CN114921527A - Probe primer group for RNA in-situ detection and application thereof - Google Patents
Probe primer group for RNA in-situ detection and application thereof Download PDFInfo
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Abstract
The invention discloses a probe primer group for RNA in-situ detection and application thereof, wherein a sample for RNA in-situ detection is a cell cultured in vitro and a cell in a tissue slice, and the probe primer group is characterized in that: comprising at least one loop-forming probe for forming a rolling circle amplification product and first to fourth detection probe sets capable of hybridizing to the rolling circle amplification product. The invention can improve the connection efficiency of the detection probe and the anchoring primer, enhance the fluorescence signal, preferably fix the GC content of the variable label sequence to 50 percent, has better specificity and can realize the in-situ detection of multiple RNAs.
Description
Technical Field
The invention belongs to the technical field of space transcriptomics, and particularly relates to a probe primer group for RNA in-situ detection and application thereof.
Background
Spatial transcriptomics technology enables to localize and differentiate active expression of functional genes in specific tissue regions, thus providing important information for basic research and clinical diagnostics. As a novel breakthrough omics research technology, the method can detect the gene activity condition in different microenvironments in a tissue sample and draw a spatial map of activity gene expression. Current spatial transcriptomics methods are mainly classified into two major categories, sequencing-based methods and microscopic imaging-based methods. The former is a series of methods based on primer coding spatial information, and the spatial position information of genes can be obtained by adding a position information tag to cDNA and sequencing. The latter is to perform in situ sequencing or multiple rounds of single molecule fluorescence in situ hybridization and imaging on the original position of RNA in the cell or tissue, so as to obtain a string of signals coded by different fluorescence colors to detect different genes and directly obtain the spatial position information of the detected genes.
The in situ sequencing technology is a targeted space transcriptomics technology, and a known gene is targeted by using probe cyclization and rolling circle amplification, so that the number of detected genes is small. However, because of its detection, single cell, and even sub-cell resolution, it is helpful to confirm the identification of spatially-characterized gene expression region markers and cell types. The in situ sequencing technology achieves the purpose of signal Amplification through Rolling Circle Amplification (RCA). RCA is a multi-primer in vitro nucleic acid exponential amplification technology provided by referring to the rolling circle replication mode of pathogenic organisms, can directly amplify specific DNA and RNA molecules and simultaneously realize the amplification of target nucleic acid signals, and the sensitivity of the RCA can reach one copy number of nucleic acid molecules. DNA barcodes corresponding to different genes are contained in the RCA amplification product, and the types of genes detected by the RCA product can be known through sequencing chemical decoding.
Disclosure of Invention
The invention aims to provide a probe primer group for RNA in-situ detection.
The invention also aims to provide an RNA in-situ detection kit.
Still another object of the present invention is to provide the use of the probe primer set for in situ detection of RNA for non-diagnostic therapeutic purposes.
It is still another object of the present invention to provide a method for in situ detection of RNA for non-diagnostic therapeutic purposes.
The technical scheme of the invention is as follows:
a probe primer set for in situ detection of RNA in a sample of ex vivo cultured cells and cells in a tissue section, comprising at least one loop-forming probe for forming a rolling circle amplification product and first to fourth detection probe sets capable of hybridizing to the rolling circle amplification product;
each of the loop forming probes corresponds to a target sequence, the sequence of the 5 'end and the 3' end of the loop forming probe is specifically hybridized and complemented with the target sequence, and a first label sequence and a second label sequence are arranged between the 5 'end and the 3' end of the loop forming probe, the first label sequence is positioned at the 3 'end of the sequence of the 5' end which is specifically hybridized and complemented with the target sequence, and the second label sequence is positioned at the 5 'end of the sequence of the 3' end which is specifically hybridized and complemented with the target sequence; the first label sequence consists of a first fixed label sequence and a first variable label sequence, the second label sequence consists of a second fixed label sequence and a second variable label sequence, and the first variable label sequence and the second variable label sequence are four-base-length sequences consisting of double-base label sequences;
a first detection probe group consisting of a plurality of first detection probe subgroups, wherein the length of each first detection probe is the same as that of the first tag sequence, the first detection probe in a first detection probe subgroup is completely the same as one of the two-base tag sequences of the first fixed tag sequence and the first variable tag sequence, the remaining two bases are degenerate sequences, the first detection probe corresponding to the different one of the two-base tag sequences is modified with different report labels, and the report labels modified by the first detection probe in the same first detection probe subgroup are the same;
the second detection probe group consists of a plurality of second detection probe subgroups, wherein the length of each second detection probe is the same as that of the first label sequence, the second detection probe in one second detection probe subgroup is completely the same as the other two-base label sequence of the first fixed label sequence and the first variable label sequence, the rest two bases are degenerate sequences, the second detection probe corresponding to the other different two-base label sequence is modified with different report labels, and the report labels modified by the second detection probe in the same second detection probe subgroup are the same;
a third detection probe group, which consists of a plurality of third detection probe subgroups, wherein the length of each third detection probe is the same as that of the first label sequence, the third detection probe in a third detection probe subgroup is completely the same as one double-base label sequence of the second fixed label sequence and the second variable label sequence, the rest two bases are degenerate sequences, the third detection probes corresponding to different one of the double-base label sequences are modified with different report labels, and the report labels modified by the third detection probes in the same third detection probe subgroup are the same;
and the fourth detection probe group consists of a plurality of fourth detection probe subgroups, wherein the length of each fourth detection probe is the same as that of the second label sequence, the fourth detection probe in one fourth detection probe subgroup is completely the same as the other two-base label sequence of the second fixed label sequence and the second variable label sequence, the rest two bases are degenerate sequences, the fourth detection probe corresponding to the other different two-base label sequence is modified with different report labels, and the report labels modified by the fourth detection probe in the same fourth detection probe subgroup are the same.
Preferably, the first variable tag sequence consists of a first double-base tag sequence and a second double-base tag sequence, the first double-base tag sequence is cw, as, ts or gw, the second variable tag sequence consists of a third double-base tag sequence and a fourth double-base tag sequence, the third double-base tag sequence is cw, as, ts or gw, the fourth double-base tag sequence is cw, as, ts or gw,
the first detection probe group consists of a plurality of first detection probe subgroups, wherein the length of each first detection probe is the same as that of the first label sequence, the first detection probe in one first detection probe subgroup is completely the same as the first fixed label sequence and the first double-base label sequence in the first variable label sequence, the rest two bases are cw, as, ts or gw, different report labels are modified on the first detection probes corresponding to the different first double-base label sequences, and the report labels modified by the first detection probe in the same first detection probe subgroup are the same;
the second detection probe group consists of a plurality of second detection probe subgroups, wherein the length of each second detection probe is the same as that of the first label sequence, the second detection probe in one second detection probe subgroup is completely the same as the second double-base label sequence in the first fixed label sequence and the first variable label sequence, the rest two bases are cw, as, ts or gw, different report labels are modified by the second detection probes corresponding to the different second double-base label sequences, and the report labels modified by the second detection probes in the same second detection probe subgroup are the same;
a third detection probe group, which consists of a plurality of third detection probe subgroups, wherein the length of each third detection probe is the same as that of the second label sequence, the third detection probe in a third detection probe subgroup is completely the same as the third double-base label sequence of the second fixed label sequence and the second variable label sequence, the remaining two bases are cw, as, ts or gw, the third detection probes corresponding to the different third double-base label sequences are modified with different report labels, and the report labels modified by the third detection probes in the same third detection probe subgroup are the same;
and the fourth detection probe group consists of a plurality of fourth detection probe subgroups, wherein the length of each fourth detection probe is the same as that of the second label sequence, the fourth detection probes in one fourth detection probe subgroup are completely the same as the fourth double-base label sequences of the second fixed label sequence and the second variable label sequence, the rest two bases are cw, as, ts or gw, the fourth detection probes corresponding to the different fourth double-base label sequences are modified with different report labels, and the report labels modified by the fourth detection probes in the same fourth detection probe subgroup are the same.
In a preferred embodiment of the invention, the loop-forming probe is a double-ligated probe or a padlock probe.
Further preferably, the ring-forming probe is a double-ligation probe, and the kit further comprises a splint primer for forming a ring after hybridization of the double-ligation probe.
Still further preferably, the kit further comprises a first anchor primer and a second anchor primer, wherein the first anchor primer is capable of strictly complementary pairing with the rolling circle amplification product and is capable of being linked to the hybridized first and second detection probes under the action of a DNA polymerase, and the second anchor primer is capable of strictly complementary pairing with the rolling circle amplification product and is capable of being linked to the hybridized third and fourth detection probes under the action of a DNA polymerase.
In a preferred embodiment of the invention, the reporter label is a fluorescent label, a chromogenic label, a radioactive label, a magnetic label or a luminescent density label.
The other technical scheme of the invention is as follows:
an RNA in situ detection kit, which is provided with at least one loop-forming probe for forming a rolling circle amplification product and first to fourth detection probe sets capable of hybridizing with the rolling circle amplification product;
each ring-forming probe corresponds to a target sequence, the sequence of the 5 'end and the 3' end of the ring-forming probe is specifically hybridized and complemented with the target sequence, and a first label sequence and a second label sequence are arranged between the 5 'end and the 3' end of the ring-forming probe, the first label sequence is positioned at the 3 'end of the sequence of the 5' end specifically hybridized and complemented with the target sequence, and the second label sequence is positioned at the 5 'end of the sequence of the 3' end specifically hybridized and complemented with the target sequence; the first tag sequence consists of a first fixed tag sequence and a first variable tag sequence, the second tag sequence consists of a second fixed tag sequence and a second variable tag sequence, and the first variable tag sequence and the second variable tag sequence are four-base-length sequences consisting of double-base tag sequences;
the first detection probe group consists of a plurality of first detection probe subgroups, wherein the length of each first detection probe is the same as that of the first label sequence, the first detection probe in one first detection probe subgroup is completely the same as one double-base label sequence of the first fixed label sequence and the first variable label sequence, the rest two bases are degenerate sequences, the first detection probes corresponding to different one double-base label sequences are modified with different report labels, and the report labels modified by the first detection probes in the same first detection probe subgroup are the same;
the second detection probe group consists of a plurality of second detection probe subgroups, wherein the length of each second detection probe is the same as that of the first label sequence, the second detection probe in one second detection probe subgroup is completely the same as the other two-base label sequence of the first fixed label sequence and the first variable label sequence, the rest two bases are degenerate sequences, the second detection probe corresponding to the other different two-base label sequence is modified with different report labels, and the report labels modified by the second detection probe in the same second detection probe subgroup are the same;
a third detection probe group, which consists of a plurality of third detection probe subgroups, wherein the length of each third detection probe is the same as that of the first label sequence, the third detection probe in a third detection probe subgroup is completely the same as one double-base label sequence of the second fixed label sequence and the second variable label sequence, the rest two bases are degenerate sequences, the third detection probes corresponding to different one of the double-base label sequences are modified with different report labels, and the report labels modified by the third detection probes in the same third detection probe subgroup are the same;
and the fourth detection probe group consists of a plurality of fourth detection probe subgroups, wherein the length of each fourth detection probe is the same as that of the second label sequence, the fourth detection probes in one fourth detection probe subgroup are completely the same as the other two-base label sequence of the second fixed label sequence and the second variable label sequence, the rest two bases are degenerate sequences, the fourth detection probes corresponding to different two-base label sequences are modified with different report labels, and the report labels modified by the fourth detection probes in the same fourth detection probe subgroup are the same.
Preferably, the first variable tag sequence consists of a first double-base tag sequence and a second double-base tag sequence, the first double-base tag sequence is cw, as, ts or gw, the second variable tag sequence consists of a third double-base tag sequence and a fourth double-base tag sequence, the third double-base tag sequence is cw, as, ts or gw, the fourth double-base tag sequence is cw, as, ts or gw,
the first detection probe group consists of a plurality of first detection probe subgroups, wherein the length of each first detection probe is the same as that of the first label sequence, the first detection probe in one first detection probe subgroup is completely the same as the first fixed label sequence and the first double-base label sequence in the first variable label sequence, the rest two bases are cw, as, ts or gw, different report labels are modified on the first detection probes corresponding to the different first double-base label sequences, and the report labels modified by the first detection probe in the same first detection probe subgroup are the same;
the second detection probe group consists of a plurality of second detection probe subgroups, wherein the length of each second detection probe is the same as that of the first label sequence, the second detection probe in one second detection probe subgroup is completely the same as the second double-base label sequence in the first fixed label sequence and the first variable label sequence, the rest two bases are cw, as, ts or gw, different report labels are modified by the second detection probes corresponding to the different second double-base label sequences, and the report labels modified by the second detection probes in the same second detection probe subgroup are the same;
a third detection probe group, which consists of a plurality of third detection probe subgroups, wherein the length of each third detection probe is the same as that of the second label sequence, the third detection probe in a third detection probe subgroup is completely the same as the third double-base label sequence of the second fixed label sequence and the second variable label sequence, the remaining two bases are cw, as, ts or gw, the third detection probes corresponding to the different third double-base label sequences are modified with different report labels, and the report labels modified by the third detection probes in the same third detection probe subgroup are the same;
and the fourth detection probe group consists of a plurality of fourth detection probe subgroups, wherein the length of each fourth detection probe is the same as that of the second label sequence, the fourth detection probes in one fourth detection probe subgroup are completely the same as the fourth double-base label sequences of the second fixed label sequence and the second variable label sequence, the rest two bases are cw, as, ts or gw, the fourth detection probes corresponding to the different fourth double-base label sequences are modified with different report labels, and the report labels modified by the fourth detection probes in the same fourth detection probe subgroup are the same.
In a preferred embodiment of the invention, the loop-forming probe is a double-ligated probe or a padlock probe.
Further preferably, the ring-forming probe is a double-ligation probe, and further comprises a splint primer for forming a ring after hybridization by matching with the double-ligation probe.
Still further preferably, the kit further comprises a first anchor primer and a second anchor primer, wherein the first anchor primer is capable of strictly complementary pairing with the rolling circle amplification product and is capable of being linked to the hybridized first and second detection probes under the action of a DNA polymerase, and the second anchor primer is capable of strictly complementary pairing with the rolling circle amplification product and is capable of being linked to the hybridized third and fourth detection probes under the action of a DNA polymerase.
In a preferred embodiment of the invention, the reporter label is a fluorescent label, a chromogenic label, a radioactive label, a magnetic label or a luminescent density label.
The invention adopts another technical scheme as follows:
use of a probe primer set for in situ detection of RNA for non-diagnostic therapeutic purposes, the sample for in situ detection of RNA being cells cultured ex vivo and cells in a tissue section, the probe primer set comprising at least one loop-forming probe for forming a rolling circle amplification product and a first to fourth detection probe set capable of hybridising to the rolling circle amplification product;
each of the loop forming probes corresponds to a target sequence, the sequence of the 5 'end and the 3' end of the loop forming probe is specifically hybridized and complemented with the target sequence, and a first label sequence and a second label sequence are arranged between the 5 'end and the 3' end of the loop forming probe, the first label sequence is positioned at the 3 'end of the sequence of the 5' end which is specifically hybridized and complemented with the target sequence, and the second label sequence is positioned at the 5 'end of the sequence of the 3' end which is specifically hybridized and complemented with the target sequence; the first label sequence consists of a first fixed label sequence and a first variable label sequence, the second label sequence consists of a second fixed label sequence and a second variable label sequence, and the first variable label sequence and the second variable label sequence are four-base-length sequences consisting of double-base label sequences;
the first detection probe group consists of a plurality of first detection probe subgroups, wherein the length of each first detection probe is the same as that of the first label sequence, the first detection probe in one first detection probe subgroup is completely the same as one double-base label sequence of the first fixed label sequence and the first variable label sequence, the rest two bases are degenerate sequences, the first detection probes corresponding to different one double-base label sequences are modified with different report labels, and the report labels modified by the first detection probes in the same first detection probe subgroup are the same;
the second detection probe group consists of a plurality of second detection probe subgroups, wherein the length of each second detection probe is the same as that of the first label sequence, the second detection probe in one second detection probe subgroup is completely the same as the other two-base label sequence of the first fixed label sequence and the first variable label sequence, the rest two bases are degenerate sequences, the second detection probe corresponding to the other different two-base label sequence is modified with different report labels, and the report labels modified by the second detection probe in the same second detection probe subgroup are the same;
a third detection probe group, which consists of a plurality of third detection probe subgroups, wherein the length of each third detection probe is the same as that of the first label sequence, the third detection probe in a third detection probe subgroup is completely the same as one double-base label sequence of the second fixed label sequence and the second variable label sequence, the rest two bases are degenerate sequences, the third detection probes corresponding to different one of the double-base label sequences are modified with different report labels, and the report labels modified by the third detection probes in the same third detection probe subgroup are the same;
and the fourth detection probe group consists of a plurality of fourth detection probe subgroups, wherein the length of each fourth detection probe is the same as that of the second label sequence, the fourth detection probes in one fourth detection probe subgroup are completely the same as the other two-base label sequence of the second fixed label sequence and the second variable label sequence, the rest two bases are degenerate sequences, the fourth detection probes corresponding to different two-base label sequences are modified with different report labels, and the report labels modified by the fourth detection probes in the same fourth detection probe subgroup are the same.
Preferably, the first variable tag sequence consists of a first double-base tag sequence and a second double-base tag sequence, the first double-base tag sequence is cw, as, ts or gw, the second variable tag sequence consists of a third double-base tag sequence and a fourth double-base tag sequence, the third double-base tag sequence is cw, as, ts or gw, the fourth double-base tag sequence is cw, as, ts or gw,
the first detection probe group consists of a plurality of first detection probe subgroups, wherein the length of each first detection probe is the same as that of the first label sequence, the first detection probe in one first detection probe subgroup is completely the same as the first fixed label sequence and the first double-base label sequence in the first variable label sequence, the rest two bases are cw, as, ts or gw, different report labels are modified on the first detection probes corresponding to the different first double-base label sequences, and the report labels modified by the first detection probe in the same first detection probe subgroup are the same;
the second detection probe group consists of a plurality of second detection probe subgroups, wherein the length of each second detection probe is the same as that of the first label sequence, the second detection probe in one second detection probe subgroup is completely the same as the second double-base label sequence in the first fixed label sequence and the first variable label sequence, the rest two bases are cw, as, ts or gw, different report labels are modified by the second detection probes corresponding to the different second double-base label sequences, and the report labels modified by the second detection probes in the same second detection probe subgroup are the same;
a third detection probe group, which consists of a plurality of third detection probe subgroups, wherein the length of each third detection probe is the same as that of the second label sequence, the third detection probe in a third detection probe subgroup is completely the same as the third double-base label sequence of the second fixed label sequence and the second variable label sequence, the remaining two bases are cw, as, ts or gw, the third detection probes corresponding to the different third double-base label sequences are modified with different report labels, and the report labels modified by the third detection probes in the same third detection probe subgroup are the same;
and the fourth detection probe group consists of a plurality of fourth detection probe subgroups, wherein the length of each fourth detection probe is the same as that of the second label sequence, the fourth detection probes in one fourth detection probe subgroup are completely the same as the fourth double-base label sequences of the second fixed label sequence and the second variable label sequence, the rest two bases are cw, as, ts or gw, the fourth detection probes corresponding to the different fourth double-base label sequences are modified with different report labels, and the report labels modified by the fourth detection probes in the same fourth detection probe subgroup are the same.
In a preferred embodiment of the invention, the loop-forming probe is a double-ligated probe or a padlock probe.
Further preferably, the ring-forming probe is a double-ligation probe, and further comprises a splint primer for forming a ring after hybridization by matching with the double-ligation probe.
Still further preferably, the kit further comprises a first anchor primer and a second anchor primer, wherein the first anchor primer is capable of strictly complementary pairing with the rolling circle amplification product and is capable of being linked to the hybridized first and second detection probes under the action of a DNA polymerase, and the second anchor primer is capable of strictly complementary pairing with the rolling circle amplification product and is capable of being linked to the hybridized third and fourth detection probes under the action of a DNA polymerase.
In a preferred embodiment of the invention, the reporter label is a fluorescent label, a chromogenic label, a radioactive label, a magnetic label or a luminescent density label.
The invention also provides another technical scheme as follows:
an in situ detection method of RNA for non-diagnostic therapeutic purposes, comprising the following steps:
(1) designing at least one looping probe:
each of the loop forming probes corresponds to a target sequence, the sequence of the 5 'end and the 3' end of the loop forming probe is specifically hybridized and complemented with the target sequence, and a first label sequence and a second label sequence are arranged between the 5 'end and the 3' end of the loop forming probe, the first label sequence is positioned at the 3 'end of the sequence of the 5' end which is specifically hybridized and complemented with the target sequence, and the second label sequence is positioned at the 5 'end of the sequence of the 3' end which is specifically hybridized and complemented with the target sequence; the first tag sequence consists of a first fixed tag sequence and a first variable tag sequence, the second tag sequence consists of a second fixed tag sequence and a second variable tag sequence, and the first variable tag sequence and the second variable tag sequence are four-base-length sequences consisting of double-base tag sequences;
(2) designing first to fourth detection probe sets:
the first detection probe group consists of a plurality of first detection probe subgroups, wherein the length of each first detection probe is the same as that of the first label sequence, the first detection probe in one first detection probe subgroup is completely the same as one double-base label sequence of the first fixed label sequence and the first variable label sequence, the rest two bases are degenerate sequences, the first detection probes corresponding to different one double-base label sequences are modified with different report labels, and the report labels modified by the first detection probes in the same first detection probe subgroup are the same;
a second detection probe group consisting of a plurality of second detection probe subgroups, wherein the length of each second detection probe is the same as that of the first label sequence, the second detection probes in a second detection probe subgroup are completely the same as the other two-base label sequence of the first fixed label sequence and the first variable label sequence, the rest two bases are degenerate sequences, the second detection probes corresponding to the different other two-base label sequences are modified with different report labels, and the report labels modified by the second detection probes in the same second detection probe subgroup are the same;
a third detection probe group, which consists of a plurality of third detection probe subgroups, wherein the length of each third detection probe is the same as that of the first label sequence, the third detection probe in a third detection probe subgroup is completely the same as one double-base label sequence of the second fixed label sequence and the second variable label sequence, the rest two bases are degenerate sequences, the third detection probes corresponding to different one of the double-base label sequences are modified with different report labels, and the report labels modified by the third detection probes in the same third detection probe subgroup are the same;
a fourth detection probe group, which consists of a plurality of fourth detection probe subgroups, wherein the length of each fourth detection probe is the same as that of the second label sequence, the fourth detection probe in one fourth detection probe subgroup is completely the same as the other two-base label sequence of the second fixed label sequence and the second variable label sequence, the rest two bases are degenerate sequences, the fourth detection probe corresponding to the other different two-base label sequence is modified with different report labels, and the report labels modified by the fourth detection probe in the same fourth detection probe subgroup are the same;
(3) after the at least one circularization probe is specifically combined with at least one target sequence in a sample to be detected, the 3 ' end and the 5 ' end of the at least one circularization probe are adjacent end to end, the circularization probe and the 5 ' end are connected through DNA ligase to form at least one circular template, and then rolling circle amplification is carried out to obtain at least one rolling circle amplification product;
(4) hybridizing the at least one rolling circle amplification product with a first detection probe set for detection to obtain a first signal;
(5) removing the first signal, and hybridizing the first signal with a second detection probe group for detection to obtain a second signal;
(6) removing the second signal, and hybridizing the second signal with a second detection probe group for detection to obtain a third signal;
(7) removing the third signal, and hybridizing the third signal with the second detection probe group for detection to obtain a fourth signal;
(8) according to the permutation and combination of the first to the fourth signals, at least one signal code corresponding to the at least one target sequence is obtained.
Preferably, the first variable tag sequence consists of a first double-base tag sequence and a second double-base tag sequence, the first double-base tag sequence is cw, as, ts or gw, the second double-base tag is cw, as, ts or gw, the second variable tag sequence consists of a third double-base tag sequence and a fourth double-base tag sequence, the third double-base tag sequence is cw, as, ts or gw, the fourth double-base tag is cw, as, ts or gw,
the first detection probe group consists of a plurality of first detection probe subgroups, wherein the length of each first detection probe is the same as that of the first label sequence, the first detection probe in one first detection probe subgroup is completely the same as the first fixed label sequence and the first double-base label sequence in the first variable label sequence, the rest two bases are cw, as, ts or gw, different report labels are modified on the first detection probes corresponding to the different first double-base label sequences, and the report labels modified by the first detection probe in the same first detection probe subgroup are the same;
the second detection probe group consists of a plurality of second detection probe subgroups, wherein the length of each second detection probe is the same as that of the first label sequence, the second detection probe in one second detection probe subgroup is completely the same as the second double-base label sequence in the first fixed label sequence and the first variable label sequence, the rest two bases are cw, as, ts or gw, different report labels are modified by the second detection probes corresponding to the different second double-base label sequences, and the report labels modified by the second detection probes in the same second detection probe subgroup are the same;
a third detection probe group, which consists of a plurality of third detection probe subgroups, wherein the length of each third detection probe is the same as that of the second label sequence, the third detection probe in a third detection probe subgroup is completely the same as the third double-base label sequence of the second fixed label sequence and the second variable label sequence, the remaining two bases are cw, as, ts or gw, the third detection probes corresponding to the different third double-base label sequences are modified with different report labels, and the report labels modified by the third detection probes in the same third detection probe subgroup are the same;
and the fourth detection probe group consists of a plurality of fourth detection probe subgroups, wherein the length of each fourth detection probe is the same as that of the second label sequence, the fourth detection probe in one fourth detection probe subgroup is completely the same as the fourth double-base label sequence of the second fixed label sequence and the second variable label sequence, the rest two bases are cw, as, ts or gw, different report markers are modified on the fourth detection probes corresponding to the different fourth double-base label sequences, and the report markers modified on the fourth detection probes in the same fourth detection probe subgroup are the same.
In a preferred embodiment of the invention, the loop-forming probe is a double-ligated probe or a padlock probe.
Further preferably, the loop-forming probe is a double-ligation probe, and the step (3) is: specifically combining the at least one circularity probe with at least one target sequence in a sample to be detected, adding a splint sequence to enable the 3 ' end and the 5 ' end of the at least one circularity probe to be adjacent end to end, connecting the circularity probe and the 5 ' end by DNA ligase to form at least one circular template, and then carrying out rolling circle amplification to obtain at least one rolling circle amplification product.
Still more preferably, a first anchor primer is further added in the steps (4) and (5) for hybridization, and the first anchor primer is strictly complementary to the rolling circle amplification product and is connected with the hybridized first and second detection probes under the action of DNA polymerase.
Still more preferably, a second anchor primer is added in the steps (6) and (7) for hybridization, and the second anchor primer is strictly complementary and paired with the rolling circle amplification product and is connected with the hybridized third and fourth detection probes under the action of DNA polymerase.
In a preferred embodiment of the invention, the reporter label is a fluorescent label, a chromogenic label, a radioactive label, a magnetic label or a luminescent density label.
The invention has the beneficial effects that: the invention can improve the connection efficiency of the detection probe and the anchoring primer, enhance the fluorescent signal, optimally fix the GC content of the variable label sequence to be 50 percent, have better specificity and realize the in-situ detection of multiple RNAs.
Drawings
FIG. 1 is a schematic diagram of the reaction principle of the probe decoding process of the present invention.
Fig. 2 is a schematic diagram of an embodiment of the present invention.
FIG. 3 is a graph showing the results of the experiment in example 1 of the present invention, on a scale: 100 μm.
Fig. 4 is a graph of the experimental results of example 2 of the present invention, scale: 10 μm.
Fig. 5 is a graph of the experimental results of example 3 of the present invention, scale: 10 μm.
FIG. 6 is a schematic comparison of two-base coding and three-base coding in example 4 of the present invention.
FIG. 7 is a graph showing the results of the experiment in example 4 of the present invention, on a scale: 50 μm.
FIG. 8 is a graph showing the results of cryosectioning the mouse brain coronal section using the two-base coding of example 5 of the present invention.
Detailed Description
The technical solution of the present invention will be further illustrated and described below with reference to the accompanying drawings by means of specific embodiments.
The following examples employ double ligation probes.
The explanations for single base coding, double base coding and three base coding are as follows:
(1) in single base coding:
each of the loop forming probes corresponds to a target sequence, the sequence of the 5 'end and the 3' end of the loop forming probe is specifically hybridized and complemented with the target sequence, and a first label sequence and a second label sequence are arranged between the 5 'end and the 3' end of the loop forming probe, the first label sequence is positioned at the 3 'end of the sequence of the 5' end which is specifically hybridized and complemented with the target sequence, and the second label sequence is positioned at the 5 'end of the sequence of the 3' end which is specifically hybridized and complemented with the target sequence; the first label sequence consists of a first fixed label sequence and a first variable label sequence, the second label sequence consists of a second fixed label sequence and a second variable label sequence, the lengths of the first variable label sequence and the second variable label sequence are both four bases, and the tail end of the first variable label sequence far away from the first fixed label sequence and the tail end of the second variable label sequence far away from the second fixed label sequence are both sequences with the length of two bases consisting of two single-base label sequences.
Taking the first detection probe set and the first detection probe set as examples, the following steps are carried out:
the first detection probe group consists of a plurality of first detection probe subgroups, wherein the length of each first detection probe is the same as that of the first label sequence, the first detection probe in one first detection probe subgroup is completely the same as one single-base label sequence of the first fixed label sequence and the first variable label sequence, different report labels are modified on the first detection probes corresponding to different single-base label sequences, and the report labels modified on the first detection probes in the same first detection probe subgroup are the same;
and the second detection probe group consists of a plurality of second detection probe subgroups, wherein the length of each second detection probe is the same as that of the first label sequence, the second detection probe in one second detection probe subgroup is completely the same as the other single-base label sequence in the first fixed label sequence and the first variable label sequence, the second detection probes corresponding to the other different single-base label sequence are modified with different report labels, and the report labels modified by the second detection probes in the same second detection probe subgroup are the same.
(2) In double-base coding:
each ring-forming probe corresponds to a target sequence, the sequence of the 5 'end and the 3' end of the ring-forming probe is specifically hybridized and complemented with the target sequence, and a first label sequence and a second label sequence are arranged between the 5 'end and the 3' end of the ring-forming probe, the first label sequence is positioned at the 3 'end of the sequence of the 5' end specifically hybridized and complemented with the target sequence, and the second label sequence is positioned at the 5 'end of the sequence of the 3' end specifically hybridized and complemented with the target sequence; the first label sequence consists of a first fixed label sequence and a first variable label sequence, the second label sequence consists of a second fixed label sequence and a second variable label sequence, and the first variable label sequence and the second variable label sequence are four-base-length sequences consisting of double-base label sequences;
take the first and second detection probe sets as examples:
the first detection probe group consists of a plurality of first detection probe subgroups, wherein the length of each first detection probe is the same as that of the first label sequence, the first detection probe in one first detection probe subgroup is completely the same as one double-base label sequence of the first fixed label sequence and the first variable label sequence, the rest two bases are degenerate sequences, the first detection probes corresponding to different one of the double-base label sequences are modified with different report labels, and the report labels modified by the first detection probes in the same first detection probe subgroup are the same.
The second detection probe group consists of a plurality of second detection probe subgroups, wherein the length of each second detection probe is the same as that of the first label sequence, the second detection probes in one second detection probe subgroup are completely the same as the other two-base label sequence of the first fixed label sequence and the first variable label sequence, the rest two bases are degenerate sequences, the second detection probes corresponding to the other different two-base label sequences are modified with different report labels, and the report labels modified by the second detection probes in the same second detection probe subgroup are the same.
Preferably, the first variable tag sequence consists of a first double-base tag sequence and a second double-base tag sequence, the first double-base tag sequence is cw, as, ts or gw, the second double-base tag is cw, as, ts or gw, the second variable tag sequence consists of a third double-base tag sequence and a fourth double-base tag sequence, the third double-base tag sequence is cw, as, ts or gw, the fourth double-base tag is cw, as, ts or gw, w represents a/t, and s represents g/c;
take the first and second detection probe sets as examples:
a first detection probe group consisting of a plurality of first detection probe subgroups, wherein the length of each first detection probe is the same as that of the first label sequence, the first detection probe in a first detection probe subgroup is completely the same as the first fixed label sequence and the first double-base label sequence in the first variable label sequence, the remaining two bases are cw, as, ts or gw, the first detection probes corresponding to different first double-base label sequences are modified with different report labels, and the report labels modified by the first detection probe in the same first detection probe subgroup are the same;
the second detection probe group consists of a plurality of second detection probe subgroups, wherein the length of each second detection probe is the same as that of the first label sequence, the second detection probe in one second detection probe subgroup is completely the same as the second double-base label sequence in the first fixed label sequence and the first variable label sequence, the rest two bases are cw, as, ts or gw, different report labels are modified by the second detection probes corresponding to the different second double-base label sequences, and the report labels modified by the second detection probes in the same second detection probe subgroup are the same;
the third and fourth detection probe sets and the corresponding third and fourth double-base tag sequences of the second variable tag sequence are arranged according to the above:
more preferably, in an embodiment, the first variable tag sequence comprises a first two-base tag sequence and a second two-base tag sequence, the first two-base tag sequence is ct, ag, tc or ga, the second variable tag sequence comprises a third two-base tag sequence and a fourth two-base tag sequence, the third two-base tag sequence is ct, ag, tc or ga, the fourth two-base tag sequence is ct, ag, tc or ga,
further, taking the first detection probe set and the second detection probe set as examples:
the first detection probe group consists of a plurality of first detection probe subgroups, wherein the length of each first detection probe is the same as that of the first label sequence, the first detection probe in one first detection probe subgroup is completely the same as the first fixed label sequence and the first double-base label sequence in the first variable label sequence, the rest two bases are ct, ag, tc or ga, different report labels are modified corresponding to the first detection probes of the different first double-base label sequences, and the report labels modified by the first detection probes in the same first detection probe subgroup are the same;
the second detection probe group consists of a plurality of second detection probe subgroups, wherein the length of each second detection probe is the same as that of the first label sequence, the second detection probes in one second detection probe subgroup are completely the same as the second double-base label sequences in the first fixed label sequence and the first variable label sequence, the rest two bases are ct, ag, tc or ga, different report labels are modified corresponding to the second detection probes of the different second double-base label sequences, and the report labels modified by the second detection probes in the same second detection probe subgroup are the same;
the third and fourth detection probe sets and the corresponding third and fourth double-base tag sequences of the second variable tag sequence are arranged according to the above:
(3) in three base coding:
each of the loop forming probes corresponds to a target sequence, the sequence of the 5 'end and the 3' end of the loop forming probe is specifically hybridized and complemented with the target sequence, and a first label sequence and a second label sequence are arranged between the 5 'end and the 3' end of the loop forming probe, the first label sequence is positioned at the 3 'end of the sequence of the 5' end which is specifically hybridized and complemented with the target sequence, and the second label sequence is positioned at the 5 'end of the sequence of the 3' end which is specifically hybridized and complemented with the target sequence; the first tag sequence consists of a first fixed tag sequence and a first variable tag sequence, the second tag sequence consists of a second fixed tag sequence and a second variable tag sequence, and the first variable tag sequence and the second variable tag sequence are six-base-length sequences consisting of two three-base tag sequences;
taking the first detection probe set and the second detection probe set as examples:
the first detection probe group consists of a plurality of first detection probe subgroups, wherein the length of each first detection probe is the same as that of the first label sequence, the first detection probes in a first detection probe subgroup are completely the same as the first fixed label sequence and one of the three-base label sequences of the first variable label sequence, the first detection probes corresponding to different one of the three-base label sequences are modified with different report labels, and the report labels modified by the first detection probes in the same first detection probe subgroup are the same.
The second detection probe group consists of a plurality of second detection probe subgroups, wherein the length of each second detection probe is the same as that of the first label sequence, the second detection probes in one second detection probe subgroup are completely the same as the other three-base label sequence of the first fixed label sequence and the first variable label sequence, the second detection probes corresponding to the other three-base label sequence are modified with different report labels, and the report labels modified by the second detection probes in the same second detection probe subgroup are the same.
Example 1 comparison of Single-base-encoded sequencing with double-base-encoded sequencing on MCF-7 cell-crawlers
Firstly, cell culture and fixation:
after human breast adenocarcinoma cells MCF-7 are cultured for 24-48h by RPMI 1640 (containing 10% FBS) (after human breast adenocarcinoma cells are cultured for 24-48h by DMEM (containing 10% FBS)), cell suspension is treated by trypsin, and the cell suspension is plated on a sterile slide glass with polylysine on the surface and is cultured for 12-24h again. Washing with DEPC-PBS for 3 × 3 min; fixing with 4% PFA prepared by DEPC-PBS at room temperature for 30 min; after washing twice again with DEPC-PBS, the reaction mixture was washed with gradient ethanol: 70%, 85%, 100% dehydration treatment, each for 5 min. And (5) air drying.
(II) pretreatment of cell sample
The cell membranes of the cells were permeabilized and punched out, and 0.1M HCl was added to the samples and incubated for 5min at room temperature. After washing twice with DEPC-PBS containing Tween 20 at a volume concentration ratio of 0.1% (v/v), the cells were washed twice with DEPC-PBS.
(III) in-situ nucleic acid detection, which specifically comprises the following steps:
(1) recognition and hybridization of the probe and the target gene:
the hybridization reaction mixture containing 10% formamide, 6 XSSC and 0.1. mu.M probe at final concentration was added to the sample and incubated at 37 ℃ for 3-4h to allow the probe to be directly complementary to the mRNA molecule for in situ specific hybridization. After the reaction was completed, the reaction mixture was washed with DEPC-PBS-Tween 20 three times for 3min each, and then washed with DEPC-PBS three times. The 5' ends of the two double-connection probes hybridized with the target gene are respectively provided with a recognition sequence which is specifically complementary with the target sequence.
The sequences of the SINGLE-base-encoded sequencing DOUBLE-ligated probe (beginning with SINGLE) and the DOUBLE-base-encoded DOUBLE-ligated probe of the present invention (beginning with DOUBLE) as a comparison are shown in Table 1 below (SEQ ID NO.0001 to SEQ ID NO.0072 in the order of the sequence numbering from the top to the bottom)
TABLE 1
(2) The double-connection probe is used for connecting in the first step:
after washing three times with DEPC-PBS-Tween 20, 50. mu.L of ligation mixture containing 25% glycerol at final concentration, 0.2. mu.g/. mu.L BSA, 1 XSsprintR buffer (NEB), 0.1U/. mu.L SplintR ligand (NEB), and 1U/. mu.L RiboLock RNase inhibitor (Thermo) was added to the sample and incubated at 37 ℃ for 30 min.
(3) Identifying and hybridizing the splint primer:
after three washes with DEPC-PBS-Tween 20, 50 μ L of hybridization mix containing final concentrations of 6 XSSC, 10% formamide, and 0.5 μ M splint primers was added to the samples and incubated at 30 ℃ for 30 min. The splint primer sequence is as follows: 5'-ggctccactaaatagacgca-3' (SEQ ID NO.0073), Reverse primer.
(4) Probe closed loop and rolling circle amplification:
after washing three times with DEPC-PBS-Tween 20, 50. mu.L of a hybridization mixture containing 5% glycerol, 0.2. mu.g/. mu.L BSA, 1 XPhi 29 polymerase buffer (Thermo), 1mM dNTPs, 0.1U/. mu.L LT 4DNA ligase (Thermo) and 1U/. mu.L Phi29 DNA polymerase (Thermo) was added to the sample, and the mixture was incubated at 37 ℃ for 3h and transferred to 30 ℃ for reaction overnight.
The (fourth) four rounds of in situ RNA sequencing are shown in FIG. 1 and FIG. 2, and the specific principle is as follows:
(1) anchor primer hybridization and fluorescent probe ligation
mu.L of a sequencing reaction mixture containing the specific primers at a final concentration of 0.2. mu.M, 0.2. mu.M each of the fluorescent probes, 1 XT 4DNA ligase buffer, 1mM ATP and 0.1 u/. mu. L T4DNA ligase was added to the sample and incubated at 30 ℃ for 45 min. After washing three times with DEPC-PBS-Tween 20, the air was dried. Finally, Gold antipide mount (Fermantas) containing 0.5. mu.g/mL DAPI was added and incubated at room temperature for 10min before fluorescence microscopy and photography.
(2) Elution of fluorescent probes
The photographed slide is incubated in 5xssc or PBS, and the coverslip and mounting medium are washed away. Then, elution buffer was added to the reaction zone at a final concentration of 80% formamide, 0.1% Triton-X, and incubated at 37 ℃ for 10min, repeated 6 times. The samples were finally washed three times with DEPC-PBS-Tween 20.
(3) Repeating the steps (1) and (2) for three times, wherein the sequences of the anchor primer and the fluorescent probe are shown in the following table 2 (the sequences are numbered from top to bottom as SEQ ID NO.0074 to SEQ ID NO.0107, wherein SEQ ID NO. 0074-:
TABLE 2
The experimental results are shown in FIG. 3, wherein A is a graph of the results of 4 rounds of sequencing by double-base coding, B is a graph of the results of 4 rounds of sequencing by single-base coding, and C is a graph of the cumulative signal intensity of a signal point 30% before the cumulative intensity of a single signal point by double-base coding and single-base coding. Numbers 1 to 4 are first to fourth round of intensity comparison, FAM, TXR, CY3 and CY5 represent different channels, respectively, with two-base signal intensity on the left and single base on the right. It can be seen that the overall intensity of the double base is significantly higher than that of the single base signal
Example 2 in situ detection of IncRNA NEAT1 on MCF-7 cell slide test samples to examine the differences in specificity between single-base and double-base coded probes
Firstly, culturing and fixing MCF-7 cells:
human breast adenocarcinoma cells MCF-7 were cultured in DMEM (containing 10% FBS) for 24-48h, then trypsinized to suspension cells, plated on sterile glass slides with polylysine on the surface, and re-cultured for 12-24 h.
Washing with DEPC-PBS for 3 × 3 min; fixing with 4% PFA prepared by DEPC-PBS at room temperature for 30 min; after washing twice again with DEPC-PBS, the reaction mixture was washed with gradient ethanol: dehydrating 70%, 85% and 100% for 5min respectively, air drying, and storing at-80 deg.C.
(II) pretreatment of MCF-7 cell slide:
taking out the cell climbing sheet from-80 ℃ to unfreeze at room temperature, wherein the unfreezing time cannot exceed 10 minutes; 1ml of 0.1MHCl is dripped on the slide to cover the tissue; pouring off the liquid on the slide after 5 min; dropping PBS on the slide, and washing for 2 times, each time for 2 min; after the slide is dried, the immunohistochemical strokes are applied to a reaction tank in a selected range; after the reaction pool was drawn, the reaction pool was rinsed 3 times with DEPC-PBS-Tween 20.
(III) in-situ RNA detection:
(1) double ligation probe hybridization:
50 mu L of hybridization mixture containing 6 XSSC, 10% formamide and 0.1 mu M of each double-linked probe is dripped on the glass slide, and the mixture is incubated for 2 to 3 hours at 37 ℃ so that each double-linked probe pair is fully hybridized with the target sequence of the corresponding gene. The double-ligation probe sequence is shown in the following table 3 (shown in sequence from top to bottom as SEQ ID NO. 0108-0115):
TABLE 3
(2) The double-connection probe is used for connecting in the first step:
after washing three times with DEPC-PBS-Tween 20, 50. mu.L of ligation mixture containing 25% glycerol, 0.2. mu.g/. mu.L BSA, 1 XSplntR buffer (NEB), 0.1U/. mu.L LSplintR ligand (NEB) and 1U/. mu.L RiboLock RNase inhibitor (Thermo) was added to the sample and incubated at 37 ℃ for 30 min.
(3) Splint primer recognition hybridization:
after three washes with DEPC-PBS-Tween 20, 50 μ L of hybridization mix containing final concentrations of 6 XSSC, 10% formamide, and 0.5 μ M splint primers was added to the samples and incubated at 30 ℃ for 30 min. The splint primer sequence is as follows: 5'-ggctccactaaatagacgca-3' (SEQ ID NO.0073), Reverse primer.
(4) Probe closed loop formation and rolling circle amplification:
after washing three times with DEPC-PBS-Tween 20, 50. mu.L of a hybridization mixture containing 5% glycerol, 0.2. mu.g/. mu.L BSA, 1 XPhi 29 polymerase buffer (Thermo), 1mM dNTPs, 0.1U/. mu.L LT 4DNA ligase (Thermo), and 1U/. mu.L Phi29 DNA polymerase (Thermo) was added to the sample, and incubated at 37 ℃ for 3h and transferred to 30 ℃ for reaction overnight.
(5) Third round of anchored primer hybridization and fluorescent Probe ligation
To the sample, 50. mu.L of a sequencing reaction mixture containing the anchor primer at a final concentration of 0.2. mu.M, each fluorescent probe at a final concentration of 0.2. mu.M, 1 XT 4DNA ligase buffer, 1mM ATP and 0.1 u/. mu. L T4DNA ligase was added, and the mixture was incubated at 30 ℃ for 45 min. After washing three times with DEPC-PBS-Tween 20, air-dried. Finally, Gold antipade Mount Encapsulated tablets (Fermantas) containing 0.5. mu.g/mL DAPI were added and incubated at room temperature for 10min for fluorescence microscopy and photographed. The sequences of the above-mentioned anchor primer and fluorescent probe are shown in Table 2 of example 1.
The results are shown in FIG. 4, wherein A is the third round in situ detection results of single-base coded probes DLP-NEAT1-L1 and DLP-NEAT1-R1, 1: MCF-7 nuclei, 2: signal of NEAT1 in MCF-7, 3: the presence of a false detection signal in another fluorescence channel; b is the third round of in situ detection results of single-base coded probes DLP-NEAT1-L2 and DLP-NEAT1-R2, 1: MCF-7 cell nuclei; 2: signal point for NEAT1 in MCF-7; 3: there is a false detection signal in the other fluorescence channel. C is the third round of in situ detection results of single-base coded probes DLP-NEAT1-L3 and DLP-NEAT1-R3, 1: MCF-7 nucleus, 2: signal of NEAT1 in MCF-7, 3: there is a false detection signal in the other fluorescence channel. D is the third round in situ detection result of the double-base coded probes DLP-NEAT1-L-1 and DLP-NEAT1-R-1, 1: MCF-7 nuclei, 2: signal of NEAT1 in MCF-7, 3: no signal was detected in the remaining fluorescence channels. E is the in-situ detection statistical result of the three single-base coded probes and the double-base coded probes. When RNA in-situ detection is carried out in MCF-7 cell slide, the single base coding mode can cause the phenomenon of mismatching of some detection probes, and the double base coding mode can effectively reduce the mismatching of part of detection probes and avoid the generation of error signals.
EXAMPLE 3 in situ detection of lncRNA NEAT1 for Experimental samples in Paraffin tissue sections of Breast cancer examination of the differences in specificity between Single-base and double-base coded probes
Firstly, paraffin tissue pretreatment:
baking paraffin tissue slices in an oven at 65 deg.C for 30min, immediately soaking in a staining jar containing xylene for 10min, and replacing xylene and soaking for 10 min. And taking out the glass slide from the second staining jar containing the dimethylbenzene, immediately putting the glass slide into the staining jar containing the absolute ethyl alcohol, soaking for 2min, replacing the absolute ethyl alcohol, and soaking for 2 min. Taking out, immediately placing the glass slide into a staining jar containing 95% ethanol, soaking for 2min, replacing 95% ethanol, and soaking for 2 min. Taking out, immediately placing the glass slide into a staining jar containing 85% ethanol, soaking for 2min, replacing 85% ethanol, and soaking for 2 min. Taking out, immediately placing the glass slide into a staining jar containing 70% ethanol, soaking for 2min, replacing 70% ethanol, and soaking for 2 min.
Taking out the glass slide from the ethanol solution, and soaking the glass slide in DEPC-H 2 Soaking in O for 5min, soaking in DEPC-PBS for 2min, and washing off residual ethanol. 4% PFA in DEPC-PBS was dropped onto the slide, completely covering the tissue sample, incubated at room temperature for 10min and washed away with DEPC-PBS. 30mL of 0.1M HCl was placed in a staining jar and preheated to 37 ℃, 30uL of 0.1mg/mL pepsin was added, the mixture was mixed well and immediately the slide was immersed in the mixture and incubated at 37 ℃ for 30 min. Taking out the glass slide and soaking the glass slide in DEPC-H 2 Soaking in O for 5min, and soaking in DEPC-PBS for 2 min. Respectively passing through gradient ethanol: dehydrating 70%, 85% and 100% for 2min respectively, and air drying. And finally washing the mixture with DEPC-PBS-Tween 20 for three times for later use.
(II) in-situ nucleic acid detection:
(1) double ligation probe hybridization:
50 mu L of hybridization mixture containing final concentration of 6 XSSC, 10% formamide and 0.1 mu M of each double-linked probe is dripped on the glass slide, and the mixture is incubated for 2 to 3 hours at 37 ℃ so that each recognition probe pair is fully hybridized with the target sequence of the corresponding gene. The ligation probe sequences are shown in Table 3(1) of example 2:
(2) the double-connection probe is used for connecting in the first step:
after washing three times with DEPC-PBS-Tween 20, 50. mu.L of ligation mixture containing 25% glycerol, 0.2. mu.g/. mu.L BSA, 1 XSSplintR buffer (NEB), 0.1U/. mu.L SplintR ligand (NEB) and 1U/. mu.L RiboLock RNase inhibitor (Thermo) was added to the sample and incubated at 37 ℃ for 30 min.
(3) Identifying and hybridizing the splint primer:
after three washes with DEPC-PBS-Tween 20, 50 μ L of hybridization mix containing final concentrations of 6 XSSC, 10% formamide, and 0.5 μ M splint primers was added to the samples and incubated at 30 ℃ for 30 min. The splint primer sequence is as follows: 5'-ggctccactaaatagacgca-3' (SEQ ID NO.0073), Reverse primer.
(4) Probe closed loop and rolling circle amplification:
after washing three times with DEPC-PBS-Tween 20, 50. mu.L of a hybridization mixture containing 5% glycerol, 0.2. mu.g/. mu.L BSA, 1 XPhi 29 polymerase buffer (Thermo), 1mM dNTPs, 0.1U/. mu.L LT 4DNA ligase (Thermo), and 1U/. mu.L Phi29 DNA polymerase (Thermo) was added to the sample, and incubated at 37 ℃ for 3h and transferred to 30 ℃ for reaction overnight.
(5) Third round of Anchor primer hybridization and fluorescent Probe ligation
To the sample, 50. mu.L of a sequencing reaction mixture containing the anchor primer at a final concentration of 0.2. mu.M, each fluorescent probe at a final concentration of 0.2. mu.M, 1 XT 4 DNAligase buffer, 1mM ATP, and 0.1 u/. mu. L T4DNA ligase was added, and the mixture was incubated at 30 ℃ for 45 min. After washing three times with DEPC-PBS-Tween 20, air-dried. Finally adding Gold antipade Mount Encapsulated tablet (Fermantas) containing 0.5 μ g/mLDAPI, incubating at room temperature for 10min, detecting with fluorescence microscope and taking pictures. The sequences of the above-mentioned anchor primer and fluorescent probe are shown in Table 2 of example 1.
The experimental results are shown in fig. 5, and the third round of in situ detection results of the single-base coded probe and the double-base coded probe on the breast cancer tissue are as follows: a is single base coded probes DLP-NEAT1-L1 and DLP-NEAT1-R1, and the third round of in-situ detection result is as follows, 1: breast cancer tissue, 2: NEAT1 signal in breast cancer tissue, 3: there is a false detection signal in the other fluorescence channel. B is a third round in-situ detection result of double-base coded probes DLP-NEAT1-L-1 and DLP-NEAT1-R-1, 1: breast cancer tissue, 2: NEAT1 signal in breast cancer tissue, 3: no signal was detected in the remaining fluorescence channels. C is the statistical result of in-situ detection of the single-base coded probe and the double-base coded probe. As can be seen from the figure, when RNA in-situ detection is carried out in paraffin tissue sections, the single-base coding mode can cause the mismatching phenomenon of some detection probes, and the double-base coding mode can effectively reduce the mismatching phenomenon of part of detection probes and avoid the generation of wrong signals.
Example 4 comparative experiments of two-base-encoded sequencing and three-base-encoded sequencing on MCF-7 cell crawlers
The principles of two-base coding and three-base coding of the present invention are left for the specific process shown in FIG. 6.
Firstly, culturing and fixing MCF-7 cells:
human breast adenocarcinoma cells MCF-7 were cultured in DMEM (containing 10% FBS) for 24-48h, then trypsinized to suspension cells, plated on sterile glass slides with polylysine on the surface, and re-cultured for 12-24 h. Washing with DEPC-PBS for 3 × 3 min; fixing with 4% PFA prepared by DEPC-PBS at room temperature for 30 min; after washing twice again with DEPC-PBS, the reaction mixture was washed with gradient ethanol: dehydrating 70%, 85% and 100% for 5min respectively, air drying, and placing at-80 deg.C for use.
(II) pretreatment of MCF-7 cell slide:
taking out the cell climbing sheet from-80 ℃ to be unfrozen at room temperature, wherein the unfrozen time cannot exceed 10 minutes; 1ml of 0.1MHCl is dripped on the slide to cover the tissue; pouring off the liquid on the slide after 5 min; dropping PBS on the slide, and washing for 2 times, each time for 2 min; after the slide is dried, the immunohistochemical strokes are applied to a reaction tank in a selected range; after the reaction pool was drawn, the reaction pool was rinsed 3 times with DEPC-PBS-Tween 20.
(III) in-situ RNA detection:
(1) double ligation probe hybridization:
50 mu L of hybridization mixture containing 6 XSSC, 10% formamide and 0.05 mu M of each double-linked probe is dripped on the glass slide, and the mixture is incubated for 2 to 3 hours at 37 ℃ so that each recognition probe pair is fully hybridized with the target sequence of the corresponding gene.
The sequences of the three-base-encoded sequencing DOUBLE-ligated probe (beginning with TRIPLE) and the two-base-encoded DOUBLE-ligated probe of the present invention (beginning with DOUBLE) for comparison are shown in Table 4 below (the sequences are numbered sequentially from top to bottom as SEQ ID NO.0116 to SEQ ID NO. 0183):
TABLE 4
(2) The double-connection probe is used for connecting in the first step:
after washing three times with DEPC-PBS-Tween 20, 50. mu.L of ligation mixture containing 25% glycerol, 0.2. mu.g/. mu.L BSA, 1 XSSplintR buffer (NEB), 0.1U/. mu.L SplintR ligand (NEB) and 1U/. mu.L RiboLock RNase inhibitor (Thermo) was added to the sample and incubated at 37 ℃ for 30 min.
(3) Identifying and hybridizing the splint primer:
after washing three times with DEPC-PBS-Tween 20, 50. mu.L of hybridization mixture containing the final concentration of 6 XSSC, 10% formamide and 0.5. mu.M splint primer was added to the sample and incubated at 30 ℃ for 30 min. The splint primer sequence is as follows: 5'-ggctccactaaatagacgca-3' (SEQ ID NO.0073), Reverse primer.
(4) Probe closed loop and rolling circle amplification:
after washing three times with DEPC-PBS-Tween 20, 50. mu.L of a hybridization mixture containing 5% glycerol, 0.2. mu.g/. mu.L BSA, 1 XPhi 29 polymerase buffer (Thermo), 1mM dNTPs, 0.1U/. mu.L LT 4DNA ligase (Thermo) and 1U/. mu.L Phi29 DNA polymerase (Thermo) was added to the sample, and the mixture was incubated at 37 ℃ for 3h and transferred to 30 ℃ for reaction overnight.
(5) Anchor primer hybridization and fluorescent probe ligation
mu.L of a sequencing reaction mixture containing the anchor primers at a final concentration of 0.2. mu.M, 0.2. mu.M each of the fluorescent probes, 1 XT 4DNA ligase buffer, 1mM ATP and 0.1 u/. mu. L T4DNA ligase was added to the sample and incubated at 30 ℃ for 45 min. After washing three times with DEPC-PBS-Tween 20, the air was dried. Finally, Gold antipide mount (Fermantas) containing 0.5. mu.g/mL DAPI was added and incubated at room temperature for 10min before fluorescence microscopy and photography. The sequences of the above-mentioned anchor primer and fluorescent probe are shown in the following Table 5 (SEQ ID NO.0074, 0076-:
TABLE 5
The experimental results are shown in FIG. 7, wherein A is a graph of the sequencing results of the two-base coding, B is a graph of the sequencing results of the three-base coding, and C is a graph of comparing fluorescence signals of 30% of the cumulative fluorescence intensity of the single signal points of the two-base coding and the three-single-base coding, wherein the left curve of each graph represents the two-base, and the right curve represents the three-base, and the fluorescence intensity of the two-base is higher than that of the three-base coding.
Example 5 double-base coding sequencing method for in-situ detection of 36 mRNA species for experimental samples by mouse coronal plane frozen section
Firstly, acquiring and embedding mouse brain tissues: the age of the mice was recorded and weighed, and the mice were sacrificed by quickly disconnecting the neck. The skin of the outer skin of the mouse is cut to the head from the opening of the abdomen by taking the dissecting scissors, the meninges of the head is carefully cut, and if the head of the mouse is cut off from the neck according to the circumstances due to inconvenient operation, the brain of the mouse can be conveniently taken. And (3) washing the blood stain of the brain tissue with normal saline or PBS, and taking gauze to suck the redundant water stain of the brain tissue after washing. A small amount of OCT is added into an embedding box, brain tissues are placed into the embedding box according to a specific direction, and the OCT is added until the brain tissues are covered. Quickly freezing with dry ice, and transferring to a refrigerator of-80 deg.C for storage. In order to obtain a tissue with the RNA as complete as possible, heart perfusion of the mouse is not needed before the mouse brain tissue is taken, PFA fixation and sucrose dehydration are not needed after the mouse brain is taken, and the mouse brain is quickly and timely embedded after the mouse brain is taken, so that the mouse brain liquefaction and RNA degradation caused by a long time are prevented.
(II) preparation of mouse brain sections: fixing an OCT embedding block containing mouse brain on a cold head of a freezing microtome according to a certain direction, wherein the thickness of a mouse brain slice is 10-12 mu m, adsorbing a tissue slice in the direction that the front surface of a glass slide faces downwards and the back surface of the glass slide faces upwards after cutting, pressing the adsorption position of the tissue on the back surface of the glass slide by using fingers after adsorption to fix the tissue, placing the slice at a low temperature of-80 ℃ for storage after adsorption, and uniformly transferring the slice temporarily stored in dry ice to a refrigerator of-80 ℃. The glass slide is precooled at least half an hour in advance, so that the phenomenon that the tissue is easily wrinkled when the temperature of the glass slide is too high is prevented.
(III) pretreatment of frozen tissue sections: taking out the frozen slices from-80 ℃ and unfreezing at room temperature, wherein the unfreezing time cannot exceed 10 minutes; 1ml of 4% PFA is dripped on the glass slide to cover the tissue, so that the dripping is avoided from directly flushing the tissue during dripping, and the tissue is prevented from falling off the slide; pouring off the liquid on the slide after 5 min; dropping PBS on the slide, and washing for 2 times, each time for 2 min; after the slide is dried, an immunohistochemical pen is used for painting a reaction tank on the periphery of the tissue, and the distance between the painted reaction tank and the tissue is a certain distance; after the reaction pool was drawn, the reaction pool was rinsed 3 times with DEPC-PBS-Tween 20.
(IV) in-situ RNA detection:
(1) double ligation probe hybridization:
50 mu L of hybridization mixture containing 6 XSSC, 10% formamide and 0.05 mu M of each double-linked probe is dripped on the glass slide, and the mixture is incubated for 2 to 3 hours at 37 ℃ so that each recognition probe pair is fully hybridized with the target sequence of the corresponding gene. The sequence of the double-connection probe is shown in the following table 6 (the sequence is numbered from top to bottom and is SEQ ID NO.0188 to SEQ ID NO. 0547):
TABLE 6
(2) The double-connection probe is used for connecting in the first step:
after washing three times with DEPC-PBS-Tween 20, 50. mu.L of ligation mixture containing 25% glycerol at the final concentration, 0.2. mu.g/. mu.L BSA, 1 XSsprintR buffer (NEB), 0.1U/. mu.L SplintR ligand (NEB) and 1U/. mu.L RiboLock RNase inhibitor (Thermo) was added to the sample and incubated at 37 ℃ for 30 min.
(3) Identifying and hybridizing the splint primer:
after washing three times with DEPC-PBS-Tween 20, 50. mu.L of hybridization mixture containing the final concentration of 6 XSSC, 10% formamide and 0.5. mu.M splint primer was added to the sample and incubated at 30 ℃ for 30 min. The splint primer sequence is as follows: 5'-ggctccactaaatagacgca-3' (SEQ ID NO.0073), Reverse primer.
(4) Probe closed loop and rolling circle amplification:
after washing three times with DEPC-PBS-Tween 20, 50. mu.L of a hybridization mixture containing 5% glycerol, 0.2. mu.g/. mu.L BSA, 1 XPhi 29 polymerase buffer (Thermo), 1mM dNTPs, 0.1U/. mu.L LT 4DNA ligase (Thermo), and 1U/. mu.L Phi29 DNA polymerase (Thermo) was added to the sample, and incubated at 37 ℃ for 3h and transferred to 30 ℃ for reaction overnight.
The concrete principle of the four-round in-situ RNA sequencing is shown in figure 1, and the concrete steps are as follows: :
(1) anchor primer hybridization and fluorescent probe ligation
To the sample, 50. mu.L of a sequencing reaction mixture containing the anchor primer at a final concentration of 0.2. mu.M, each fluorescent probe at a final concentration of 0.2. mu.M, 1 XT 4DNA ligase buffer, 1mM ATP and 0.1 u/. mu. L T4DNA ligase was added, and the mixture was incubated at 30 ℃ for 45 min. After washing three times with DEPC-PBS-Tween 20, the air was dried. Finally, Gold antipide mount (Fermantas) containing 0.5. mu.g/mL DAPI was added and incubated at room temperature for 10min before fluorescence microscopy and photography.
(2) Elution of fluorescent probes
The above photographed slides were incubated in 5 × ssc or PBS, and the coverslip and mounting medium were washed away. Then, elution buffer was added to the reaction zone at a final concentration of 80% formamide, 0.1% Triton-X, and incubated at 37 ℃ for 10min, repeated 6 times. The samples were finally washed three times with DEPC-PBS-Tween 20.
(3) Steps (1) and (2) were repeated three more times, and the sequences of the anchor primer and the fluorescent probe were as shown in Table 2 of example 1.
(4) Analysis of Gene
The color sequence of four rounds is obtained for each signal point by integrating the signals of the four rounds of sequencing, and the signal of which gene is the point can be obtained according to the base sequence on the previously designed probe, as shown in FIG. 8.
The above description is only a preferred embodiment of the present invention, and therefore should not be taken as limiting the scope of the invention, which is defined by the appended claims.
Sequence listing
<110> university of Huaqiao
<120> probe primer group for RNA in-situ detection and application thereof
<160> 547
<170> SIPOSequenceListing 1.0
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cgagaccacg ctcaaaaaaa aaattcctat cacgctgcgt ctatt 45
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ctgtgctcgc ggggcgttcc tatctcgatg cgtctatt 38
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tagtggagcc acctctatcc tttcggcaaa ggcgaggct 39
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ctcatcggat tttgcagttc ctatctcgtt gcgtctatt 39
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cgagaccacg ctcaaaaaaa aaattcactc agacttgcgt ctatt 45
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tagtggagcc ctgaactcac ttaaaaaaag gtccagttag cagtc 45
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tcgctctgtc tcgctcaaaa aaattcactc actagtgcgt ctatt 45
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tagtggagcc cttcactcac ttaaaaaaag gtcagattgc gttgc 45
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cgagtccaag gtgaacaaaa aaattcactc acttctgcgt ctatt 45
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tagtggagcc tcctactcac ttaaaaaaat attgcaagaa aaaga 45
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aggtgagctc cgactcaaaa aaattcactc acttctgcgt ctatt 45
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tagtggagcc agctactcac ttaaaaaaac cgtccttgct gaaac 45
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cgctggatag cctccaaaaa aaattcactc atcagtgcgt ctatt 45
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tagtggagcc tcagactcac ttaaaaaaac tgaatctttg gagta 45
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agccacacag tgctttaaaa aaattcactc agatctgcgt ctatt 45
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tagtggagcc agagactcac ttaaaaaaat gaacaccgct tattc 45
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tcctccccgc actcgtaaaa aaattcactc aagcttgcgt ctatt 45
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tagtggagcc gagaactcac ttaaaaaaag cctcccatct caaac 45
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tgcgtcggcg tggcagaaaa aaattcactc aaggatgcgt ctatt 45
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tagtggagcc aggaactcac ttaaaaaaac gtgcagagag gggtc 45
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ttgttcccca gaaaagaaaa aaattcactc actcttgcgt ctatt 45
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 54
tagtggagcc gaagactcac ttaaaaaaag acttggagtc acctc 45
<210> 55
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 55
ccttgttctt cttcttcaaa aaattcactc atcagtgcgt ctatt 45
<210> 56
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 56
tagtggagcc gagaactcac ttaaaaaaat ttactgcaac accac 45
<210> 57
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 57
tggaataaat ctgcgtgttc actcaaggat gcgtctatt 39
<210> 58
<211> 37
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 58
tagtggagcc cttcactcac ttccagttgt tgtttca 37
<210> 59
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 59
agagctcttg tgtgtgttca ctcacttctg cgtctatt 38
<210> 60
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 60
tagtggagcc ctgaactcac ttgtgatgtt ggagataa 38
<210> 61
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 61
ctgtgctcgc ggggcgttca ctcagaagtg cgtctatt 38
<210> 62
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 62
tagtggagcc agctactcac tttcggcaaa ggcgaggct 39
<210> 63
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 63
attctcctcg gtgtccttca ctcatctctg cgtctatt 38
<210> 64
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 64
tagtggagcc ctagactcac ttggtgttcg cctcttgac 39
<210> 65
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 65
tttgccatcc actatcttca ctcaagtctg cgtctatt 38
<210> 66
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 66
tagtggagcc tcctactcac tttggtctca gacaccac 38
<210> 67
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 67
gtagctctcg aacatgtttc actcagactt gcgtctatt 39
<210> 68
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 68
tagtggagcc gagaactcac ttgcctaagg ttgttgat 38
<210> 69
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 69
ctcatcggat tttgcagttc actcagatct gcgtctatt 39
<210> 70
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 70
tagtggagcc ctctactcac ttctttactt ggcggcagt 39
<210> 71
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 71
agttctcgaa gtctgacttc actcaagctt gcgtctatt 39
<210> 72
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 72
tagtggagcc gactactcac ttgctccaaa ttccctgg 38
<210> 73
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 73
ggctccacta aatagacgca 20
<210> 74
<211> 15
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 74
tgcgtctatt tagtg 15
<210> 75
<211> 15
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 75
ctatttagtg gagcc 15
<210> 76
<211> 9
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 76
actcaagnn 9
<210> 77
<211> 9
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 77
actcatcnn 9
<210> 78
<211> 9
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 78
actcactnn 9
<210> 79
<211> 9
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 79
actcagann 9
<210> 80
<211> 9
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 80
actcannag 9
<210> 82
<211> 9
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 82
actcannct 9
<210> 81
<211> 9
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 81
actcanntc 9
<210> 83
<211> 9
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 83
actcannga 9
<210> 84
<211> 9
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 84
nnagactca 9
<210> 85
<211> 9
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 85
nntcactca 9
<210> 86
<211> 9
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 86
nnctactca 9
<210> 87
<211> 9
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 87
nngaactca 9
<210> 88
<211> 9
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 88
agnnactca 9
<210> 89
<211> 9
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 89
tcnnactca 9
<210> 90
<211> 9
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 90
ctnnactca 9
<210> 91
<211> 9
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 91
gannactca 9
<210> 92
<211> 9
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 92
ctatcnnan 9
<210> 93
<211> 9
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 93
ctatcnntn 9
<210> 94
<211> 9
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 94
ctatcnncn 9
<210> 95
<211> 9
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 95
ctatcnngn 9
<210> 96
<211> 9
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 96
ctatcnnna 9
<210> 97
<211> 9
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 97
ctatcnnnt 9
<210> 98
<211> 9
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 98
ctatcnnnc 9
<210> 99
<211> 9
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 99
ctatcnnng 9
<210> 100
<211> 9
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 100
annnctatc 9
<210> 101
<211> 9
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 101
tnnnctatc 9
<210> 102
<211> 9
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 102
cnnnctatc 9
<210> 103
<211> 9
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 103
gnnnctatc 9
<210> 104
<211> 9
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 104
nannctatc 9
<210> 105
<211> 9
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 105
ntnnctatc 9
<210> 106
<211> 9
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 106
ncnnctatc 9
<210> 107
<211> 9
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 107
ngnnctatc 9
<210> 108
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 108
taccaccccc accaccaaaa aaattcctat ctcgttgcgt ctatt 45
<210> 109
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 109
tagtggagcc aactctatcc ttaaaaaaat tcaacctgca tttcc 45
<210> 110
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 110
taccaccccc accaccaaaa aaattcctat ctctttgcgt ctatt 45
<210> 111
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 111
tagtggagcc aactctatcc ttaaaaaaat tcaacctgca tttcc 45
<210> 112
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 112
taccaccccc accaccaaaa aaattcctat ctcgttgcgt ctatt 45
<210> 113
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 113
tagtggagcc tactctatcc ttaaaaaaat tcaacctgca tttcc 45
<210> 114
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 114
taccaccccc accaccaaaa aaattcactc agatctgcgt ctatt 45
<210> 115
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 115
tagtggagcc agagactcac ttaaaaaaat tcaacctgca tttcc 45
<210> 116
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 116
agagctcttg tgtgtgttct cactatcatg cgtctatt 38
<210> 117
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 117
tagtggagcc ctagatactc ttgtgatgtt ggagataa 38
<210> 118
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 118
ctgtgctcgc ggggcgttct cagatagttg cgtctatt 38
<210> 119
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 119
tagtggagcc agtctaactc tttcggcaaa ggcgaggct 39
<210> 120
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 120
attctcctcg gtgtccttct catcatcatg cgtctatt 38
<210> 121
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 121
tagtggagcc ctaagtactc ttggtgttcg cctcttgac 39
<210> 122
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 122
tttgccatcc actatcttct caagttcatg cgtctatt 38
<210> 123
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 123
tagtggagcc tcactaactc tttggtctca gacaccac 38
<210> 124
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 124
gtagctctcg aacatgtttc tcagatctat gcgtctatt 39
<210> 125
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 125
tagtggagcc gatgatactc ttgcctaagg ttgttgat 38
<210> 126
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 126
ctcatcggat tttgcagttc tcagattcat gcgtctatt 39
<210> 127
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 127
tagtggagcc ctactaactc ttctttactt ggcggcagt 39
<210> 128
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 128
agttctcgaa gtctgacttc tcaagtctat gcgtctatt 39
<210> 129
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 129
tagtggagcc gatctaactc ttgctccaaa ttccctgg 38
<210> 130
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 130
ttgttcccca gaaaagaaaa aaattctcac tactatgcgt ctatt 45
<210> 131
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 131
tagtggagcc gatagtactc ttaaaaaaag acttggagtc acctc 45
<210> 132
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 132
ccttgttctt cttcttcaaa aaattctcat caagttgcgt ctatt 45
<210> 133
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 133
tagtggagcc gatgatactc ttaaaaaaat ttactgcaac accac 45
<210> 134
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 134
cgagaccacg ctcaaaaaaa aaattctcag atctatgcgt ctatt 45
<210> 135
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 135
tagtggagcc ctagatactc ttaaaaaaag gtccagttag cagtc 45
<210> 136
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 136
tcgctctgtc tcgctcaaaa aaattctcac taagttgcgt ctatt 45
<210> 137
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 137
tagtggagcc ctatcaactc ttaaaaaaag gtcagattgc gttgc 45
<210> 138
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 138
cgagtccaag gtgaacaaaa aaattctcac tatcatgcgt ctatt 45
<210> 139
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 139
tagtggagcc tcactaactc ttaaaaaaat attgcaagaa aaaga 45
<210> 140
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 140
aggtgagctc cgactcaaaa aaattctcac tatcatgcgt ctatt 45
<210> 141
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 141
tagtggagcc agtctaactc ttaaaaaaac cgtccttgct gaaac 45
<210> 142
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 142
cgctggatag cctccaaaaa aaattctcat caagttgcgt ctatt 45
<210> 143
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 143
tagtggagcc tcaagtactc ttaaaaaaac tgaatctttg gagta 45
<210> 144
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 144
agccacacag tgctttaaaa aaattctcag attcatgcgt ctatt 45
<210> 145
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 145
tagtggagcc agtagtactc ttaaaaaaat gaacaccgct tattc 45
<210> 146
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 146
tcctccccgc actcgtaaaa aaattctcaa gtctatgcgt ctatt 45
<210> 147
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 147
tagtggagcc gatgatactc ttaaaaaaag cctcccatct caaac 45
<210> 148
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 148
tgcgtcggcg tggcagaaaa aaattctcaa gtgattgcgt ctatt 45
<210> 149
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 149
tagtggagcc agtgatactc ttaaaaaaac gtgcagagag gggtc 45
<210> 150
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 150
cgagaccacg ctcaaaaaaa aaattcactc agacttgcgt ctatt 45
<210> 151
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 151
tagtggagcc ctgaactcac ttaaaaaaag gtccagttag cagtc 45
<210> 152
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 152
tcgctctgtc tcgctcaaaa aaattcactc actagtgcgt ctatt 45
<210> 153
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 153
tagtggagcc cttcactcac ttaaaaaaag gtcagattgc gttgc 45
<210> 154
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 154
cgagtccaag gtgaacaaaa aaattcactc acttctgcgt ctatt 45
<210> 155
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 155
tagtggagcc tcctactcac ttaaaaaaat attgcaagaa aaaga 45
<210> 156
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 156
aggtgagctc cgactcaaaa aaattcactc acttctgcgt ctatt 45
<210> 157
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 157
tagtggagcc agctactcac ttaaaaaaac cgtccttgct gaaac 45
<210> 158
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 158
cgctggatag cctccaaaaa aaattcactc atcagtgcgt ctatt 45
<210> 159
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 159
tagtggagcc tcagactcac ttaaaaaaac tgaatctttg gagta 45
<210> 160
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 160
agccacacag tgctttaaaa aaattcactc agatctgcgt ctatt 45
<210> 161
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 161
tagtggagcc agagactcac ttaaaaaaat gaacaccgct tattc 45
<210> 162
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 162
tcctccccgc actcgtaaaa aaattcactc aagcttgcgt ctatt 45
<210> 163
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 163
tagtggagcc gagaactcac ttaaaaaaag cctcccatct caaac 45
<210> 164
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 164
tgcgtcggcg tggcagaaaa aaattcactc aaggatgcgt ctatt 45
<210> 165
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 165
tagtggagcc aggaactcac ttaaaaaaac gtgcagagag gggtc 45
<210> 166
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 166
ttgttcccca gaaaagaaaa aaattcactc actcttgcgt ctatt 45
<210> 167
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 167
tagtggagcc gaagactcac ttaaaaaaag acttggagtc acctc 45
<210> 168
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 168
ccttgttctt cttcttcaaa aaattcactc atcagtgcgt ctatt 45
<210> 169
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 169
tagtggagcc gagaactcac ttaaaaaaat ttactgcaac accac 45
<210> 170
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 170
agagctcttg tgtgtgttca ctcacttctg cgtctatt 38
<210> 171
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 171
tagtggagcc ctgaactcac ttgtgatgtt ggagataa 38
<210> 172
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 172
ctgtgctcgc ggggcgttca ctcagaagtg cgtctatt 38
<210> 173
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 173
tagtggagcc agctactcac tttcggcaaa ggcgaggct 39
<210> 174
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 174
attctcctcg gtgtccttca ctcatctctg cgtctatt 38
<210> 175
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 175
tagtggagcc ctagactcac ttggtgttcg cctcttgac 39
<210> 176
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 176
tttgccatcc actatcttca ctcaagtctg cgtctatt 38
<210> 177
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 177
tagtggagcc tcctactcac tttggtctca gacaccac 38
<210> 178
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 178
gtagctctcg aacatgtttc actcagactt gcgtctatt 39
<210> 179
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 179
tagtggagcc gagaactcac ttgcctaagg ttgttgat 38
<210> 180
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 180
ctcatcggat tttgcagttc actcagatct gcgtctatt 39
<210> 181
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 181
tagtggagcc ctctactcac ttctttactt ggcggcagt 39
<210> 182
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 182
agttctcgaa gtctgacttc actcaagctt gcgtctatt 39
<210> 183
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 183
tagtggagcc gactactcac ttgctccaaa ttccctgg 38
<210> 184
<211> 9
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 184
tcaagtnnn 9
<210> 185
<211> 9
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 185
tcatcannn 9
<210> 186
<211> 9
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 186
tcactannn 9
<210> 187
<211> 9
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 187
tcagatnnn 9
<210> 188
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 188
cgtgctgtct gcgtataaaa aaattcactc agacttgcgt ctatt 45
<210> 189
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 189
tagtggagcc ctgaactcac ttaaaaaaaa cacagggaca ggaaa 45
<210> 190
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 190
tgccaattcc caattaaaaa aaattcactc agacttgcgt ctatt 45
<210> 191
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 191
tagtggagcc ctgaactcac ttaaaaaaag attttgcggt tggtc 45
<210> 192
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 192
atctggttgc agctctaaaa aaattcactc agacttgcgt ctatt 45
<210> 193
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 193
tagtggagcc ctgaactcac ttaaaaaaag aggtaggaag catgc 45
<210> 194
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 194
cctttcccca gacagtaaaa aaattcactc agacttgcgt ctatt 45
<210> 195
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 195
tagtggagcc ctgaactcac ttaaaaaaac tgggatgcaa acgac 45
<210> 196
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 196
aatggagagg gcacacaaaa aaattcactc agacttgcgt ctatt 45
<210> 197
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 197
tagtggagcc ctgaactcac ttaaaaaaag ttttctctaa gagcc 45
<210> 198
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 198
tggcgatgct caaagtaaaa aaattcactc atccttgcgt ctatt 45
<210> 199
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 199
tagtggagcc tcctactcac ttaaaaaaag tccatctgca ggccc 45
<210> 200
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 200
acgtcatcac cagctcaaaa aaattcactc atccttgcgt ctatt 45
<210> 201
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 201
tagtggagcc tcctactcac ttaaaaaaaa cgacgtacaa gatac 45
<210> 202
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 202
agcgccttgg ccaccaaaaa aaattcactc atccttgcgt ctatt 45
<210> 203
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 203
tagtggagcc tcctactcac ttaaaaaaac aacggatagg acagc 45
<210> 204
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 204
cgatgacgaa gatggcaaaa aaattcactc atccttgcgt ctatt 45
<210> 205
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 205
cgatgacgaa gatggcaaaa aaattcactc atccttgcgt ctatt 45
<210> 206
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 206
ctgaatggct gtgaaaaaaa aaattcactc atccttgcgt ctatt 45
<210> 207
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 207
tagtggagcc tcctactcac ttaaaaaaac tgtagattcc aagca 45
<210> 208
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 208
aaacccctct tcaaacaaaa aaattcactc agaagtgcgt ctatt 45
<210> 209
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 209
tagtggagcc agctactcac ttaaaaaaag gtcgtggggc aggtc 45
<210> 210
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 210
ccaggacact ccactgaaaa aaattcactc agaagtgcgt ctatt 45
<210> 211
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 211
tagtggagcc agctactcac ttaaaaaaat agctgagcag gaata 45
<210> 212
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 212
tccaccttgt tggtttaaaa aaattcactc agaagtgcgt ctatt 45
<210> 213
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 213
tagtggagcc agctactcac ttaaaaaaac tgttctttgt acgtc 45
<210> 214
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 214
tgtaacgtgt ccttggaaaa aaattcactc agaagtgcgt ctatt 45
<210> 215
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 215
tagtggagcc agctactcac ttaaaaaaat actattacaa aatcc 45
<210> 216
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 216
caagatacat ttacaaaaaa aaattcactc agaagtgcgt ctatt 45
<210> 217
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 217
tagtggagcc agctactcac ttaaaaaaaa cagagtcagc agcac 45
<210> 218
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 218
tgctgcggag atcttaaaaa aaattcactc acttctgcgt ctatt 45
<210> 219
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 219
tagtggagcc ctgaactcac ttaaaaaaat gcagctggag tgctc 45
<210> 220
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 220
atttcctggt tcgtggaaaa aaattcactc acttctgcgt ctatt 45
<210> 221
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 221
tagtggagcc ctgaactcac ttaaaaaaat ggttatcgtg aatcc 45
<210> 222
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 222
ccatgcgtac cacagcaaaa aaattcactc acttctgcgt ctatt 45
<210> 223
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 223
tagtggagcc ctgaactcac ttaaaaaaat gtttcccact ggaga 45
<210> 224
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 224
caccaacaac ctgttgaaaa aaattcactc acttctgcgt ctatt 45
<210> 225
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 225
tagtggagcc ctgaactcac ttaaaaaaaa agcaccaagc atgtc 45
<210> 226
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 226
ccatgctaca aagtgtaaaa aaattcactc acttctgcgt ctatt 45
<210> 227
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 227
tagtggagcc ctgaactcac ttaaaaaaaa gggggagggt tgttc 45
<210> 228
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 228
agaaagcacg cagcataaaa aaattcactc actagtgcgt ctatt 45
<210> 229
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 229
tagtggagcc agtcactcac ttaaaaaaaa atgtcgaccc tcttc 45
<210> 230
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 230
atgttagggg cgctctaaaa aaattcactc actagtgcgt ctatt 45
<210> 231
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 231
tagtggagcc agtcactcac ttaaaaaaac agcagccgca gccgc 45
<210> 232
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 232
cgcagcctgg tcgtgcaaaa aaattcactc actagtgcgt ctatt 45
<210> 233
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 233
tagtggagcc agtcactcac ttaaaaaaac gcggaaaaag ttagc 45
<210> 234
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 234
agtttcctgt tgctgtaaaa aaattcactc actagtgcgt ctatt 45
<210> 235
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 235
tagtggagcc agtcactcac ttaaaaaaac ccgataatct ccatc 45
<210> 236
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 236
tcttccactg cagctcaaaa aaattcactc actagtgcgt ctatt 45
<210> 237
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 237
tagtggagcc agtcactcac ttaaaaaaac ctcaagaatg aattc 45
<210> 238
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 238
ttatgcagcc gatgaaaaaa aaattcactc aagtctgcgt ctatt 45
<210> 239
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 239
tagtggagcc gatcactcac ttaaaaaaac ttttgatctt gctgc 45
<210> 240
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 240
ggatcgagac cagaaaaaaa aaattcactc aagtctgcgt ctatt 45
<210> 241
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 241
tagtggagcc gatcactcac ttaaaaaaaa ttgtaatgag cggtg 45
<210> 242
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 242
ccgtgtagat cgtgtcaaaa aaattcactc aagtctgcgt ctatt 45
<210> 243
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 243
tagtggagcc gatcactcac ttaaaaaaac ggtccatgtc gcagc 45
<210> 244
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 244
ccatccatcc atccataaaa aaattcactc aagtctgcgt ctatt 45
<210> 245
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 245
tagtggagcc gatcactcac ttaaaaaaaa gtattaacat cccta 45
<210> 246
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 246
taagtggtgg gggaataaaa aaattcactc aagtctgcgt ctatt 45
<210> 247
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 247
tagtggagcc gatcactcac ttaaaaaaaa aatttcagct gtctc 45
<210> 248
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 248
tctgaggggc tccaggaaaa aaattcactc aaggatgcgt ctatt 45
<210> 249
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 249
tagtggagcc ctgaactcac ttaaaaaaac tctcacagcc tgcac 45
<210> 250
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 250
cgaagttgga gacagcaaaa aaattcactc aaggatgcgt ctatt 45
<210> 251
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 251
tagtggagcc ctgaactcac ttaaaaaaac ggtacagatc gtagc 45
<210> 252
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 252
cagaggggca acaaagaaaa aaattcactc aaggatgcgt ctatt 45
<210> 253
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 253
tagtggagcc ctgaactcac ttaaaaaaat cccataggac ttctc 45
<210> 254
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 254
caagccgtat cgtaagaaaa aaattcactc aaggatgcgt ctatt 45
<210> 255
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 255
tagtggagcc ctgaactcac ttaaaaaaaa gttgagatca gagtc 45
<210> 256
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 256
ctcgaaagca gccctgaaaa aaattcactc aaggatgcgt ctatt 45
<210> 257
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 257
tagtggagcc ctgaactcac ttaaaaaaac cccctcttca ttcca 45
<210> 258
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 258
ccttgcccag ttcaccaaaa aaattcactc aagtctgcgt ctatt 45
<210> 259
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 259
tagtggagcc gactactcac ttaaaaaaat gctgttcctt cttga 45
<210> 260
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 260
tctgggttcc catctgaaaa aaattcactc aagtctgcgt ctatt 45
<210> 261
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 261
tagtggagcc gactactcac ttaaaaaaac tggcctgagg ggtgc 45
<210> 262
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 262
gtcctgaggg ctgatgaaaa aaattcactc aagtctgcgt ctatt 45
<210> 263
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 263
tagtggagcc gactactcac ttaaaaaaag catggccata atagg 45
<210> 264
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 264
ttgctctcca aggagaaaaa aaattcactc aagtctgcgt ctatt 45
<210> 265
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 265
tagtggagcc gactactcac ttaaaaaaag tagcacacgg gcccc 45
<210> 266
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 266
cgtactttat taaggtaaaa aaattcactc aagtctgcgt ctatt 45
<210> 267
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 267
tagtggagcc gactactcac ttaaaaaaat ggggagacag agcta 45
<210> 268
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 268
aaatcgttgc agcaagaaaa aaattcactc acttctgcgt ctatt 45
<210> 269
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 269
tagtggagcc ctagactcac ttaaaaaaat catcttccca ggagc 45
<210> 270
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 270
tagaaagctt cactgcaaaa aaattcactc acttctgcgt ctatt 45
<210> 271
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 271
tagtggagcc ctagactcac ttaaaaaaac ctgcaggctc tgtgc 45
<210> 272
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 272
tcagaaccga ggctataaaa aaattcactc acttctgcgt ctatt 45
<210> 273
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 273
tagtggagcc ctagactcac ttaaaaaaag agaaggtccc ggtcc 45
<210> 274
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 274
ccttcaaacc cacagcaaaa aaattcactc acttctgcgt ctatt 45
<210> 275
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 275
tagtggagcc ctagactcac ttaaaaaaag tttgagacct taata 45
<210> 276
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 276
ccatcacctc ctcaaaaaaa aaattcactc acttctgcgt ctatt 45
<210> 277
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 277
tagtggagcc ctagactcac ttaaaaaaag accactcagt ctcac 45
<210> 278
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 278
aaccttccag tctgggaaaa aaattcactc agatctgcgt ctatt 45
<210> 279
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 279
tagtggagcc agctactcac ttaaaaaaaa ggcagtcttt ggacc 45
<210> 280
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 280
tggttcttgt acaagcaaaa aaattcactc agatctgcgt ctatt 45
<210> 281
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 281
tagtggagcc agctactcac ttaaaaaaac cttgaggacg gctac 45
<210> 282
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 282
tttgccccaa gactgtaaaa aaattcactc agatctgcgt ctatt 45
<210> 283
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 283
tagtggagcc agctactcac ttaaaaaaag aaggtttttc tgtcc 45
<210> 284
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 284
cagagcctgt gacttgaaaa aaattcactc agatctgcgt ctatt 45
<210> 285
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 285
tagtggagcc agctactcac ttaaaaaaaa ggatggcagg gaagc 45
<210> 286
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 286
cggccagtcg tttgtaaaaa aaattcactc agatctgcgt ctatt 45
<210> 287
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 287
tagtggagcc agctactcac ttaaaaaaaa ttctgcagcg gtgca 45
<210> 288
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 288
ccttccatct gctccaaaaa aaattcactc acttctgcgt ctatt 45
<210> 289
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 289
tagtggagcc cttcactcac ttaaaaaaat ccgtggttcc ataga 45
<210> 290
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 290
ctgcacctgc tcgtccaaaa aaattcactc acttctgcgt ctatt 45
<210> 291
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 291
tagtggagcc cttcactcac ttaaaaaaac attccagtgt gtaaa 45
<210> 292
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 292
agtaactggt ggattgaaaa aaattcactc acttctgcgt ctatt 45
<210> 293
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 293
tagtggagcc cttcactcac ttaaaaaaat aacccaccca tctgc 45
<210> 294
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 294
caggcaggga agggtcaaaa aaattcactc acttctgcgt ctatt 45
<210> 295
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 295
tagtggagcc cttcactcac ttaaaaaaag tagtggttgg agaaa 45
<210> 296
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 296
ccatgagatc tttcgtaaaa aaattcactc acttctgcgt ctatt 45
<210> 297
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 297
tagtggagcc cttcactcac ttaaaaaaat tctgctcgat gttgc 45
<210> 298
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 298
tcgctgtcct gcctgaaaaa aaattcactc aagtctgcgt ctatt 45
<210> 299
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 299
tagtggagcc ctgaactcac ttaaaaaaac ggagcttctg gaatc 45
<210> 300
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 300
ttgcactgct ccatgtaaaa aaattcactc aagtctgcgt ctatt 45
<210> 301
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 301
tagtggagcc ctgaactcac ttaaaaaaac cgcgttactt ggggc 45
<210> 302
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 302
aaagagggac ccattcaaaa aaattcactc aagtctgcgt ctatt 45
<210> 303
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 303
tagtggagcc ctgaactcac ttaaaaaaaa ctgccctcgg aaggc 45
<210> 304
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 304
cgctgaagca aggcaaaaaa aaattcactc aagtctgcgt ctatt 45
<210> 305
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 305
tagtggagcc ctgaactcac ttaaaaaaag ttctcccagc tgtcc 45
<210> 306
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 306
aggtacaagc cagtccaaaa aaattcactc aagtctgcgt ctatt 45
<210> 307
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 307
tagtggagcc ctgaactcac ttaaaaaaat gggctcgcag gtggc 45
<210> 308
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 308
catctggagg caacataaaa aaattcactc acttctgcgt ctatt 45
<210> 309
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 309
tagtggagcc gaagactcac ttaaaaaaat cttctagtcc tgttc 45
<210> 310
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 310
ttatctggac cccaacaaaa aaattcactc acttctgcgt ctatt 45
<210> 311
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 311
tagtggagcc gaagactcac ttaaaaaaag tttccccagt gcacc 45
<210> 312
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 312
agttcacagc tggcttaaaa aaattcactc acttctgcgt ctatt 45
<210> 313
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 313
tagtggagcc gaagactcac ttaaaaaaac attctcacac aaagc 45
<210> 314
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 314
tttgggaaag gatgaaaaaa aaattcactc acttctgcgt ctatt 45
<210> 315
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 315
tagtggagcc gaagactcac ttaaaaaaac agcagatagg aaggc 45
<210> 316
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 316
cagggacaag attggcaaaa aaattcactc acttctgcgt ctatt 45
<210> 317
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 317
tagtggagcc gaagactcac ttaaaaaaag tgcaatctgt gggca 45
<210> 318
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 318
cattgtcgca caccagaaaa aaattcactc acttctgcgt ctatt 45
<210> 319
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 319
tagtggagcc gatcactcac ttaaaaaaat tacagagccc agagc 45
<210> 320
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 320
caagtccaga cgcatgaaaa aaattcactc acttctgcgt ctatt 45
<210> 321
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 321
tagtggagcc gatcactcac ttaaaaaaat gagatctcgg ccagc 45
<210> 322
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 322
gaatgatttg gaaaggaaaa aaattcactc acttctgcgt ctatt 45
<210> 323
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 323
tagtggagcc gatcactcac ttaaaaaaat caaagctttg ggcag 45
<210> 324
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 324
cgcatgcata tccatgaaaa aaattcactc acttctgcgt ctatt 45
<210> 325
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 325
tagtggagcc gatcactcac ttaaaaaaat gcaagttaaa agtca 45
<210> 326
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 326
tgataggcaa aggaacaaaa aaattcactc acttctgcgt ctatt 45
<210> 327
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 327
tagtggagcc gatcactcac ttaaaaaaat ctgtagttcc cattc 45
<210> 328
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 328
ccttgcctag ggagataaaa aaattcactc aagagtgcgt ctatt 45
<210> 329
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 329
tagtggagcc ctgaactcac ttaaaaaaac ctacacaaat atgaa 45
<210> 330
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 330
agcatccagg actttgaaaa aaattcactc aagagtgcgt ctatt 45
<210> 331
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 331
tagtggagcc ctgaactcac ttaaaaaaac agggctgcct cggac 45
<210> 332
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 332
cgatttggtg tccagtaaaa aaattcactc aagagtgcgt ctatt 45
<210> 333
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 333
tagtggagcc ctgaactcac ttaaaaaaac aagtgtcttc cagta 45
<210> 334
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 334
atcttcctga gctgctaaaa aaattcactc aagagtgcgt ctatt 45
<210> 335
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 335
tagtggagcc ctgaactcac ttaaaaaaaa agaatgcttc acggc 45
<210> 336
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 336
tactgtgcat ctacagaaaa aaattcactc aagagtgcgt ctatt 45
<210> 337
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 337
tagtggagcc ctgaactcac ttaaaaaaat gctctgggaa aacac 45
<210> 338
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 338
tggaagaagt tcctccaaaa aaattcactc aagtctgcgt ctatt 45
<210> 339
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 339
tagtggagcc ctagactcac ttaaaaaaaa gccagaggaa ggttc 45
<210> 340
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 340
cacttgtaga tggccgaaaa aaattcactc aagtctgcgt ctatt 45
<210> 341
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 341
tagtggagcc ctagactcac ttaaaaaaag aagttgtccg tgatc 45
<210> 342
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 342
tccagtgagc tcagctaaaa aaattcactc aagtctgcgt ctatt 45
<210> 343
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 343
tagtggagcc ctagactcac ttaaaaaaag cttagctcca gaccc 45
<210> 344
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 344
ttgggcttga gggaacaaaa aaattcactc aagtctgcgt ctatt 45
<210> 345
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 345
tagtggagcc ctagactcac ttaaaaaaag tgcttttgcc aatcc 45
<210> 346
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 346
cccagttaag tcttagaaaa aaattcactc aagtctgcgt ctatt 45
<210> 347
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 347
tagtggagcc ctagactcac ttaaaaaaaa gccgggcatc ctttc 45
<210> 348
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 348
cgtagaggag gtctgtaaaa aaattcactc aagtctgcgt ctatt 45
<210> 349
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 349
tagtggagcc cttcactcac ttaaaaaaaa tgtcatttga gccca 45
<210> 350
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 350
tcaggtcctt gctcctaaaa aaattcactc aagtctgcgt ctatt 45
<210> 351
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 351
tagtggagcc cttcactcac ttaaaaaaac tgcggaaagc atgtc 45
<210> 352
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 352
cttggtcagt ttcctcaaaa aaattcactc aagtctgcgt ctatt 45
<210> 353
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 353
tagtggagcc cttcactcac ttaaaaaaaa gtggtactgt ttacc 45
<210> 354
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 354
aattcctagc ccctggaaaa aaattcactc aagtctgcgt ctatt 45
<210> 355
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 355
tagtggagcc cttcactcac ttaaaaaaag taggacgaag ctgac 45
<210> 356
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 356
attttactgg aaggccaaaa aaattcactc aagtctgcgt ctatt 45
<210> 357
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 357
tagtggagcc cttcactcac ttaaaaaaac aatagctgtg ttaac 45
<210> 358
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 358
ttcaggcagg gctgccaaaa aaattcactc atcagtgcgt ctatt 45
<210> 359
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 359
tagtggagcc tcgaactcac ttaaaaaaac tccttggctg tttcc 45
<210> 360
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 360
ataatagggt gtgccaaaaa aaattcactc atcagtgcgt ctatt 45
<210> 361
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 361
tagtggagcc tcgaactcac ttaaaaaaac atttcttgac acatc 45
<210> 362
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 362
gcctcttccc atcattaaaa aaattcactc atcagtgcgt ctatt 45
<210> 363
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 363
tagtggagcc tcgaactcac ttaaaaaaat cagctctgcc cagag 45
<210> 364
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 364
tacccttgca gaaagcaaaa aaattcactc atcagtgcgt ctatt 45
<210> 365
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 365
tagtggagcc tcgaactcac ttaaaaaaaa attttcctcg attgc 45
<210> 366
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 366
atagcacacg cagaaaaaaa aaattcactc atcagtgcgt ctatt 45
<210> 367
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 367
tagtggagcc tcgaactcac ttaaaaaaat ctagtttaca catcc 45
<210> 368
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 368
aaagcggagg ttctgcaaaa aaattcactc agaagtgcgt ctatt 45
<210> 369
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 369
tagtggagcc agtcactcac ttaaaaaaac aacatcgtag agatc 45
<210> 370
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 370
tctaccgcaa aggaaaaaaa aaattcactc agaagtgcgt ctatt 45
<210> 371
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 371
tagtggagcc agtcactcac ttaaaaaaag aaggtactct ctgac 45
<210> 372
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 372
ctggatttgg gtgtgaaaaa aaattcactc agaagtgcgt ctatt 45
<210> 373
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 373
tagtggagcc agtcactcac ttaaaaaaat ctataccaca gtccc 45
<210> 374
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 374
gccatgagga aggtgtaaaa aaattcactc agaagtgcgt ctatt 45
<210> 375
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 375
tagtggagcc agtcactcac ttaaaaaaac tgggtgtgaa tggtg 45
<210> 376
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 376
tgtgcacaca tgtaccaaaa aaattcactc agaagtgcgt ctatt 45
<210> 377
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 377
tagtggagcc agtcactcac ttaaaaaaat gccaatgtca ccatc 45
<210> 378
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 378
tggattttag ggcccaaaaa aaattcactc aagagtgcgt ctatt 45
<210> 379
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 379
tagtggagcc gactactcac ttaaaaaaat ttctctgggg agcgc 45
<210> 380
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 380
atctatggtt ccccgaaaaa aaattcactc aagagtgcgt ctatt 45
<210> 381
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 381
tagtggagcc gactactcac ttaaaaaaac ccagttcagt ctatc 45
<210> 382
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 382
agtcgagaaa ggggtgaaaa aaattcactc aagagtgcgt ctatt 45
<210> 383
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 383
tagtggagcc gactactcac ttaaaaaaac tcagcactgg gtatc 45
<210> 384
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 384
gacaccgctc actcataaaa aaattcactc aagagtgcgt ctatt 45
<210> 385
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 385
tagtggagcc gactactcac ttaaaaaaag acaggcatca tccag 45
<210> 386
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 386
aaaatgcaaa gcagtcaaaa aaattcactc aagagtgcgt ctatt 45
<210> 387
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 387
tagtggagcc gactactcac ttaaaaaaaa taggtagaag agccc 45
<210> 388
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 388
taaaaccact tccaagaaaa aaattcactc actgatgcgt ctatt 45
<210> 389
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 389
tagtggagcc gatcactcac ttaaaaaaac acgacacatt ctcac 45
<210> 390
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 390
gtattttaag ccacagaaaa aaattcactc actgatgcgt ctatt 45
<210> 391
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 391
tagtggagcc gatcactcac ttaaaaaaaa ttgctagtgg gcatg 45
<210> 392
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 392
tgacaatggg aaacacaaaa aaattcactc actgatgcgt ctatt 45
<210> 393
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 393
tagtggagcc gatcactcac ttaaaaaaaa gtgggatctt caggc 45
<210> 394
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 394
tgaacctact gcccttaaaa aaattcactc actgatgcgt ctatt 45
<210> 395
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 395
tagtggagcc gatcactcac ttaaaaaaat agagacattc ttgcc 45
<210> 396
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 396
ctgtcccttt cttagtaaaa aaattcactc actgatgcgt ctatt 45
<210> 397
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 397
tagtggagcc gatcactcac ttaaaaaaat ggttgattga tagcc 45
<210> 398
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 398
tacggactca tctgcaaaaa aaattcactc actcttgcgt ctatt 45
<210> 399
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 399
tagtggagcc tcctactcac ttaaaaaaat tttggccatg ctgcc 45
<210> 400
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 400
atggtctccg tcaggcaaaa aaattcactc actcttgcgt ctatt 45
<210> 401
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 401
tagtggagcc tcctactcac ttaaaaaaag gcaacgtttg aggtc 45
<210> 402
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 402
gatgtgcatt gctaggaaaa aaattcactc actcttgcgt ctatt 45
<210> 403
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 403
tagtggagcc tcctactcac ttaaaaaaaa ctataggcaa aatgg 45
<210> 404
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 404
caaaacttga agctcaaaaa aaattcactc actcttgcgt ctatt 45
<210> 405
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 405
tagtggagcc tcctactcac ttaaaaaaag tgaacccttc ctcca 45
<210> 406
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 406
accacaaagc cctatcaaaa aaattcactc actcttgcgt ctatt 45
<210> 407
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 407
tagtggagcc tcctactcac ttaaaaaaac caggggtaat cattc 45
<210> 408
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 408
cgagtagccc acagggaaaa aaattcactc actgatgcgt ctatt 45
<210> 409
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 409
tagtggagcc tcagactcac ttaaaaaaat ttcacagtgg atgcc 45
<210> 410
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 410
caccttgaag gtcttcaaaa aaattcactc actgatgcgt ctatt 45
<210> 411
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 411
tagtggagcc tcagactcac ttaaaaaaac gtcgaggctg taagc 45
<210> 412
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 412
tgtgggctac cttgtaaaaa aaattcactc actgatgcgt ctatt 45
<210> 413
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 413
tagtggagcc tcagactcac ttaaaaaaat gcacaccatc atcac 45
<210> 414
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 414
ctctgaaatc ccctggaaaa aaattcactc actgatgcgt ctatt 45
<210> 415
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 415
tagtggagcc tcagactcac ttaaaaaaaa aagggtgaag ggcaa 45
<210> 416
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 416
cagtgcaagg gtcaagaaaa aaattcactc actgatgcgt ctatt 45
<210> 417
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 417
tagtggagcc tcagactcac ttaaaaaaat tgtcaactga ggctc 45
<210> 418
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 418
ctttgctcag ctggaaaaaa aaattcactc atcgatgcgt ctatt 45
<210> 419
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 419
tagtggagcc cttcactcac ttaaaaaaat ctgttttgcg gctga 45
<210> 420
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 420
tgacaggtgg tccaggaaaa aaattcactc atcgatgcgt ctatt 45
<210> 421
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 421
tagtggagcc cttcactcac ttaaaaaaaa gggaaggcaa tagac 45
<210> 422
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 422
aggagatgct tgcaaaaaaa aaattcactc atcgatgcgt ctatt 45
<210> 423
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 423
tagtggagcc cttcactcac ttaaaaaaag cacatcctgg tcttc 45
<210> 424
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 424
cacaaggttg tcatggaaaa aaattcactc atcgatgcgt ctatt 45
<210> 425
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 425
tagtggagcc cttcactcac ttaaaaaaat cagctccttg gaaac 45
<210> 426
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 426
gaagcttatg aactgaaaaa aaattcactc atcgatgcgt ctatt 45
<210> 427
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 427
tagtggagcc cttcactcac ttaaaaaaag ctccagagct ctatg 45
<210> 428
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 428
cctcaagctc caacagaaaa aaattcactc agatctgcgt ctatt 45
<210> 429
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 429
tagtggagcc agtcactcac ttaaaaaaat ccgtttgttt ctctc 45
<210> 430
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 430
acgttggggt cgtcttaaaa aaattcactc agatctgcgt ctatt 45
<210> 431
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 431
tagtggagcc agtcactcac ttaaaaaaac tcacttctga tttcc 45
<210> 432
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 432
ttgaacgtcc tcccttaaaa aaattcactc agatctgcgt ctatt 45
<210> 433
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 433
tagtggagcc agtcactcac ttaaaaaaaa ttaaactcgc tggtc 45
<210> 434
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 434
tgacttcgtg caagttaaaa aaattcactc agatctgcgt ctatt 45
<210> 435
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 435
tagtggagcc agtcactcac ttaaaaaaat ccaccacgaa ctctc 45
<210> 436
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 436
tacgagatcc aaaatgaaaa aaattcactc agatctgcgt ctatt 45
<210> 437
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 437
tagtggagcc agtcactcac ttaaaaaaaa gccatggtgg tcatc 45
<210> 438
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 438
cccaatgtag aggaagaaaa aaattcactc aaggatgcgt ctatt 45
<210> 439
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 439
tagtggagcc cttcactcac ttaaaaaaaa cagggacacg atgtc 45
<210> 440
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 440
tgaatgagga tgtccgaaaa aaattcactc aaggatgcgt ctatt 45
<210> 441
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 441
tagtggagcc cttcactcac ttaaaaaaag gacaccagct ttgtc 45
<210> 442
<211> 44
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 442
agatttctcc accgtaaaaa aattcactca aggatgcgtc tatt 44
<210> 443
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 443
tagtggagcc cttcactcac ttaaaaaaat ttctacccac agctc 45
<210> 444
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 444
ctccaagagc ttccttaaaa aaattcactc aaggatgcgt ctatt 45
<210> 445
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 445
tagtggagcc cttcactcac ttaaaaaaat aatcccaggc tggtc 45
<210> 446
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 446
tcactctcat ccgaggaaaa aaattcactc aaggatgcgt ctatt 45
<210> 447
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 447
tagtggagcc cttcactcac ttaaaaaaat gtagtgatga gtctc 45
<210> 448
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 448
cggatatcaa ggaatcaaaa aaattcactc aagagtgcgt ctatt 45
<210> 449
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 449
tagtggagcc cttcactcac ttaaaaaaaa ttgtccttgg tgaga 45
<210> 450
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 450
cacatggcaa aacattaaaa aaattcactc aagagtgcgt ctatt 45
<210> 451
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 451
tagtggagcc cttcactcac ttaaaaaaat cacttgttca ggttc 45
<210> 452
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 452
acacacccaa aaaaggaaaa aaattcactc aagagtgcgt ctatt 45
<210> 453
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 453
tagtggagcc cttcactcac ttaaaaaaaa gctgcattcc taagc 45
<210> 454
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 454
caaatgcttt tcccccaaaa aaattcactc aagagtgcgt ctatt 45
<210> 455
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 455
tagtggagcc cttcactcac ttaaaaaaaa agtgacaaag ttttc 45
<210> 456
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 456
aaggagtttt ctcagcaaaa aaattcactc aagagtgcgt ctatt 45
<210> 457
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 457
tagtggagcc cttcactcac ttaaaaaaac ttgacccctt gatgc 45
<210> 458
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 458
ccatcaggga cggagaaaaa aaattcactc atcgatgcgt ctatt 45
<210> 459
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 459
tagtggagcc agctactcac ttaaaaaaag gtttagacga gaatc 45
<210> 460
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 460
tgtagaagtg tcgcctaaaa aaattcactc atcgatgcgt ctatt 45
<210> 461
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 461
tagtggagcc agctactcac ttaaaaaaag cgcagcccaa acatc 45
<210> 462
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 462
aggaaccatt tctgctaaaa aaattcactc atcgatgcgt ctatt 45
<210> 463
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 463
tagtggagcc agctactcac ttaaaaaaag tgtgatgagg tgtcc 45
<210> 464
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 464
cgtttggccc ttctgcaaaa aaattcactc atcgatgcgt ctatt 45
<210> 465
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 465
tagtggagcc agctactcac ttaaaaaaag tgatgcttgg gacta 45
<210> 466
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 466
ggaacgtgtg tgtgtgaaaa aaattcactc atcgatgcgt ctatt 45
<210> 467
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 467
tagtggagcc agctactcac ttaaaaaaat gtaggtggtt gaatg 45
<210> 468
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 468
ctgttatccc tcccacaaaa aaattcactc atcgatgcgt ctatt 45
<210> 469
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 469
tagtggagcc ctagactcac ttaaaaaaag gtgctgcaga attgc 45
<210> 470
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 470
aaatagcgag cagactaaaa aaattcactc atcgatgcgt ctatt 45
<210> 471
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 471
tagtggagcc ctagactcac ttaaaaaaat gagtagccat gtacc 45
<210> 472
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 472
cactgatgga gacctcaaaa aaattcactc atcgatgcgt ctatt 45
<210> 473
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 473
tagtggagcc ctagactcac ttaaaaaaat ctaaggtcgt agagc 45
<210> 474
<211> 44
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 474
gcttgaagca ggtcaaaaaa aattcactca tcgatgcgtc tatt 44
<210> 475
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 475
tagtggagcc ctagactcac ttaaaaaaaa tcctgttgga cgttg 45
<210> 476
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 476
taactggggt tacattaaaa aaattcactc atcgatgcgt ctatt 45
<210> 477
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 477
tagtggagcc ctagactcac ttaaaaaaaa ttcttcacac gtcac 45
<210> 478
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 478
gatgtgtgcc tggtggaaaa aaattcactc actgatgcgt ctatt 45
<210> 479
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 479
tagtggagcc tctcactcac ttaaaaaaac aggtggcgct tgaag 45
<210> 480
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 480
catgttttct ggggggaaaa aaattcactc actgatgcgt ctatt 45
<210> 481
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 481
tagtggagcc tctcactcac ttaaaaaaag caggctcact tggtc 45
<210> 482
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 482
atttgatgga ggcacaaaaa aaattcactc actgatgcgt ctatt 45
<210> 483
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 483
tagtggagcc tctcactcac ttaaaaaaaa tctccagggg tcaac 45
<210> 484
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 484
cgtccaaaca gggcacaaaa aaattcactc actgatgcgt ctatt 45
<210> 485
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 485
tagtggagcc tctcactcac ttaaaaaaac tctctctcaa gtgaa 45
<210> 486
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 486
aggggaagcc aacagaaaaa aaattcactc actgatgcgt ctatt 45
<210> 487
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 487
tagtggagcc tctcactcac ttaaaaaaaa agggtttcct gtacc 45
<210> 488
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 488
gaacgggttg gactaaaaaa aaattcactc atcgatgcgt ctatt 45
<210> 489
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 489
tagtggagcc agagactcac ttaaaaaaac agggccttta tggag 45
<210> 490
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 490
ctccagcgat tcaaccaaaa aaattcactc atcgatgcgt ctatt 45
<210> 491
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 491
tagtggagcc agagactcac ttaaaaaaaa gaactggatc tcctc 45
<210> 492
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 492
ttcgagtcct taatgaaaaa aaattcactc atcgatgcgt ctatt 45
<210> 493
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 493
tagtggagcc agagactcac ttaaaaaaag tccttgtgct cctgc 45
<210> 494
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 494
tgccaagtgc tgagaaaaaa aaattcactc atcgatgcgt ctatt 45
<210> 495
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 495
tagtggagcc agagactcac ttaaaaaaac ttatgccata gatcc 45
<210> 496
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 496
tacctgccca ccaatcaaaa aaattcactc atcgatgcgt ctatt 45
<210> 497
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 497
tagtggagcc agagactcac ttaaaaaaat gctcaatgtc ttccc 45
<210> 498
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 498
aggccatcca cagacgaaaa aaattcactc aagagtgcgt ctatt 45
<210> 499
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 499
tagtggagcc gagaactcac ttaaaaaaag ctgtgatgaa cagac 45
<210> 500
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 500
ctgttcctgg gtagcgaaaa aaattcactc aagagtgcgt ctatt 45
<210> 501
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 501
tagtggagcc gagaactcac ttaaaaaaat cggattggga atgtc 45
<210> 502
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 502
catctctgtg gctgtgaaaa aaattcactc aagagtgcgt ctatt 45
<210> 503
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 503
tagtggagcc gagaactcac ttaaaaaaag aacagtaact gtggc 45
<210> 504
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 504
aagttcaagg agccctaaaa aaattcactc aagagtgcgt ctatt 45
<210> 505
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 505
tagtggagcc gagaactcac ttaaaaaaac agtcctcgaa ggagc 45
<210> 506
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 506
tcctggagtg ctgtgaaaaa aaattcactc aagagtgcgt ctatt 45
<210> 507
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 507
tagtggagcc gagaactcac ttaaaaaaat cagagatcca cctgc 45
<210> 508
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 508
cttggatttg gggaagaaaa aaattcactc atcagtgcgt ctatt 45
<210> 509
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 509
tagtggagcc cttcactcac ttaaaaaaat ctggtggtag cgtta 45
<210> 510
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 510
ccaatttcaa gtgagaaaaa aaattcactc atcagtgcgt ctatt 45
<210> 511
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 511
tagtggagcc cttcactcac ttaaaaaaat acatcaaacc tgtgc 45
<210> 512
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 512
cgacaatggg aaatgtaaaa aaattcactc atcagtgcgt ctatt 45
<210> 513
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 513
tagtggagcc cttcactcac ttaaaaaaat gtggtgagtg agaaa 45
<210> 514
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 514
tttcaggcac aggaccaaaa aaattcactc atcagtgcgt ctatt 45
<210> 515
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 515
tagtggagcc cttcactcac ttaaaaaaag tagaaccaag ccccc 45
<210> 516
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 516
acagtagctt cgggtcaaaa aaattcactc atcagtgcgt ctatt 45
<210> 517
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 517
tagtggagcc cttcactcac ttaaaaaaag tgttcattca cacac 45
<210> 518
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 518
tgccggaact ccatggaaaa aaattcactc atcgatgcgt ctatt 45
<210> 519
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 519
tagtggagcc ctgaactcac ttaaaaaaac ttccgaaact cctcc 45
<210> 520
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 520
atggcaagca gggtataaaa aaattcactc atcgatgcgt ctatt 45
<210> 521
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 521
tagtggagcc ctgaactcac ttaaaaaaaa tttgctccag atgcc 45
<210> 522
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 522
cccagagatg gcaaagaaaa aaattcactc atcgatgcgt ctatt 45
<210> 523
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 523
tagtggagcc ctgaactcac ttaaaaaaaa gtggttcacg ttaaa 45
<210> 524
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 524
ctttcagggg agtctgaaaa aaattcactc atcgatgcgt ctatt 45
<210> 525
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 525
tagtggagcc ctgaactcac ttaaaaaaaa acaagcggag aacga 45
<210> 526
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 526
cgtcacagag acagacaaaa aaattcactc atcgatgcgt ctatt 45
<210> 527
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 527
tagtggagcc ctgaactcac ttaaaaaaag gcagggcagg attta 45
<210> 528
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 528
tcaaggaaac tgtcccaaaa aaattcactc aagcttgcgt ctatt 45
<210> 529
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 529
tagtggagcc gatcactcac ttaaaaaaat ccacgctgag agttc 45
<210> 530
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 530
cgaaagattt tccactaaaa aaattcactc aagcttgcgt ctatt 45
<210> 531
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 531
tagtggagcc gatcactcac ttaaaaaaag gttcgtggaa aggcc 45
<210> 532
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 532
tcatccactg gacattaaaa aaattcactc aagcttgcgt ctatt 45
<210> 533
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 533
tagtggagcc gatcactcac ttaaaaaaat tccaactgct cttgc 45
<210> 534
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 534
tactcaccag cccctaaaaa aaattcactc aagcttgcgt ctatt 45
<210> 535
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 535
tagtggagcc gatcactcac ttaaaaaaaa agaaagctat taccc 45
<210> 536
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 536
atggattgat tctggaaaaa aaattcactc aagcttgcgt ctatt 45
<210> 537
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 537
tagtggagcc gatcactcac ttaaaaaaat caaaagcgaa ctcac 45
<210> 538
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 538
ctctcctcgg tgaatcaaaa aaattcactc agaagtgcgt ctatt 45
<210> 539
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 539
tagtggagcc tcgaactcac ttaaaaaaat tctcttcttt ccagc 45
<210> 540
<211> 46
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 540
tcgtgtgtga gtccttgaaa aaaattcact cagaagtgcg tctatt 46
<210> 541
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 541
tagtggagcc tcgaactcac ttaaaaaaac cataatgggt agttc 45
<210> 542
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 542
ttggcggtgt gcctgtaaaa aaattcactc agaagtgcgt ctatt 45
<210> 543
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 543
tagtggagcc tcgaactcac ttaaaaaaac atgctctctg gctcc 45
<210> 544
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 544
agagggacag cacgagaaaa aaattcactc agaagtgcgt ctatt 45
<210> 545
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 545
tagtggagcc tcgaactcac ttaaaaaaaa ccatgagaag tggcc 45
<210> 546
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 546
ctggagggca aacactaaaa aaattcactc agaagtgcgt ctatt 45
<210> 547
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 547
tagtggagcc tcgaactcac ttaaaaaaaa ggtcgttcag tcaca 45
Claims (21)
1. A probe primer group for RNA in-situ detection, wherein a sample for RNA in-situ detection is cells cultured in vitro and cells in a tissue section, and the probe primer group is characterized in that: comprising at least one loop-forming probe for forming a rolling circle amplification product and first to fourth detection probe sets capable of hybridizing to the rolling circle amplification product;
each of the loop forming probes corresponds to a target sequence, the sequence of the 5 'end and the 3' end of the loop forming probe is specifically hybridized and complemented with the target sequence, and a first label sequence and a second label sequence are arranged between the 5 'end and the 3' end of the loop forming probe, the first label sequence is positioned at the 3 'end of the sequence of the 5' end which is specifically hybridized and complemented with the target sequence, and the second label sequence is positioned at the 5 'end of the sequence of the 3' end which is specifically hybridized and complemented with the target sequence; the first tag sequence consists of a first fixed tag sequence and a first variable tag sequence, the second tag sequence consists of a second fixed tag sequence and a second variable tag sequence, and the first variable tag sequence and the second variable tag sequence are four-base-length sequences consisting of double-base tag sequences;
a first detection probe group consisting of a plurality of first detection probe subgroups, wherein the length of each first detection probe is the same as that of the first tag sequence, the first detection probe in a first detection probe subgroup is completely the same as one of the two-base tag sequences of the first fixed tag sequence and the first variable tag sequence, the remaining two bases are degenerate sequences, the first detection probe corresponding to the different one of the two-base tag sequences is modified with different report labels, and the report labels modified by the first detection probe in the same first detection probe subgroup are the same;
the second detection probe group consists of a plurality of second detection probe subgroups, wherein the length of each second detection probe is the same as that of the first label sequence, the second detection probe in one second detection probe subgroup is completely the same as the other two-base label sequence of the first fixed label sequence and the first variable label sequence, the rest two bases are degenerate sequences, the second detection probe corresponding to the other different two-base label sequence is modified with different report labels, and the report labels modified by the second detection probe in the same second detection probe subgroup are the same;
a third detection probe group, which consists of a plurality of third detection probe subgroups, wherein the length of each third detection probe is the same as that of the first label sequence, the third detection probe in a third detection probe subgroup is completely the same as one double-base label sequence of the second fixed label sequence and the second variable label sequence, the rest two bases are degenerate sequences, the third detection probes corresponding to different one of the double-base label sequences are modified with different report labels, and the report labels modified by the third detection probes in the same third detection probe subgroup are the same;
and the fourth detection probe group consists of a plurality of fourth detection probe subgroups, wherein the length of each fourth detection probe is the same as that of the second label sequence, the fourth detection probes in one fourth detection probe subgroup are completely the same as the other two-base label sequence of the second fixed label sequence and the second variable label sequence, the rest two bases are degenerate sequences, the fourth detection probes corresponding to different two-base label sequences are modified with different report labels, and the report labels modified by the fourth detection probes in the same fourth detection probe subgroup are the same.
2. The probe-primer set of claim 1, wherein: the first variable label sequence consists of a first double-base label sequence and a second double-base label sequence, the first double-base label sequence is cw, as, ts or gw, the second variable label sequence consists of a third double-base label sequence and a fourth double-base label sequence, the third double-base label sequence is cw, as, ts or gw, the fourth double-base label sequence is cw, as, ts or gw,
the first detection probe group consists of a plurality of first detection probe subgroups, wherein the length of each first detection probe is the same as that of the first label sequence, the first detection probe in one first detection probe subgroup is completely the same as the first fixed label sequence and the first double-base label sequence in the first variable label sequence, the rest two bases are cw, as, ts or gw, different report labels are modified on the first detection probes corresponding to the different first double-base label sequences, and the report labels modified by the first detection probe in the same first detection probe subgroup are the same;
the second detection probe group consists of a plurality of second detection probe subgroups, wherein the length of each second detection probe is the same as that of the first label sequence, the second detection probe in one second detection probe subgroup is completely the same as the second double-base label sequence in the first fixed label sequence and the first variable label sequence, the rest two bases are cw, as, ts or gw, different report labels are modified by the second detection probes corresponding to the different second double-base label sequences, and the report labels modified by the second detection probes in the same second detection probe subgroup are the same;
a third detection probe group, which consists of a plurality of third detection probe subgroups, wherein the length of each third detection probe is the same as that of the second label sequence, the third detection probe in a third detection probe subgroup is completely the same as the third double-base label sequence of the second fixed label sequence and the second variable label sequence, the rest two bases are cw, as, ts or gw, different report labels are modified on the third detection probes corresponding to the different third double-base label sequences, and the report labels modified by the third detection probes in the same third detection probe subgroup are the same;
and the fourth detection probe group consists of a plurality of fourth detection probe subgroups, wherein the length of each fourth detection probe is the same as that of the second label sequence, the fourth detection probes in one fourth detection probe subgroup are completely the same as the fourth double-base label sequences of the second fixed label sequence and the second variable label sequence, the rest two bases are cw, as, ts or gw, the fourth detection probes corresponding to the different fourth double-base label sequences are modified with different report labels, and the report labels modified by the fourth detection probes in the same fourth detection probe subgroup are the same.
3. The probe-primer set according to claim 1 or 2, wherein: the ring-forming probe is a double-connection probe or a locking probe.
4. The probe primer set of claim 3, wherein: the ring-forming probe is a double-connection probe, and also comprises a splint primer which is matched with the double-connection probe to form a ring after hybridization.
5. The probe primer set of claim 4, wherein: the kit also comprises a first anchor primer and a second anchor primer, wherein the first anchor primer can be strictly complementary and paired with the rolling circle amplification product and can be connected with the hybridized first detection probe and second detection probe under the action of DNA polymerase, and the second anchor primer can be strictly complementary and paired with the rolling circle amplification product and can be connected with the hybridized third detection probe and fourth detection probe under the action of DNA polymerase.
6. An RNA in-situ detection kit, which is characterized in that: having at least one loop-forming probe for forming a rolling circle amplification product and first to fourth detection probe sets capable of hybridizing to the rolling circle amplification product;
each of the loop forming probes corresponds to a target sequence, the sequence of the 5 'end and the 3' end of the loop forming probe is specifically hybridized and complemented with the target sequence, and a first label sequence and a second label sequence are arranged between the 5 'end and the 3' end of the loop forming probe, the first label sequence is positioned at the 3 'end of the sequence of the 5' end which is specifically hybridized and complemented with the target sequence, and the second label sequence is positioned at the 5 'end of the sequence of the 3' end which is specifically hybridized and complemented with the target sequence; the first tag sequence consists of a first fixed tag sequence and a first variable tag sequence, the second tag sequence consists of a second fixed tag sequence and a second variable tag sequence, and the first variable tag sequence and the second variable tag sequence are four-base-length sequences consisting of double-base tag sequences;
the first detection probe group consists of a plurality of first detection probe subgroups, wherein the length of each first detection probe is the same as that of the first label sequence, the first detection probe in one first detection probe subgroup is completely the same as one double-base label sequence of the first fixed label sequence and the first variable label sequence, the rest two bases are degenerate sequences, the first detection probes corresponding to different one double-base label sequences are modified with different report labels, and the report labels modified by the first detection probes in the same first detection probe subgroup are the same;
the second detection probe group consists of a plurality of second detection probe subgroups, wherein the length of each second detection probe is the same as that of the first label sequence, the second detection probe in one second detection probe subgroup is completely the same as the other two-base label sequence of the first fixed label sequence and the first variable label sequence, the rest two bases are degenerate sequences, the second detection probe corresponding to the other different two-base label sequence is modified with different report labels, and the report labels modified by the second detection probe in the same second detection probe subgroup are the same;
a third detection probe group, consisting of a plurality of third detection probe subgroups, wherein the length of each third detection probe is the same as that of the first tag sequence, the third detection probes in a third detection probe subgroup are completely the same as one of the double-base tag sequences of the second fixed tag sequence and the second variable tag sequence, the remaining two bases are degenerate sequences, the third detection probes corresponding to different one of the double-base tag sequences are modified with different report labels, and the report labels modified by the third detection probes in the same third detection probe subgroup are the same;
and the fourth detection probe group consists of a plurality of fourth detection probe subgroups, wherein the length of each fourth detection probe is the same as that of the second label sequence, the fourth detection probes in one fourth detection probe subgroup are completely the same as the other two-base label sequence of the second fixed label sequence and the second variable label sequence, the rest two bases are degenerate sequences, the fourth detection probes corresponding to different two-base label sequences are modified with different report labels, and the report labels modified by the fourth detection probes in the same fourth detection probe subgroup are the same.
7. The in situ RNA detection kit of claim 6, wherein: the first variable label sequence consists of a first double-base label sequence and a second double-base label sequence, the first double-base label sequence is cw, as, ts or gw, the second variable label sequence consists of a third double-base label sequence and a fourth double-base label sequence, the third double-base label sequence is cw, as, ts or gw, the fourth double-base label sequence is cw, as, ts or gw,
the first detection probe group consists of a plurality of first detection probe subgroups, wherein the length of each first detection probe is the same as that of the first label sequence, the first detection probe in one first detection probe subgroup is completely the same as the first fixed label sequence and the first double-base label sequence in the first variable label sequence, the rest two bases are cw, as, ts or gw, different report labels are modified on the first detection probes corresponding to the different first double-base label sequences, and the report labels modified by the first detection probe in the same first detection probe subgroup are the same;
the second detection probe group consists of a plurality of second detection probe subgroups, wherein the length of each second detection probe is the same as that of the first label sequence, the second detection probe in one second detection probe subgroup is completely the same as the second double-base label sequence in the first fixed label sequence and the first variable label sequence, the rest two bases are cw, as, ts or gw, different report labels are modified by the second detection probes corresponding to the different second double-base label sequences, and the report labels modified by the second detection probes in the same second detection probe subgroup are the same;
a third detection probe group, which consists of a plurality of third detection probe subgroups, wherein the length of each third detection probe is the same as that of the second label sequence, the third detection probe in a third detection probe subgroup is completely the same as the third double-base label sequence of the second fixed label sequence and the second variable label sequence, the rest two bases are cw, as, ts or gw, different report labels are modified on the third detection probes corresponding to the different third double-base label sequences, and the report labels modified by the third detection probes in the same third detection probe subgroup are the same;
and the fourth detection probe group consists of a plurality of fourth detection probe subgroups, wherein the length of each fourth detection probe is the same as that of the second label sequence, the fourth detection probe in one fourth detection probe subgroup is completely the same as the fourth double-base label sequence of the second fixed label sequence and the second variable label sequence, the rest two bases are cw, as, ts or gw, different report markers are modified on the fourth detection probes corresponding to the different fourth double-base label sequences, and the report markers modified on the fourth detection probes in the same fourth detection probe subgroup are the same.
8. The kit for in situ detection of RNA as claimed in claim 6 or 7, wherein: the loop-forming probe is a double-connection probe or a padlock probe.
9. The in situ RNA detection kit of claim 8, wherein: the ring-forming probe is a double-connection probe, and also comprises a splint primer which is matched with the double-connection probe to form a ring after hybridization.
10. The in situ RNA detection kit of claim 9, wherein: the kit also comprises a first anchor primer and a second anchor primer, wherein the first anchor primer can be strictly complementary and paired with the rolling circle amplification product and can be connected with the first detection probe and the second detection probe after hybridization under the action of DNA polymerase, and the second anchor primer can be strictly complementary and paired with the rolling circle amplification product and can be connected with the third detection probe and the fourth detection probe after hybridization under the action of DNA polymerase.
11. The application of the probe primer group in the RNA in-situ detection of non-diagnostic treatment purposes, wherein the RNA in-situ detection samples are cells cultured in vitro and cells in tissue slices, and the method is characterized in that: the probe primer set comprises at least one loop-forming probe for forming a rolling circle amplification product and first to fourth detection probe sets capable of hybridizing with the rolling circle amplification product;
each of the loop forming probes corresponds to a target sequence, the sequence of the 5 'end and the 3' end of the loop forming probe is specifically hybridized and complemented with the target sequence, and a first label sequence and a second label sequence are arranged between the 5 'end and the 3' end of the loop forming probe, the first label sequence is positioned at the 3 'end of the sequence of the 5' end which is specifically hybridized and complemented with the target sequence, and the second label sequence is positioned at the 5 'end of the sequence of the 3' end which is specifically hybridized and complemented with the target sequence; the first tag sequence consists of a first fixed tag sequence and a first variable tag sequence, the second tag sequence consists of a second fixed tag sequence and a second variable tag sequence, and the first variable tag sequence and the second variable tag sequence are four-base-length sequences consisting of double-base tag sequences;
the first detection probe group consists of a plurality of first detection probe subgroups, wherein the length of each first detection probe is the same as that of the first label sequence, the first detection probe in one first detection probe subgroup is completely the same as one double-base label sequence of the first fixed label sequence and the first variable label sequence, the rest two bases are degenerate sequences, the first detection probes corresponding to different one double-base label sequences are modified with different report labels, and the report labels modified by the first detection probes in the same first detection probe subgroup are the same;
a second detection probe group consisting of a plurality of second detection probe subgroups, wherein the length of each second detection probe is the same as that of the first label sequence, the second detection probes in a second detection probe subgroup are completely the same as the other two-base label sequence of the first fixed label sequence and the first variable label sequence, the rest two bases are degenerate sequences, the second detection probes corresponding to the different other two-base label sequences are modified with different report labels, and the report labels modified by the second detection probes in the same second detection probe subgroup are the same;
a third detection probe group, consisting of a plurality of third detection probe subgroups, wherein the length of each third detection probe is the same as that of the first tag sequence, the third detection probes in a third detection probe subgroup are completely the same as one of the double-base tag sequences of the second fixed tag sequence and the second variable tag sequence, the remaining two bases are degenerate sequences, the third detection probes corresponding to different one of the double-base tag sequences are modified with different report labels, and the report labels modified by the third detection probes in the same third detection probe subgroup are the same;
and the fourth detection probe group consists of a plurality of fourth detection probe subgroups, wherein the length of each fourth detection probe is the same as that of the second label sequence, the fourth detection probes in one fourth detection probe subgroup are completely the same as the other two-base label sequence of the second fixed label sequence and the second variable label sequence, the rest two bases are degenerate sequences, the fourth detection probes corresponding to different two-base label sequences are modified with different report labels, and the report labels modified by the fourth detection probes in the same fourth detection probe subgroup are the same.
12. The use of claim 11, wherein: the first variable label sequence consists of a first double-base label sequence and a second double-base label sequence, the first double-base label sequence is cw, as, ts or gw, the second variable label sequence consists of a third double-base label sequence and a fourth double-base label sequence, the third double-base label sequence is cw, as, ts or gw, the fourth double-base label sequence is cw, as, ts or gw, the first detection probe group consists of a plurality of first detection probe subgroups, wherein the length of each first detection probe is the same as that of the first label sequence, the first detection probe in a first detection probe subgroup is completely the same as the first fixed label sequence and the first double-base label sequence in the first variable label sequence, the rest two bases are cw, as, ts or gw, and the rest two bases are different from that of the first detection probe corresponding to the different first double-base label sequences have different report labels, and the reporter label modified by the first detection probe in the same first detection probe subgroup is the same;
a second detection probe group consisting of a plurality of second detection probe subgroups, wherein the length of each second detection probe is the same as that of the first label sequence, the second detection probe in a second detection probe subgroup is completely the same as the second double-base label sequence in the first fixed label sequence and the first variable label sequence, the remaining two bases are cw, as, ts or gw, the second detection probes corresponding to different second double-base label sequences are modified with different report labels, and the report labels modified by the second detection probes in the same second detection probe subgroup are the same;
a third detection probe group, which consists of a plurality of third detection probe subgroups, wherein the length of each third detection probe is the same as that of the second label sequence, the third detection probe in a third detection probe subgroup is completely the same as the third double-base label sequence of the second fixed label sequence and the second variable label sequence, the rest two bases are cw, as, ts or gw, different report labels are modified on the third detection probes corresponding to the different third double-base label sequences, and the report labels modified by the third detection probes in the same third detection probe subgroup are the same;
and the fourth detection probe group consists of a plurality of fourth detection probe subgroups, wherein the length of each fourth detection probe is the same as that of the second label sequence, the fourth detection probe in one fourth detection probe subgroup is completely the same as the fourth double-base label sequence of the second fixed label sequence and the second variable label sequence, the rest two bases are cw, as, ts or gw, different report markers are modified on the fourth detection probes corresponding to the different fourth double-base label sequences, and the report markers modified on the fourth detection probes in the same fourth detection probe subgroup are the same.
13. Use according to claim 11 or 12, characterized in that: the ring-forming probe is a double-connection probe or a locking probe.
14. The use of claim 13, wherein: the ring-forming probe is a double-connection probe, and also comprises a splint primer which is matched with the double-connection probe to form a ring after hybridization.
15. The use of claim 14, wherein: the kit also comprises a first anchor primer and a second anchor primer, wherein the first anchor primer can be strictly complementary and paired with the rolling circle amplification product and can be connected with the first detection probe and the second detection probe after hybridization under the action of DNA polymerase, and the second anchor primer can be strictly complementary and paired with the rolling circle amplification product and can be connected with the third detection probe and the fourth detection probe after hybridization under the action of DNA polymerase.
16. An in situ detection method for RNA for non-diagnostic therapeutic purposes, characterized in that: the method comprises the following steps:
(1) designing at least one looping probe:
each ring-forming probe corresponds to a target sequence, the sequence of the 5 'end and the 3' end of the ring-forming probe is specifically hybridized and complemented with the target sequence, and a first label sequence and a second label sequence are arranged between the 5 'end and the 3' end of the ring-forming probe, the first label sequence is positioned at the 3 'end of the sequence of the 5' end specifically hybridized and complemented with the target sequence, and the second label sequence is positioned at the 5 'end of the sequence of the 3' end specifically hybridized and complemented with the target sequence; the first label sequence consists of a first fixed label sequence and a first variable label sequence, the second label sequence consists of a second fixed label sequence and a second variable label sequence, and the first variable label sequence and the second variable label sequence are four-base-length sequences consisting of double-base label sequences;
(2) designing first to fourth detection probe sets:
the first detection probe group consists of a plurality of first detection probe subgroups, wherein the length of each first detection probe is the same as that of the first label sequence, the first detection probe in one first detection probe subgroup is completely the same as one double-base label sequence of the first fixed label sequence and the first variable label sequence, the rest two bases are degenerate sequences, the first detection probes corresponding to different one double-base label sequences are modified with different report labels, and the report labels modified by the first detection probes in the same first detection probe subgroup are the same;
the second detection probe group consists of a plurality of second detection probe subgroups, wherein the length of each second detection probe is the same as that of the first label sequence, the second detection probe in one second detection probe subgroup is completely the same as the other two-base label sequence of the first fixed label sequence and the first variable label sequence, the rest two bases are degenerate sequences, the second detection probe corresponding to the other different two-base label sequence is modified with different report labels, and the report labels modified by the second detection probe in the same second detection probe subgroup are the same;
a third detection probe group, which consists of a plurality of third detection probe subgroups, wherein the length of each third detection probe is the same as that of the first label sequence, the third detection probe in a third detection probe subgroup is completely the same as one double-base label sequence of the second fixed label sequence and the second variable label sequence, the rest two bases are degenerate sequences, the third detection probes corresponding to different one of the double-base label sequences are modified with different report labels, and the report labels modified by the third detection probes in the same third detection probe subgroup are the same;
a fourth detection probe group, which consists of a plurality of fourth detection probe subgroups, wherein the length of each fourth detection probe is the same as that of the second tag sequence, the fourth detection probe in a fourth detection probe subgroup is completely the same as the other two-base tag sequence of the second fixed tag sequence and the second variable tag sequence, the remaining two bases are degenerate sequences, the fourth detection probe corresponding to the different other two-base tag sequence is modified with different report labels, and the report labels modified by the fourth detection probe in the same fourth detection probe subgroup are the same;
(3) after the at least one circularization probe is specifically combined with at least one target sequence in a sample to be detected, the 3 ' end and the 5 ' end of the at least one circularization probe are adjacent end to end, the circularization probe and the 5 ' end are connected through DNA ligase to form at least one circular template, and then rolling circle amplification is carried out to obtain at least one rolling circle amplification product;
(4) hybridizing the at least one rolling circle amplification product with a first detection probe set for detection to obtain a first signal;
(5) removing the first signal, and hybridizing the first signal with a second detection probe group for detection to obtain a second signal;
(6) removing the second signal, and hybridizing the second signal with a second detection probe group for detection to obtain a third signal;
(7) removing the third signal, and hybridizing the third signal with the second detection probe group for detection to obtain a fourth signal;
(8) according to the permutation and combination of the first to the fourth signals, at least one signal code corresponding to the at least one target sequence is obtained.
17. The method of claim 16 for in situ detection of RNA for non-diagnostic therapeutic purposes, wherein: the first variable label sequence consists of a first double-base label sequence and a second double-base label sequence, the first double-base label sequence is cw, as, ts or gw, the second variable label sequence consists of a third double-base label sequence and a fourth double-base label sequence, the third double-base label sequence is cw, as, ts or gw, the fourth double-base label sequence is cw, as, ts or gw,
the first detection probe group consists of a plurality of first detection probe subgroups, wherein the length of each first detection probe is the same as that of the first label sequence, the first detection probe in one first detection probe subgroup is completely the same as the first fixed label sequence and the first double-base label sequence in the first variable label sequence, the rest two bases are cw, as, ts or gw, different report labels are modified on the first detection probes corresponding to the different first double-base label sequences, and the report labels modified by the first detection probe in the same first detection probe subgroup are the same;
a second detection probe group consisting of a plurality of second detection probe subgroups, wherein the length of each second detection probe is the same as that of the first label sequence, the second detection probe in a second detection probe subgroup is completely the same as the second double-base label sequence in the first fixed label sequence and the first variable label sequence, the remaining two bases are cw, as, ts or gw, the second detection probes corresponding to different second double-base label sequences are modified with different report labels, and the report labels modified by the second detection probes in the same second detection probe subgroup are the same;
a third detection probe group, which consists of a plurality of third detection probe subgroups, wherein the length of each third detection probe is the same as that of the second label sequence, the third detection probe in a third detection probe subgroup is completely the same as the third double-base label sequence of the second fixed label sequence and the second variable label sequence, the remaining two bases are cw, as, ts or gw, the third detection probes corresponding to the different third double-base label sequences are modified with different report labels, and the report labels modified by the third detection probes in the same third detection probe subgroup are the same;
and the fourth detection probe group consists of a plurality of fourth detection probe subgroups, wherein the length of each fourth detection probe is the same as that of the second label sequence, the fourth detection probes in one fourth detection probe subgroup are completely the same as the fourth double-base label sequences of the second fixed label sequence and the second variable label sequence, the rest two bases are cw, as, ts or gw, the fourth detection probes corresponding to the different fourth double-base label sequences are modified with different report labels, and the report labels modified by the fourth detection probes in the same fourth detection probe subgroup are the same.
18. A method of in situ detection of RNA for non-diagnostic therapeutic purposes according to claim 16 or 17, wherein: the ring-forming probe is a double-connection probe or a locking probe.
19. The method of claim 18 for in situ detection of RNA for non-diagnostic therapeutic purposes, wherein: the cyclization probe is a double-connection probe, and the step (3) is as follows: specifically combining the at least one circularity probe with at least one target sequence in a sample to be detected, adding a splint sequence to enable the 3 ' end and the 5 ' end of the at least one circularity probe to be adjacent end to end, connecting the circularity probe and the 5 ' end by DNA ligase to form at least one circular template, and then carrying out rolling circle amplification to obtain at least one rolling circle amplification product.
20. The method of claim 19 for in situ detection of RNA for non-diagnostic therapeutic purposes, wherein: and (5) adding a first anchor primer for hybridization, wherein the first anchor primer is strictly complementary and paired with the rolling circle amplification product and is connected with the hybridized first detection probe and the hybridized second detection probe under the action of DNA polymerase.
21. The method of claim 19 for in situ detection of RNA for non-diagnostic therapeutic purposes, wherein: and (3) adding a second anchor primer for hybridization in the steps (6) and (7), wherein the second anchor primer is strictly complementary and paired with the rolling circle amplification product and is connected with the hybridized third and fourth detection probes under the action of DNA polymerase.
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| PCT/CN2023/094251 WO2023221933A1 (en) | 2022-05-16 | 2023-05-15 | Probe primer set used for rna in-situ detection, and application thereof |
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| CN109797201B (en) * | 2019-03-12 | 2023-01-24 | 厦门先能生物科技有限公司 | In-situ multiple nucleic acid detection method |
| CN111534573A (en) * | 2020-05-26 | 2020-08-14 | 深圳百纳心致生命科学有限公司 | Probe composition and application thereof in nucleic acid in-situ detection |
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