATGGTGCCGTACCTGCGAGC-3' CCTF 49: (SEQ ID. NO: 232)
5'-CCTGGCGCGGCCTCAGACATAATCGACT-3'
[0172] 2. PCR Amplification and Determination of SCO Inherent
Dissociation
[0173] PCR reaction was performed using the following CFX96
Real-time PCR (Bio-Rad USA) with 20 .mu. of total reaction solution
obtained by adding three kinds of the specific forward primers and
three kinds of reverse primers of each target sequence, as
mentioned in the above primer design, and three kinds of SCO to be
0.15 .mu.M, respectively, and adding PspGI (NEB, USA) 2 U, PCR
buffer (1.times.), MgCl.sub.2 2.5 mM, dNTP 200 .mu.M, h-Taq DNA
polymerase (Solgent, Korea) 1.6 U, and contained the template DNA
diluted by 10-folds with 100 pg/.mu.l genomic DNA proven by the
conventional quantitation method for each causative organism.
[0174] 95.degree. C. 15 mins,
[0175] 95.degree. C. 30 secs, 63.degree. C. min (50 cycles).
[0176] A reaction was repeatedly performed with a cycle at the
denaturation temperature of 95.degree. C. for 15 minutes once, and
with a cycle at the denaturation temperature of 95.degree. C. for
30 seconds, and an annealing temperature of 63.degree. C. for 1
minute 50 times. In addition, fluorescence signals were collected
at the annealing stage, and the data analysis was performed with
Bio-Rad CFX Manager 1.6. Cycle threshold (Ct) was started with an
algebraic amplifier using a known number of DNA concentrations to
create a standard curve for the strain.
[0177] As shown in (a) of FIG. 8, the expected fluorescence
amplification curves of SCO could be observed with each of
different graphs depending on the concentration of the template.
Also, any peak was observed when the template DNA was not added
(b). As the results showing fluorescence amplification curves and
standards of SCO represented by the experimental condition of
Polymerase Chain Reaction of NG (solid line), MG (dotted line), and
UP (circle), dilutions for genomic DNA of each causative organism
diluted by 10-folds starting from the concentration of 100 .mu.g,
graph (a) indicates the fluorescence amplification curve drawn when
the three target sequences are present at the same time by the
concentration, graph (b) is the negative result drawn when all
three target sequences are not included. When the graph
corresponding to NG in graph (a) is represented by the single
fluorescence amplification curve and thus the standard curve, it
can be represented by (c) and (d), respectively. The graph
corresponding to MG can be expressed by (e) and (f), respectively,
and the curve corresponding to UP can be represented by (g) and
(h), respectively.
[0178] Regression coefficient (r.sup.2) in the linear regression
analysis of the standard curve was represented by NG 0.9982, MG
0.999, UP 0.9992, respectively. The slope of the regression plot
was NG -3.85, MG -3.89, and UP -3.66, respectively. It could be
identified that the respective amplification efficiency
(E=10.sup.[-1/slope]-1) was 81.8% for NG, 80.7% for MG and 87.6%
for UP, respectively, and thus, they were listed in the proper
range of between 80 and 120%.
[0179] From this Example, when reading the different CCTFs by each
of causal organisms using the real-time PCR instrument, it was
demonstrated that the relative amount of CCTF to be generated by
measuring a degree of the real-time fluorescence of SCO is grasped,
and by using this, the Ct value is confirmed, and therefore, the
identifying of the target sequence is possible.
Sequence CWU 1
1
232147DNAArtificial Sequenceprimer 1tgaactatcc tggtccgacg
tttcggttgt gttgaaacac cgcccgg 47222DNAArtificial Sequenceprimer
2gctccttatt cggtttgacc gg 22341DNAArtificial Sequenceprimer
3atctatgata cctggtttag ctcctattgc caacgtattg g 41428DNAArtificial
Sequenceprimer 4tgtgtggagc atcttgtaat ctttggtc 285117DNAArtificial
Sequenceproduct 5tgaactatcc tggtccgacg tttcggttgt gttgaaacac
cgcccggaac ccgatataat 60ccgcccttca acatcagtga aaatcttttt tttaaccggt
caaaccgaat aaggagc 1176125DNAArtificial Sequenceproduct 6atctatgata
cctggtttag ctcctattgc caacgtattg gaaaaaaact ttggtattga 60aaaaggattt
atgacaacag tccactcata tacagcagac caaagattac aagatgctcc 120acaca
125713DNAArtificial SequenceCCTF 7ccaggatagt tca 13815DNAArtificial
SequenceCCTF 8ccaggtatca tagat 15947DNAArtificial Sequenceprimer
9ccactccagc cggctgacac caggacttgg tgtgacgcta tcagcat
471029DNAArtificial Sequenceprimer 10gttttcaaaa cacggtcgaa
aacaaagtc 291146DNAArtificial Sequenceprimer 11catcgccacg
agccggttaa ccaggttgaa acaccgcccg gaaccc 461223DNAArtificial
Sequenceprimer 12gctccttatt cggtttgacc ggt 231351DNAArtificial
Sequenceprimer 13actcacgcta atggagcgca ccaggtttag ctcctattgc
caacgtattg g 511428DNAArtificial Sequenceprimer 14tgtgtggagc
atcttgtaat ctttggtc 281552DNAArtificial Sequenceprimer 15gctacccagc
cggctacaag ccaggcttta tggtgcttat attggtggca tg 521624DNAArtificial
Sequenceprimer 16ctgtataacg ttgtgcagca ggtc 241747DNAArtificial
Sequenceprimer 17tgccgcgtga ttcgatccca ccaggtatgt ccggcacaac
atgcgct 471823DNAArtificial Sequenceprimer 18gagcttacga aggtcggagt
tga 231951DNAArtificial Sequenceprimer 19tctcatagct gggccgctgc
caggaagtag catatgatga agcacacaac a 512025DNAArtificial
Sequenceprimer 20taatgcaacg tgcatttgct tcaac 252155DNAArtificial
Sequenceprimer 21cagatcgttg gcactctgcg accaggttaa agtagcatat
gatcaagctc attca 552231DNAArtificial Sequenceprimer 22ttgtaatgat
acaacgagca tcatcattaa t 312351DNAArtificial Sequenceprimer
23gctcgtatgc cgctccatat accaggccaa atctggatct tcctctgcat c
512421DNAArtificial Sequenceprimer 24gagcttgagc tggacccaga g
212548DNAArtificial Sequenceprimer 25acgtgccgtg catcgttgca
ccaggcaacc ggctccattt tggtggag 482621DNAArtificial Sequenceprimer
26cgtcacgtcc ttcatcggtc c 212746DNAArtificial Sequenceprimer
27tcgcagtccc gtcgaggaac caggaggcct ggctatccgg agaaac
462820DNAArtificial Sequenceprimer 28cgttgtgttg gccgcaggtc
202944DNAArtificial Sequenceprimer 29ctcatagcta ggcgcctgcc
agggctgcac gtgggtctgt tgtg 443021DNAArtificial Sequenceprimer
30ggaaacgcag gccacgaaac c 213150DNAArtificial Sequenceprimer
31gcttcgcgtc tcaggcctgt ccagggggca ttacagtttt gcgtcatgac
503221DNAArtificial Sequenceprimer 32caagtctgag cacttgcacc g
213347DNAArtificial Sequenceprimer 33ctgttagctc tgcgagctcc
aggggagcga cacttgttgg tgttgac 473423DNAArtificial Sequenceprimer
34tgatgaaatg aagccacccg tgc 233541DNAArtificial SequenceSCO
35tcggagccag cgcggcgtaa acccactcca gccggctgac a 413644DNAArtificial
SequenceSCO 36tacaacagca gtacggagac gactcatcgc cacgagccgg ttaa
443744DNAArtificial SequenceSCO 37atttattctt actcgatgtt aaatactcac
gctaatggag cgca 443843DNAArtificial SequenceSCO 38tatatatata
tattattata aatgctaccc agccggctac aag 433944DNAArtificial
SequenceSCO 39aagaataact actacaatct actttgccgc gtgattcgat ccca
444042DNAArtificial SequenceSCO 40ttattattat tattattata tattctcata
gctgggccgc tg 424142DNAArtificial SequenceSCO 41aatcttcaat
gcttaccgta tcagatcgtt ggcactctgc ga 424242DNAArtificial SequenceSCO
42aaaataaata atataatata tgctcgtatg ccgctccata ta
424343DNAArtificial SequenceSCO 43tcggagccag cgcggcgtaa cgtacgtgcc
gtgcatcgtt gca 434443DNAArtificial SequenceSCO 44aagaataact
actacaatct actttcgcag tcccgtcgag gaa 434541DNAArtificial
SequenceSCO 45tcggagccag cgcggcgtaa tctctcatag ctaggcgcct g
414641DNAArtificial SequenceSCO 46aaaataaata atataatata gtcttcgcgt
ctcaggcctg t 414740DNAArtificial SequenceSCO 47aaaataaata
atataatata ttctgttagc tctgcgagct 404895DNAArtificial
Sequenceproduct 48ccactccagc cggctgacac caggacttgg tgtgacgcta
tcagcatgcg tatgggttac 60tatggtgact ttgttttcga ccgtgttttg aaaac
9549122DNAArtificial Sequenceproduct 49cgcccaccgc atcccgcgcc
cctccctcag caccaggttg aaacaccgcc cggaacccga 60tataatccgc ccttcaacat
cagtgaaaat ctttttttaa ccggtcaaac cgaataagga 120gc
12250135DNAArtificial Sequenceproduct 50actcacgcta atggagcgca
ccaggtttag ctcctattgc caacgtattg gaaaaaaact 60ttggtattga aaaaggattt
atgacaacag tccactcata tacagcagac caaagattac 120aagatgctcc acaca
13551251DNAArtificial Sequenceproduct 51gctacccagc cggctacaag
ccaggcttta tggtgcttat attggtggca tgcaccatga 60tcgtcctttt aaaaagtctg
cgaggattgt tggtgatgta atgagtaaat tccaccctca 120tggtgatatg
gcaatatatg acaccatgtc aagaatggct caagactttt cattaagata
180ccttttaatt gatggtcatg gtaattttgg ttctatagat ggtgatagac
ctgctgcaca 240acgttataca g 25152111DNAArtificial Sequenceproduct
52tgccgcgtga ttcgatccca ccaggtatgt ccggcacaac atgcgcttat gtccggcaca
60acatgcgctc tccgcttccc aggtcagctc aactccgacc ttcgtaagct c
11153225DNAArtificial Sequenceproduct 53tctcatagct gggccgctgc
caggaagtag catatgatga agcacacaac aaaatggcgc 60atactgtgta tttcactaat
ttctatcgtt catcaaaacc actattttta gatgaagaag 120acccaattaa
tccctgtttt caaactatta gtatgggtgg gggttatgta tctggtgaag
180tgtatcgttc tgattttgaa gttgaagcaa atgcacgttg catta
22554236DNAArtificial Sequenceproduct 54cagatcgttg gcactctgcg
accaggttaa agtagcatat gatcaagctc attcaaaaat 60ggcacatact gtctatttta
cgaattttta tcgttcatct aaacctttat ttttagatga 120agaagatcca
atcaacccct gttttcaaac aattagtatg ggtggtggat atgtttcagg
180tgaaatttat cgttctgatt ttgaaattaa tgatgatgct cgttgtatca ttacaa
23655102DNAArtificial Sequenceproduct 55gctcgtatgc cgctccatat
accaggccaa atctggatct tcctctgcat ctgcttctgg 60atcatcaagc agcagcacca
gctctgggtc cagctcaagc tc 10256187DNAArtificial Sequenceproduct
56acgtgccgtg catcgttgca ccaggcaacc ggctccattt tggtggagtc gcttgatcgt
60tttgtgatcg tttagtgtga tgatttatta tgtctagaga gttaagcgat aggcttttac
120tggtgtatca ctgtaagggc gtattggttg gatgccttgg tagacaggac
cgatgaagga 180cgtgacg 18757172DNAArtificial Sequenceproduct
57tcgcagtccc gtcgaggaac caggaggcct ggctatccgg agaaacagca cacgacttgg
60cgttctgtgt gtcgcgatgt ctctgcgcgc agtctggcat ctggggcttt tgggaagcct
120cgtgggggct gttcttgccg ccacccatcg gggacctgcg gccaacacaa cg
17258209DNAArtificial Sequenceproduct 58ctcatagcta ggcgcctgcc
agggctgcac gtgggtctgt tgtgggtaga ggtgggcggg 60gagggccccg gccccaccgc
cccccccaca ggcggcgcgt gcggagggcg gcccgtgcgt 120ccccccggtc
cccgcgggcc gcccgtggcg ctcggtgccc ccggtatggt attccgcccc
180caaccccggg tttcgtggcc tgcgtttcc 20959183DNAArtificial
Sequenceproduct 59gcttcgcgtc tcaggcctgt ccagggggca ttacagtttt
gcgtcatgac ggctttgaag 60ctgacgacct cattgcaacc ctagcaaaac gagttgcggc
tgagcactgt catgttgtga 120ttatctcctc agataaagat gtacttcagc
ttgtgtgtga tacggtgcaa gtgctcagac 180ttg 18360183DNAArtificial
Sequenceproduct 60ctgttagctc tgcgagctcc aggggagcga cacttgttgg
tgttgacaag ttcggtaaca 60aatactacca gaagctaggc gatactcaat acggtatgca
cagatgggta gagtatgctt 120caaaggatcg ttacaacgca tctcaagtac
cagctgaatg gcacgggtgg cttcatttca 180tca 1836124DNAArtificial
SequenceCCTF 61cctggtgtca gccggctgga gtgg 246225DNAArtificial
SequenceCCTF 62cctggttaac cggctcgtgg cgatg 256325DNAArtificial
SequenceCCTF 63cctggtgcgc tccattagcg tgagt 256425DNAArtificial
SequenceCCTF 64cctggcttgt agccggctgg gtagc 256525DNAArtificial
SequenceCCTF 65cctggtggga tcgaatcacg cggca 256624DNAArtificial
SequenceCCTF 66cctggcagcg gcccagctat gaga 246726DNAArtificial
SequenceCCTF 67cctggtcgca gagtgccaac gatctg 266826DNAArtificial
SequenceCCTF 68cctggtatat ggagcggcat acgagc 266925DNAArtificial
SequenceCCTF 69cctggtgcaa cgatgcacgg cacgt 257024DNAArtificial
SequenceCCTF 70cctggttcct cgacgggact gcga 247123DNAArtificial
SequenceCCTF 71cctggcaggc gcctagctat gag 237225DNAArtificial
SequenceCCTF 72cctggacagg cctgagacgc gaagc 257323DNAArtificial
SequenceCCTF 73cctggagctc gcagagctaa cag 237453DNAArtificial
Sequenceprimer 74gcaggagcct ctcatctcgc caggctcatt tatagacarc
ttctcactaa ttc 537530DNAArtificial Sequenceprimer 75agttttttct
gatccaatyt gytctatttc 307649DNAArtificial Sequenceprimer
76tcagacggtt cgaggctccc caggargaty aagcgtggag tatayatgg
497721DNAArtificial Sequenceprimer 77tttgcgtgcy tcttcacacg c
217846DNAArtificial Sequenceprimer 78aacgcgaatc gaccggatcc
aggcgcgatg tgtttgccga taaaac 467920DNAArtificial Sequenceprimer
79cattgcgtct gccccacttg 208051DNAArtificial Sequenceprimer
80aacgcgaatc gaccggatcc aggaaacaag aacacctatg cctacatgaa c
518127DNAArtificial Sequenceprimer 81atgttaacgt ccttcctgaa gttccac
278246DNAArtificial Sequenceprimer 82tagatcggac tgcgaatcgc
cagggagatc gcratctyct gcccga 468322DNAArtificial Sequenceprimer
83rgcgtcctta gacgccatca tc 228449DNAArtificial Sequenceprimer
84atctacagcg tcgcatcacg ccaggcgcaa tctggctccc arttttgtg
498522DNAArtificial Sequenceprimer 85gcgtcaytcg acgccatcyt ca
228651DNAArtificial Sequenceprimer 86cataggtcga ggtcctcacc
cagggcaaac tccggcatct actaatagac g 518720DNAArtificial
Sequenceprimer 87aagcggtgat ccgcacagtg 208847DNAArtificial
SequenceSCO 88tcggccgatc gtccatagag tcaagctcgc aggagcctct catctcg
478945DNAArtificial SequenceSCO 89tcacgatgag cgagttgagc tacgtatcag
acggttcgag gctcc 459043DNAArtificial SequenceSCO 90tgttcaatat
ataatgataa tatgtaacgc gaatcgaccg gat 439143DNAArtificial
SequenceSCO 91tgttcaatat ataatgataa tatgtaacgc gaatcgaccg gat
439242DNAArtificial SequenceSCO 92acatttataa tacagtattt tattagatcg
gactgcgaat cg 429345DNAArtificial SequenceSCO 93agctcctgcc
agtactgcca tccatatcta cagcgtcgca tcacg 459443DNAArtificial
SequenceSCO 94tagttataat gaataactat tattcatagg tcgaggtcct cac
4395105DNAArtificial Sequenceproduct 95gcaggagcct ctcatctcgc
caggctcatt tatagacarc ttctcactaa ttcatattca 60gtagatttac atgatgaaat
agarcaratt ggatcagaaa aaact 10596114DNAArtificial Sequenceproduct
96tcagacggtt cgaggctccc caggargaty aagcgtggag tatayatgga cctgcttgtc
60tcgggggcaa gcccaggcaa tgcatggtcc catgcgtgtg aagargcacg caaa
1149782DNAArtificial Sequenceproduct 97aacgcgaatc gaccggatcc
aggcgcgatg tgtttgccga taaaacgtac caaccggagc 60cccaagtggg gcagacgcaa
tg 8298308DNAArtificial Sequenceproduct 98aacgcgaatc gaccggatcc
aggaaacaag aacacctatg cctacatgaa cggtcgggtg 60gcggttccta gcgccctcga
tacctacgta aacatcgggg cacggtggtc tccagatccc 120atggacaatg
ttaacccctt caatcaccac cgtaacgccg gtctgcgcta tcgatccatg
180ctcttgggca acgggcgtta cgtacccttc cacattcaag tcccccagaa
gttttttgcc 240attaaaaatc tcctcctctt accgggttcc tacacctacg
agtggaactt caggaaggac 300gttaacat 3089978DNAArtificial
Sequenceproduct 99tagatcggac tgcgaatcgc cagggagatc gcratctyct
gcccgaattc gtaaatgatg 60atggcgtcta aggacgcy 7810073DNAArtificial
Sequenceproduct 100atctacagcg tcgcatcacg ccaggcgcaa tctggctccc
arttttgtga atgargatgg 60cgtcgartga cgc 73101196DNAArtificial
Sequenceproduct 101cataggtcga ggtcctcacc cagggcaaac tccggcatct
actaatagac gccggccatt 60caaacatgag gattacccat gtcgaagaca acaaagaagt
tcaactcttt atgtattgat 120cttcctcgcg atctttctct cgaaatttac
caatcaattg cttctgtcgc tactggaagc 180ggtgatccgc acagtg
19610224DNAArtificial SequenceCCTF 102cctggtgtca gccggctgga gtgg
2410325DNAArtificial SequenceCCTF 103cctggttaac cggctcgtgg cgatg
2510425DNAArtificial SequenceCCTF 104cctggtgcgc tccattagcg tgagt
2510525DNAArtificial SequenceCCTF 105cctggcttgt agccggctgg gtagc
2510625DNAArtificial SequenceCCTF 106cctggtggga tcgaatcacg cggca
2510724DNAArtificial SequenceCCTF 107cctggcagcg gcccagctat gaga
2410826DNAArtificial SequenceCCTF 108cctggtcgca gagtgccaac gatctg
2610926DNAArtificial SequenceCCTF 109cctggtatat ggagcggcat acgagc
2611025DNAArtificial SequenceCCTF 110cctggtgcaa cgatgcacgg cacgt
2511124DNAArtificial SequenceCCTF 111cctggttcct cgacgggact gcga
2411223DNAArtificial SequenceCCTF 112cctggcaggc gcctagctat gag
2311325DNAArtificial SequenceCCTF 113cctggacagg cctgagacgc gaagc
2511423DNAArtificial SequenceCCTF 114cctggagctc gcagagctaa cag
2311551DNAArtificial Sequenceprimer 115ctctgatagc gactgctcgc
accaggataa tataaggggt cggtggaccg g 5111625DNAArtificial
Sequenceprimer 116ctccatgcat gattacagct gggtt 2511749DNAArtificial
Sequenceprimer 117atcggtctcc tgaaagctgc gccaggcaga aggtacagac
ggggagggc 4911822DNAArtificial Sequenceprimer 118cacctccagc
cgctccccta at 2211951DNAArtificial Sequenceprimer 119ctggcgtaga
gcacttacgc tccaggcaac gataaccgac caccacaagc a 5112023DNAArtificial
Sequenceprimer 120cggggtctgc acagaacagc ttt 2312151DNAArtificial
Sequenceprimer 121ctggcgtaga gcacttacgc tccaggagga cccagctgaa
cgaccttaca a 5112225DNAArtificial Sequenceprimer 122ctgtccaccg
tccaccgatg ttatg 2512348DNAArtificial Sequenceprimer 123ctggcgtaga
gcacttacgc tccagggctg gcaacgtaca cgacaacg 4812422DNAArtificial
Sequenceprimer 124gctgtacaac gcgaagggtg tc 2212551DNAArtificial
Sequenceprimer 125ctggcgtaga gcacttacgc tccaggtcca cctatgcacc
gaaacctcca a 5112625DNAArtificial Sequenceprimer 126tgcagtgacg
agtccccgtg tagta 2512749DNAArtificial Sequenceprimer 127ctggcgtaga
gcacttacgc tccagggact gtacaccgta tgcagcgtg 4912821DNAArtificial
Sequenceprimer 128gcgtatcagc agctcatgta a 2112949DNAArtificial
Sequenceprimer 129ctggcgtaga gcacttacgc tccaggacaa actcgacgtc
gtctcggaa 4913025DNAArtificial Sequenceprimer 130caggtcacca
caacaaaggc tccgt 2513149DNAArtificial Sequenceprimer 131atcaggacgc
agccggttct ccaggccaag gacaggtacg gctgtcatc 4913224DNAArtificial
Sequenceprimer 132ggtgcccttg aggttgtcca ggtg 2413345DNAArtificial
SequenceSCO 133gagacgttta agtccgcgac cgctctctga tagcgactgc tcgca
4513445DNAArtificial SequenceSCO 134caggcgacgt ccatatggtg
cgctatcggt ctcctgaaag ctgcg 4513539DNAArtificial SequenceSCO
135cccttaggta acgtctggct ggcgtagagc acttacgct 3913638DNAArtificial
SequenceSCO 136aaactttaat tattgtatat caggacgcag ccggttct
38137118DNAArtificial Sequenceproduct 137ctctgatagc gactgctcgc
accaggataa tataaggggt cggtggaccg gtcgatgtat 60gtcttgttgc agatcatcaa
gaacacgtag agaaacccag ctgtaatcat gcatggag 118138236DNAArtificial
Sequenceproduct 138atcggtctcc tgaaagctgc gccaggcacg acaggaacga
ctccaacgac gcagagaaac 60acaagtataa tattaagtat gcatggacct aaggcaacat
tgcaagacat tgtattgcat 120ttagagcccc aaaatgaaat tccggttgac
cttctatgtc acgagcaatt aagcgactca 180gaggaagaaa acgatgaaat
agatggagtt aatcatcaac atttaccagc ccgacg 236139352DNAArtificial
Sequenceproduct 139ctggcgtaga gcacttacgc tccaggacgc catgagagga
cacaagccaa cgttaaagga 60atatgtttta gatttatatc ctgaaccaac tgacctatac
tgctatgagc aattaagtga 120cagctcagat gaggatgaag gcttggaccg
gccagatgga caagcacaac cagccacagc 180tgattactac attgtaacct
gttgtcacac ttgtaacacc acagttcgtt tatgtgtcaa 240cagtacagca
agtgacctac gaaccataca gcaactactt atgggcacag tgaatattgt
300gtgccctacc tgtgcacaac aataaacatc atctacaatg gccgatcctg aa
352140419DNAArtificial Sequenceproduct 140ctggcgtaga gcacttacgc
tccaggagga cccagctgaa cgaccttaca aactgcatga 60tttgtgcaac gaggtagaag
aaagcatcca tgaaatttgt ttgaattgtg tatactgcaa 120acaagaatta
cagcggagtg aggtatatga ctttgcatgc tatgatttgt gtatagtata
180tagagaaggc cagccatatg gagtatgcat gaaatgttta aaattttatt
caaaaataag 240tgaatataga tggtatagat atagtgtgta tggagaaacg
ttagaaaaac aatgcaacaa 300acagttatgt catttattaa ttaggtgtat
tacatgtcaa aaaccgctgt gtccagttga 360aaagcaaaga catttagaag
aaaaaaaacg attccataac atcggtggac ggtggacag 419141327DNAArtificial
Sequenceproduct 141ctggcgtaga gcacttacgc tccagggctg gcaacgtaca
cgacaacgta acgaaaccca 60agtgtaataa agccatgcgt ggtaatgtac cacaattaaa
agatgtagta ttgcatttaa 120caccacagac tgaaattgac ttgcaatgct
acgagcaatt tgacagctca gaggaggagg 180atgaagtaga taatatgcgt
gaccagctac cagaaagacg ggctggacag gctacgtgtt 240acagaattga
agctccgtgt tgcaggtgtt caagtgtagt acaactggca gtggaaagca
300gtggagacac ccttcgcgtt gtacagc 327142211DNAArtificial
Sequenceproduct 142ctggcgtaga gcacttacgc tccaggtcca cctatgcacc
gaaacctcca agacctccgc 60attgtccgtg ggtgccaaag acacacacct acaaccacca
cagaaacgac gacgaccaga 120cgtcacagac tccagaaaca ccaagtaccc
caacaacctt ttgcggggac aacaatccgt 180ggacagtact acacggggac
tcgtcactgc a 211143167DNAArtificial Sequenceproduct 143ctggcgtaga
gcacttacgc tccagggtta agaccgaaaa cggtgcatat aaaggtagtt 60agaaagaaaa
gggcaacggc atggcacgct ttgaggatcc tacacaacga ccatacaaac
120tgcctgactt gagcacaaca ttgaatattc ctctgcatga tattcgc
167144179DNAArtificial Sequenceproduct 144ctggcgtaga gcacttacgc
tccaggatgg cgctatttca caaccctgag gaacggccat 60acaaattgcc agacctgtgc
aggacattgg acactacatt gcatgacgtt acaatagagt 120gtgtctattg
cagaaggcaa ctacaacgga cagaggtata tgaatttgcc tttagtgac
179145277DNAArtificial Sequenceproduct 145gctcatatgc ggcgccattt
accagggcag gttgctatca aggttacaag acaggtttaa 60ggagaccaat agaaactggg
catgtggaga cagagaagac tcttgggttt ctgataggca 120ctgactctct
ctgcctattg gtctattttc ccacccttag gctgctggtg gtctaccctt
180ggacccagag gttctttgag tcctttgggg atctgtccac tcctgatgct
gttatgggca 240accctaaggt gaaggctcat ggcaagaaag tgctcgg
27714621DNAArtificial SequenceCCTF 146tgcgagcagt cgctatcaga g
2114721DNAArtificial SequenceCCTF 147cgcagctttc aggagaccga t
2114821DNAArtificial SequenceCCTF 148agcgtaagtg ctctacgcca g
2114920DNAArtificial SequenceCCTF 149agaaccggct gcgtcctgat
2015026DNAArtificial Sequenceprimer 150ttgctatggc tgacggggaa gaatgg
2615156DNAArtificial Sequenceprimer 151gccccgttga gagcacgaat
ccaggggggt gaatcttctg cttaatgtga agacac 5615226DNAArtificial
Sequenceprimer 152gggcaccatg cagtaccaaa cggaac 2615352DNAArtificial
Sequenceprimer 153ccgtggcgcg aacttatcga ccaggatcac actgagggtc
tcccaataga gc 5215429DNAArtificial Sequenceprimer 154tcaaagacta
agtggtgcca tggatgaac 2915550DNAArtificial Sequenceprimer
155aagtgacctg ccattgcgcg ccaggtatgt ctacagcaga gggacccagc
5015649DNAArtificial Sequenceprimer 156ggcttagagc accgcgtcat
tccaggtgtc gctactggaa gcggtgatc 4915724DNAArtificial Sequenceprimer
157gcgatagcta aggtacgacg ggtc 2415829DNAArtificial Sequenceprimer
158gtagattcga tccatgctcc tctactacc 2915955DNAArtificial
Sequenceprimer 159cgtcttacat gcgcaagcgg ccaggtgata ttgagttcgg
taatgcaaga tctgc 5516027DNAArtificial Sequenceprimer 160ccatagagat
ggcaatagat gaagagc 2716152DNAArtificial Sequenceprimer
161aggcgttccg cttcaacgag ccaggttgtc agattctgta gcttgctcag tc
5216229DNAArtificial Sequenceprimer 162ggtggtgatc ccaacttgtt
atatcgaag 2916356DNAArtificial Sequenceprimer 163tccgtctgcg
aagatctgag cccaggttca atctatcrtc tgacagatct tgaagt
5616454DNAArtificial Sequenceprimer 164gtgtcacgac gcgcgaatct
ccaggagatc gtgaccagta taatagctca acac 5416528DNAArtificial
Sequenceprimer 165tttcagacaa tgcagggata acaccagc
2816650DNAArtificial Sequenceprimer 166cccagaacga tttgcggcgt
ccaggcttgg tcctctctta ggaggcaagc 5016722DNAArtificial
Sequenceprimer 167aggatgcttc ggagtacctg ag 2216845DNAArtificial
Sequenceprimer 168tgcattgccg tcgcagagac ccaggcaacg ggcacgaagc gcatc
4516928DNAArtificial Sequenceprimer 169gccctaatga taagacaggc
agttgtgg 2817052DNAArtificial Sequenceprimer 170atgcgcttgg
attgccgatg ccaggagccc tgttagttct ggatgctgaa ca 5217135DNAArtificial
SequenceSCO 171cttatagatt atattgcccc gttgagagca cgaat
3517242DNAArtificial SequenceSCO 172ctaagtaagc ctatatcgaa
ttccgtggcg cgaacttatc ga 4217342DNAArtificial SequenceSCO
173cgtactgcac tcgcctacga ctaagtgacc tgccattgcg cg
4217435DNAArtificial SequenceSCO 174cttataagtt acatggctta
gagcaccgcg tcatt 3517534DNAArtificial SequenceSCO 175ctaattgtaa
tactcgtctt acatgcgcaa gcgg 3417642DNAArtificial SequenceSCO
176ctaatcgtat gagatctatg ataggcgttc cgcttcaacg ag
4217735DNAArtificial SequenceSCO 177tcatagacat ttattccgtc
tgcgaagatc tgagc 3517842DNAArtificial SequenceSCO 178tacgaatctg
acctagtaag atgtgtcacg acgcgcgaat ct 4217942DNAArtificial
SequenceSCO 179tgccactaac aggccgctag atcccagaac gatttgcggc gt
4218041DNAArtificial SequenceSCO 180tcgagcgtgc gccagatcca
ttgcattgcc gtcgcagaga c 4118142DNAArtificial SequenceSCO
181tcgactgtgc ctgcgtccgt atatgcgctt ggattgccga tg
42182219DNAArtificial Sequenceproduct 182ttgctatggc tgacggggaa
gaatggtttg tacccaaacc tgagcatgtc ctatgtaaac 60aacaaagaga aagaagtcct
tgtgctatgg ggtgttcatc acccacctaa catagggaac 120caaagggccc
tctaccatac agaaaatgct tatgtctctg tagtgtcttc acattatagc
180agaagattca cccccctgga ttcgtgctct caacggggc
219183211DNAArtificial Sequenceproduct 183gggcaccatg cagtaccaaa
cggaacgata gtgaaaacaa tcacaaatga ccaaattgaa 60gttactaatg ctactgagtt
ggttcagaat tcctcaatag gtgaaatatg cgacagtcct 120catcagatcc
ttgatggaga gaactgcaca ctaatagatg ctctattggg agaccctcag
180tgtgatcctg gtcgataagt tcgcgccacg g 211184236DNAArtificial
Sequenceproduct 184tcaaagacta agtggtgcca tggatgaact ccacaacgaa
atactcgagc tggatgaaaa 60agtggatgac ctcagagctg acactataag ctcacaaata
gaacttgcag tcttgctttc 120caacgaagga ataataaaca gtgaagatga
gcatctattg gcacttgaga gaaaactaaa 180gaaaatgctg ggtccctctg
ctgtagacat acctggcgcg caatggcagg tcactt 236185161DNAArtificial
Sequenceproduct 185ggcttagagc accgcgtcat tccaggtgtc gctactggaa
gcggtgatcc gcacagtgac 60gactttacag caattgctta cttaagggac gaattgctcg
caaagcatcc gaccttaggt 120tctggtaatg acgaggcgac ccgtcgtacc
ttagctatcg c 161186244DNAArtificial Sequenceproduct 186gtagattcga
tccatgctcc tctactacca tggtccagcc gactgagaca agggatgata 60tataatgcca
ataaagtagc tctggcaccc caatgtctcc cagtcgacaa agatatcaga
120ttcagagttg tatttgtcaa cggaacatca ctgggtacaa tcacaattgc
caaggtccca 180aaaactcttg cagatcttgc attaccgaac tcaatatcac
ctggccgctt gcgcatgtaa 240gacg 244187179DNAArtificial
Sequenceproduct 187ccatagagat ggcaatagat gaagagccag aacaattcga
acatagagca gaccaagaac 60aagatgggga acctcaatca tctataatcc aatatgcttg
ggcagaagga aacagaagcg 120atgaccggac tgagcaagct acagaatctg
acaacctggc tcgttgaagc ggaacgcct 179188178DNAArtificial
Sequenceproduct 188ggtggtgatc ccaacttgtt atatcgaagt ttctatagaa
gaactcctga tttcctcaca 60gaggctatag ttcactctgt gttcatactt agttattata
caaaccatga tttaaaggat 120aaacttcaag atctgtcaga ygatagattg
aacctgggct cagatcttcg cagacgga 178189138DNAArtificial
Sequenceproduct 189ggtggtgatc ctaatttgtt atatcgaagc ttttatagga
gaactccaga cttccttaca 60gaagctatag tacattcagt gttcgtgttg agctattata
ctggtcacga tctcctggag 120attcgcgcgt cgtgacac 138190328DNAArtificial
Sequenceproduct 190tttcagacaa tgcagggata acaccagcaa tatcattgga
cctaatgact gatgctgaac 60tggccagagc tgtatcatac atgccaacat ctgcagggca
gataaagctg atgttggaga 120accgcgcaat ggtaaggaga aaaggatttg
gaatcctaat aggggtctac ggaagctctg 180tgatttacat ggttcaattg
ccgatctttg gtgtcataga tacaccttgt tggataatca 240aggcagctcc
ctcttgctca gaaaaaaacg ggaattatgc ttgcctccta agagaggacc
300aagcctggac gccgcaaatc gttctggg 328191187DNAArtificial
Sequenceproduct 191aggatgcttc ggagtacctg agtccgggtc tggtgcagtt
cgcccgtgca acagacacct 60acttcagtat ggggaacaag tttagaaacc ccacagtggc
gcccacccac gatgtgacca 120ccgaccgtag ccagcgactg atgctgcgct
tcgtgcccgt tgcctgggtc tctgcgacgg 180caatgca 187192290DNAArtificial
Sequenceproduct 192gccctaatga taagacaggc agttgtggtc cagtatcgtc
taatggagca aatggagtaa 60aaggattttc attcaaatac ggcaatggtg tttggatagg
gagaactaaa agcattagtt 120caagaaaagg ttttgagatg atttgggatc
cgaatggatg gactgggact gacaataaat 180tctcaataaa gcaagatatc
gtaggaataa atgagtggtc agggtatagc gggagttttg 240ttcagcatcc
agaactaaca gggctcctgg catcggcaat ccaagcgcat 29019320DNAArtificial
SequenceCCTF 193attcgtgctc tcaacggggc 2019420DNAArtificial
SequenceCCTF 194tcgataagtt cgcgccacgg 2019520DNAArtificial
SequenceCCTF 195cgcgcaatgg caggtcactt 2019621DNAArtificial
SequenceCCTF 196aatgacgcgg tgctctaagc c 2119720DNAArtificial
SequenceCCTF 197ccgcttgcgc atgtaagacg 2019820DNAArtificial
SequenceCCTF 198ctcgttgaag cggaacgcct 2019921DNAArtificial
SequenceCCTF 199gctcagatct tcgcagacgg a 2120020DNAArtificial
SequenceCCTF 200agattcgcgc gtcgtgacac 2020120DNAArtificial
SequenceCCTF 201acgccgcaaa tcgttctggg 2020220DNAArtificial
SequenceCCTF 202gtctctgcga cggcaatgca 2020320DNAArtificial
SequenceCCTF 203catcggcaat ccaagcgcat 2020457DNAArtificial
Sequenceprimer 204acgaggcctg tccgcttact agccaggctg gtcctcatcc
aacagctctt ctatcgc 5720559DNAArtificial Sequenceprimer
205ccgggtacgc taagtccgct atccaggttc tggtcctcat ccaacagctc ttctatcgt
5920627DNAArtificial Sequenceprimer 206gacccatggg actctggaga
gcgtgaa 2720754DNAArtificial Sequenceprimer 207gctcatatgc
ggcgccattt accagggcag gttgctatca aggttacaag acag
5420824DNAArtificial Sequenceprimer 208ccgagcactt tcttgccatg agcc
2420944DNAArtificial SequenceSCO 209gtagcacgct tcgaatggct
atacgaggcc tgtccgctta ctag 4421045DNAArtificial SequenceSCO
210gatacggagg tccgaaggca gtgttggtta ccctaacgcg ccgga
4521145DNAArtificial SequenceSCO 211attagtttaa ctattatatt
ttatgctcat atgcggcgcc attta 45212139DNAArtificial Sequenceproduct
212acgaggcctg tccgcttact agccaggctg gtcctcatcc aacagctctt
ctatcacgtg 60ttcgaaagtg tcagccaatg atgtcaagcc tcttgaacct gccttgggcc
cattcacgct 120ctccagagtc ccatgggtc 139213141DNAArtificial
Sequenceproduct 213ccgggtacgc taagtccgct atccaggttc tggtcctcat
ccaacagctc ttctatcacg 60tgttcgaaag tgtcagccaa tgatgtcaag cctcttgaac
ctgccttggg cccattcacg 120ctctccagag tcccatgggt c
141214277DNAArtificial Sequenceproduct 214gctcatatgc ggcgccattt
accagggcag gttgctatca aggttacaag acaggtttaa 60ggagaccaat agaaactggg
catgtggaga cagagaagac tcttgggttt ctgataggca 120ctgactctct
ctgcctattg gtctattttc ccacccttag gctgctggtg gtctaccctt
180ggacccagag gttctttgag tcctttgggg atctgtccac tcctgatgct
gttatgggca 240accctaaggt gaaggctcat ggcaagaaag tgctcgg
27721522DNAArtificial SequenceCCTF 215ctagtaagcg gacaggcctc gt
2221622DNAArtificial SequenceCCTF 216atagcggact tagcgtaccc gg
2221720DNAArtificial SequenceCCTF 217taaatggcgc cgcatatgag
2021848DNAArtificial Sequenceprimer 218ctcatcgcca cgagccggtt
aaccaggttg aaacaccgcc cggaaccc 4821923DNAArtificial Sequenceprimer
219gctccttatt cggtttgacc ggt 2322055DNAArtificial Sequenceprimer
220gctcgcaggt acggcaccat tcaccaggca gaaggtatga taacaacggt agagc
5522121DNAArtificial Sequenceprimer 221cccctttgca ccgttgaggg g
2122257DNAArtificial Sequenceprimer 222agtcgattat gtctgaggcc
gcgccaggtt aaagtagcat atgatcaagc tcattca 5722335DNAArtificial
Sequenceprimer 223gatcctgaca tataatcatt atctcctttt ataaa
3522423DNAArtificial SequenceSCO 224tctcatcgcc acgagccggt taa
2322523DNAArtificial SequenceSCO 225tgtcgcaggt acggcaccat tca
2322624DNAArtificial SequenceSCO 226tagtcgatta tgtctgaggc cgcg
24227112DNAArtificial Sequenceproduct 227ctcatcgcca cgagccggtt
aaccaggttg aaacaccgcc cggaacccga tataatccgc 60ccttcaacat cagtgaaaat
ctttttttaa ccggtcaaac cgaataagga gc 112228109DNAArtificial
Sequenceproduct 228gctcgcaggt acggcaccat tcaccaggca gaaggtatga
taacaacggt agagctttat 60atgatattaa cttagcaaaa atggaaaacc cctcaacggt
gcaaagggg 109229115DNAArtificial Sequenceproduct 229agtcgattat
gtctgaggcc gcgccagggt ttctgtacac gatccaatty acaaataaca 60tttacaattc
gtaaaatttt tttataaaag gagataatga ttatatgtca ggatc
11523027DNAArtificial SequenceSCO 230cctggttaac cggctcgtgg cgatgag
2723128DNAArtificial SequenceSCO 231cctggtgaat ggtgccgtac ctgcgagc
2823228DNAArtificial SequenceSCO 232cctggcgcgg cctcagacat aatcgact
28
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