CN110628882B

CN110628882B - Primer and kit for PCR amplification, method for detecting MSI state and application

Info

Publication number: CN110628882B
Application number: CN201910995495.9A
Authority: CN
Inventors: 胡玉刚; 汪彪; 郑文莉; 吴强
Original assignee: Naonda Nanjing Biological Technology Co ltd
Current assignee: Naonda Nanjing Biological Technology Co ltd
Priority date: 2019-10-18
Filing date: 2019-10-18
Publication date: 2020-09-01
Anticipated expiration: 2039-10-18
Also published as: CN110628882A

Abstract

The invention provides a primer and a kit for PCR amplification, a method for detecting MSI state and application. The primer comprises a target sequence amplification region and a closed region arranged at the 3 'end of the target sequence amplification region, wherein the closed region has a structure shown in R (D) n1 DsDsDdX or R (D) n2DsDsMx, wherein R represents RNA base, D represents complementary pairing base, M represents mismatch base, n1 and n2 represent the number of D base in brackets, n1 is 1 or 2, n2 is 2, s represents the 3' oxygen atom between two adjacent nucleotides for carrying out thio modification, and x represents closed modification. And finally, the thiocoupling among the three bases prevents bases with closed modification at the tail end from being cut off by enzyme, so that high-fidelity enzyme amplification can be adopted, the amplification accuracy can be ensured, and non-specific amplification and primer dimer are not generated, so that the MSI state detection accuracy is improved.

Description

Primer and kit for PCR amplification, method for detecting MSI state and application

Technical Field

The invention relates to the field of high-throughput sequencing detection, in particular to a primer and a kit for PCR amplification, a method for detecting MSI state and application.

Background

In the human genome, a number of short (1-6 bases) tandem repeat DNA sequences, the MicroSatellite Sequence (MS), are widely distributed. MicroSatellite Instability (MSI) refers to any change in the length of a MicroSatellite due to the insertion or deletion of a repeat unit from a MicroSatellite, giving rise to a new MicroSatellite allele. These sequences are frequently in a state where a small range of base deletions, insertions or substitutions are not repaired during the process of cell proliferation, resulting in DNA replication due to loss of function of the mismatch repair gene. Depending on the degree of MSI instability, a classification can be made into microsatellite high instability (MSI-H), microsatellite low instability (MSI-L) and microsatellite stability (MSS).

The traditional method for detecting the stability of the MS locus mainly adopts a method of combining PCR amplification with capillary electrophoresis, because the number of the detected loci is limited, generally only 6 common loci are detected, unstable judgment of more than or equal to 2 loci is microsatellite high unstable type (MSI-H), unstable judgment of 1 locus is microsatellite low unstable type (MSI-L), and unstable judgment of no locus is microsatellite stable type (MSS). Therefore, the accuracy and the scientificity of the detection are further improved.

Disclosure of Invention

The invention provides a primer and a kit for PCR amplification, a method for detecting MSI state and application thereof, which are used for reducing non-specific amplification during PCR amplification of a target fragment, thereby improving the accuracy of MSI state detection.

In order to achieve the above object, according to one aspect of the present invention, there is provided a primer for PCR amplification comprising a target sequence amplification region and a blocking region provided at the 3 'end of the target sequence amplification region, the blocking region having a structure represented by R (D) n1 dsdsdsdx or R (D) n2 dsdsdssdmx, wherein R represents an RNA base, D represents a complementary pairing base, M represents a mismatched base, n1 and n2 represent the number of D bases in parentheses, n1 is 1 or 2, n2 is 2, s represents a 3' oxygen atom between two adjacent nucleotides for thiomodification, and x represents a blocking modification.

Further, the blocking modification is selected from any one of: MGB modification, C3 spacer modification, 3 'phosphorylation modification, 3' digoxin modification, 3 'biotin modification and base at 3' end being dideoxy base.

Further, the primers were primers for amplifying the sites shown in Table 1.

Further, the primers are shown in SEQ ID NO 70 to SEQ ID NO 207.

According to a second aspect of the present invention, there is provided a kit comprising any one of the primers described above.

Further, the kit also comprises a sequencing platform joint universal primer.

Further, the universal primers are: 208 and 209, or 210 and 211.

According to a third aspect of the present invention, there is provided a method of detecting the status of MSI, the method comprising the steps of performing PCR amplification detection of a target MSI site, using any one of the primers described above for amplification; or using any one of the above kits for amplification.

According to a third aspect of the present invention, there is provided the use of any one of the primers described above in the preparation of a kit for detecting the status of an MSI.

Further, the kit is a kit for constructing an amplicon library.

By applying the technical scheme of the invention, in the improved primer, 4 or 5 bases behind the RNA base expressed by R can be ensured to be well recognized and cut by RNase H2, and the last three bases are connected through thio, and the thio connection can effectively prevent the sequence of the closed region at the tail end of the primer from being cut off by enzyme in the reaction, so that the amplicon of the invention can be amplified by high-fidelity amplificase without worrying about the problem that the closed region is cut off.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this application, illustrate embodiments of the invention and, together with the description, serve to explain the invention and not to limit the invention. In the drawings:

FIG. 1 shows a schematic flow diagram of amplicon library construction in a preferred embodiment of the present application;

FIG. 2 shows a library quality control map of genomic DNA in a preferred embodiment of the present application;

FIG. 3 shows amplicon balance data for a pair of tumor samples amplified using the methods of the present application in a preferred embodiment of the present application;

FIGS. 4a, 4b and 4c show the results of MSI status measurements at three sites, Bat26, Bat25 and NR27, respectively, in a pair of tumor samples using the method of the present application in a preferred embodiment of the present application, where the abscissa shows the length of the repeated bases and partial outer sequences of the amplicon and the ordinate shows the sequencing depth of the amplicon;

FIG. 5 shows a comparison of the pooling of plasma DNA and genomic DNA using the methods of the present application in a preferred embodiment of the present application;

FIG. 6 shows the results of comparing the on-target rates of amplicons and common amplicons obtained using the methods of the present application in a preferred embodiment of the present application;

FIG. 7 shows the results of comparing the hit ratios of amplicons and IDT amplicons obtained using the methods of the present application in a preferred embodiment of the present application.

Detailed Description

It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict. The present application will be described in detail with reference to examples.

As mentioned in the background art, the existing detection method for the unstable state of microsatellite has the defect of low detection accuracy, and in order to further improve the detection accuracy, the applicant of the present application aims at the existing situation, on one hand, starting from the structural characteristic that the microsatellite sequence is a repeated sequence with 1-6 bases in series, and improving the amplification specificity and the amplification efficiency of the amplified repeated sequence. On the other hand, improvement is made in terms of selection and number of microsatellite loci so as to relatively more accurately evaluate the state of the microsatellite through more loci.

In addition, in order to meet the detection flux of increased microsatellite loci, the method utilizes an amplicon to amplify and establish a library of alternative MSI loci, high-flux sequencing is carried out, whether the sequence of MSI is stable is judged by analyzing and judging sequencing data, and the result shows that the improved method can detect plasma DNA and samples with serious degradation, and primer dimer and non-specific amplification are not generated, so that the ratio of effective data in the obtained sequencing data of the amplicon is high, and the detection efficiency and accuracy are improved.

In the process of improving the amplification specificity of the target site, the applicant performed amplification by the rhAMP method of IDT, which is an RNase H2-dependent PCR amplification method (rhPCR). The amplification principle of the RNase H2-dependent PCR amplification method is: the 3' end of the target fragment amplification primer is provided with a special sequence, and the basic structure of the special sequence is RDDDDMx, wherein R represents RNA base, D represents complementary pairing base, M represents mismatch base, and x represents blocking modification. When the sequence before the RNA base in the primer is completely matched with the target fragment, the RNase H2 cuts off the RNA base, and the blocking of the end of the primer is removed, so that the primer can extend and amplify the target fragment under the action of polymerase. On the other hand, when the sequence before the RNA base in the primer does not match the target fragment, the RNase H2 cannot cut from the RNA base, so that the primer cannot extend and amplify the target fragment due to the existence of the terminal base blocking modification. This can prevent non-specific amplification of the primer and also prevent the formation of primer dimers.

However, during amplification using the above primers, it was found that there was still non-specific amplification. Applicants have attempted to modify the specific structure described above to further reduce non-specific amplification. Through research, the applicant found that the above primer must be used in combination with the DNA polymerase provided by the primer to reduce non-specific amplification, and when the primer is replaced by other polymerase with high fidelity, such as Q5 high fidelity DNA polymerase from NEB, relatively more non-specific amplification occurs. Since the high fidelity DNA polymerase has mismatch detection and repair capability, when the sequence before the RNA base in the primer is not matched with the target fragment, the RNase H2 can not cut to remove the block, but the mismatch detection and modification function of the high fidelity DNA polymerase can detect the mismatch, so that the 3 'to 5' end exonuclease activity is started to cut off the terminal base, so that the block is removed, and the amplification of the non-target fragment is started, so that the non-specific amplification is generated. Therefore, it is possible to prevent non-specific amplification by a high fidelity polymerase without initiating cleavage of the terminal-blocked base in the case where a primer is mismatched with a target sequence. Therefore, the applicant proposed an improved scheme of S modification of the linkage (oxygen atom at 3' position of ribose is changed to sulfur atom) between 3 nucleotides at the end of the special structure of the above primer, and verified the non-specificity of amplification of the primer after the improvement by experiment.

On the basis of the above-mentioned research results, the applicant has proposed the present application. In a typical embodiment, a primer for PCR amplification is provided, the primer comprising a target sequence amplification region and a blocker region disposed at the 3 'end of the target sequence amplification region, the blocker region having a structure represented by R (D) n1 dsdsdsdx or R (D) n2 dsdsdsmx, wherein R represents an RNA base, D represents a complementary pairing base, M represents a mismatch base, n1 and n2 represent the number of D bases in parentheses, n1 is 1 or 2, n2 is 2, s represents a 3' oxygen atom between two adjacent nucleotides for thio-modification, and x represents a blocking modification.

In the improved primer, 4 or 5 bases behind the RNA base represented by R can be well identified and cut by RNase H2, and the last three bases are connected through thio, so that the sequence of a closed region at the tail end of the primer can be effectively prevented from being cut off by enzyme in the reaction, and thus, the amplicon of the primer can be amplified by high-fidelity amplificator without worrying about the problem of closed cut-off.

The specific modification type of the blocking modification is not limited, and any modification that can block the 3' -end is applicable to the present application. Thus, in the present application, the blocking modifications described above include, but are not limited to, any of the following: MGB modification, C3 spacer modification, 3 'phosphorylation modification, 3' digoxin modification, 3 'biotin modification and base at 3' end being dideoxy base. The end modification matches the end of the three last base connection is thio, thus the application of the improved primers the primer can use high fidelity amplification enzyme amplification.

As mentioned above, the existing MSI detection method has few targeted sites, which results in insufficient accuracy, and therefore, in order to improve the detection accuracy, the present application selects more than 60 other sites besides 6 sites (black parts in table 1) of the traditional detection. In selecting new sites, the MSI detection characteristics are fully considered, single base repeat detection sites are preferably selected, and double base or triple base repeat sites are selected in small amounts (see Table 1 below). Thus, in a preferred embodiment of the present application, the primers can amplify more than 60 sites selected in the present application. The method has the advantages that when the tumor sample and the tissue beside the cancer are detected for comparison, the method has a sample matching function, sample confusion errors in sampling and experiment links are avoided, the detection accuracy is improved (the MSI site of the tumor is unstable, the detection sites in the traditional method are few, the tumor tissue and the blood sample or the tissue beside the cancer of a patient are different and are considered to be unstable, and if the sample confusion is wrong, the pair of samples cannot be judged.

Table 1:

wherein a represents a repeating unit, and b represents the number of repetitions of the repeating unit.

Aiming at the sites, different primers can be designed to amplify to construct sequencing detection in a library according to different universal primers of the joint used by a sequencing platform. In a preferred embodiment of the present application, the above primers are shown in SEQ ID NO:70 to SEQ ID NO:207 shown in Table 2 below.

TABLE 2.69 primer sequences corresponding to the amplicons

Note: in Table 2 above,/rA/,/rC/and/rG/each represents a nucleobase, a thio group, and x represents a spacer of block-modified C3.

In the primer in the above table 2, the reciprocal 5-base at the 3' end is the terminal closed region in the primer, wherein the linkage between the three bases after the end is replaced by-S-instead of-O-.

In a second exemplary embodiment of the present application, a kit is provided, which comprises any one of the primers described above. The primer can start amplification extension under the condition that the primer is completely matched with a target sequence, and can effectively prevent a closed sequence region at the tail end of the primer from being cut off due to mismatch detection and repair of high-fidelity DNA polymerase when mismatched bases exist with the target sequence, so that nonspecific amplification and primer dimer are greatly reduced.

The kit can further comprise a universal primer of a sequencing platform adaptor according to the requirement of convenient detection, so that the universal primer can be conveniently carried out on an Illumina or MGI sequencing platform, and only a part of sequence of the 5' end in the primer needs to be changed into a corresponding universal part of the sequencing platform adaptor during synthesis. Preferably, the universal primer can be a universal primer of MGI sequencing platforms shown by SEQ ID NO:208 and SEQ ID NO:209, or the universal primer can be a universal primer of Illumina sequencing platforms shown by SEQ ID NO:210 and SEQ ID NO: 211.

Sequencing platform for MGI:

the sequence/dnSeq-f: 208 of SEQ ID NO: TTGTCTTCCTAAGACCGCTTGGCCTCCGACTT, respectively;

(dnSeq-r/sequence: 209, SEQ ID NO: GAACGACATGGCTACGATCCGACTT are provided.

For the Illumina sequencing platform:

the sequence/dnSeq-f: 210 in SEQ ID NO: ACACTCTTTCCCTACACGACGCTCTTCCGATCT, respectively;

(dnSeq-r/sequence: 211, SEQ ID NO: GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT are provided.

In a third exemplary embodiment of the present application, a method for detecting MSI status is provided, the method comprising the steps of performing PCR amplification detection of target MSI sites, using any one of the primers described above for amplification; or using any one of the above kits for amplification.

In a preferred embodiment, an amplicon library was constructed from the improved 69 microsatellite instability sites by using the improved primers described above in this application and was detected by high throughput sequencing. Preferably, the library construction process is similar to the existing process, as shown in FIG. 1, and in the first step, specific primer amplification is performed on each site by using primers. Unlike the conventional common amplification effect, the non-specific amplification and primer dimer amplification are not generated. Since the end of the amplification primer of this step in this application is a sequence with a block (R in FIG. 1 represents an RNA base and x represents a block modification), this block sequence is recognized by RNase H2 only when bound to a perfectly matched region, and the sequence after the RNA base is cut off to unblock the amplification; secondly, sequencing primers with index are used for amplification; the purification of the reaction product is preferably carried out both after the first step and after the second step.

In a fourth exemplary embodiment of the present application, there is provided the use of any one of the primers described above in the preparation of a kit for detecting MSI status, preferably an amplicon library construction kit.

The following examples are intended to further illustrate the benefits of the present application and should be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. The reagents and consumables used in the following examples were obtained from NEB corporation, unless otherwise specified.

Example 1 genomic DNA or FFPE sample DNA library construction

This example employed three pairs of tumor samples, two of which were MSI-H (ND18161 and ND18163) and one of which was MSI-L (ND18171), where ND18162 was the control for ND 18161; ND18164 is a control for ND 18163; ND18172 is a control for ND 18171. DNA of each pair of tumor tissue and tissue beside the cancer is taken from frozen stock (certainly, the DNA can also be used for FFEP samples, and the target fragment designed by the primer is between 100 and 160bp, so the DNA is suitable for large FFPE samples), and gDNA is extracted by adopting a DNA extraction kit of Tiangen company respectively.

The experimental steps are as follows:

first, PCR amplification

1. Forward and reverse primers for multiplex amplification shown in Table 2 were prepared, each primer was prepared at a concentration of 0.1 nmol/. mu.l, and the upstream and downstream primers were mixed at equal ratios. The system for the first multiplex amplification was configured as shown in Table 3.

Table 3:

mix well and centrifuge instantaneously to place all the reaction solution at the bottom of the PCR tube.

The following reaction procedure was started on the PCR machine, the temperature of the hot lid was 105 ℃ and the reaction tube was placed into the PCR machine when the temperature was stabilized to 95 ℃.

Table 4:

second, purifying the first amplification product

2.1 Add 30. mu.l NanoPrepTM SP Beads (1.5 Xmagnetic Beads) to the PCR tube, mix well, incubate at25 ℃ for 5-10 min.

2.2 placing the PCR tube on a magnetic frame for 5min after instantaneous centrifugation until the liquid is completely clarified, sucking by using a pipettor, and removing the supernatant.

2.3 Add 150. mu.l 80% ethanol slowly along the side wall of the PCR tube, take care not to disturb the beads, let stand for 30s, pipette off the supernatant.

2.4 repeat step 3 once.

2.5PCR tubes were centrifuged instantaneously, placed on a magnetic rack and a small amount of residual ethanol was removed using a 10. mu.l tip, taking care not to attract the beads.

2.6 open the tube cap of the PCR tube, and keep standing at room temperature for about 5min until the ethanol is completely volatilized.

2.7 remove the PCR tube, add 21. mu.l TE Solution to the PCR tube, suspend the beads evenly using a pipette, incubate for 2min at25 ℃.

2.8 the PCR tube was centrifuged instantaneously and placed on a magnetic rack for 2min until the liquid was clear completely, and 20. mu.l of the supernatant was carefully transferred to a new 0.2ml PCR tube using a pipette for storage, taking care not to attract the beads.

Third, Index primer amplification

3.1 NanoPrep for MGI sequencing platform^TMM-index Primer Mix was naturally dissolved on ice (NanoPrep for Illumina sequencing platform)^TMUDI index Primer Mix), mixed well and centrifuged instantaneously for use.

3.2 the reaction system was prepared in a PCR tube placed on ice according to the system shown in Table 5 below.

Table 5:

3.3 mix well and centrifuge instantaneously to place all the reaction solution at the bottom of the PCR tube.

3.4 the reaction procedure shown in Table 6 below was started on the PCR machine, the temperature of the hot lid was 105 ℃ and the reaction tube was placed into the PCR machine when the temperature stabilized to 95 ℃.

Table 6:

the fourth step, library purification

4.1 Add 75. mu.l NanoPrepTM SP Beads (1.5 Xmagnetic Beads) to the PCR tube, mix well, incubate at25 ℃ for 5-10 min.

4.2 placing the PCR tube on a magnetic frame for 5min after instantaneous centrifugation until the liquid is completely clarified, sucking by using a pipettor, and removing the supernatant.

4.3 Add 150. mu.l 80% ethanol slowly along the side wall of the PCR tube, take care not to disturb the beads, let stand for 30s, pipette off the supernatant.

4.4 repeat step 3 once.

4.5PCR tubes were centrifuged instantaneously, placed on a magnetic rack and a small amount of residual ethanol was removed using a 10. mu.l tip, taking care not to attract the beads.

4.6 open the tube cap of the PCR tube, and keep standing at room temperature for about 5min until the ethanol is completely volatilized.

4.7 Add 20. mu.l TE Solution to the PCR tube, suspend the beads evenly using a pipette, incubate for 2min at25 ℃.

4.8 the PCR tube was centrifuged instantaneously and placed on a magnetic rack for 2min until the liquid was clarified completely, and the supernatant was carefully transferred to a new 0.2ml PCR tube using a pipettor for storage, taking care not to attract the beads.

4.9 quantification of the library using Qubit.

4.10 library fragment distribution assays were performed using the Qsep100(Bioptic) fragment analyzer instrument, see FIG. 2. As can be seen from FIG. 2, the length of the target fragment is between 200 and 350bp, which is consistent with the expected length.

Second, build the experimental data of the storehouse

The three tumor sample database is shown in table 7 below.

Table 7:

third, sequencing data analysis

Sequencing 6 libraries through an Illumina sequencing platform PE150, analyzing and comparing obtained data, and comparing the reads of 69 amplicons, wherein if the reads of one amplicon is lower, the reads are balanced in the figure 3.

According to sequencing data analysis of the amplicon, ND18161 and ND18171 are consistent with the results of traditional PCR combined capillary electrophoresis, while the results of ND18163 are obviously inconsistent, ND18163 has 3 sites judged to be unstable by detecting 6 sites by a PCR amplification combined capillary electrophoresis method, the detection of the invention finds that only one of the 6 sites is an unstable site and the other two sites are not very stable, but because the detection sites of the invention are more, the unstable ratio detected from other sites also indicates that most sites of the sample are stable, and the specific detection results are shown in Table 8 below. Because the MS sites are originally single base or a small amount of double base repetition and have slight fluctuation in the PCR amplification process, the judgment is not scientific only based on 6 sites, and meanwhile, the traditional method is greatly influenced by capillary electrophoresis, so that the method is more scientific and accurate than the traditional method combining PCR with capillary electrophoresis.

Table 8:

to further illustrate the difference between the detection method of the present application and the detection method of the conventional method, the results of capillary electrophoresis of the ND18163 site are summarized in table 9 below, the detection results of the method of the present invention corresponding to these three sites are shown in fig. 4a, 4b and 4c, the Bat26 site is determined to be an unstable site, the Bat25 and NR27 sites are near the critical point of judgment, and the judgment is that the results are greatly influenced, compared with the limited number of sites of the conventional method, the scheme of the present application expands the detection sites from 6 to more than 60, and the detection results are relatively more scientific and reliable.

Table 9:

example 2 pooling of plasma DNA

Firstly, experimental steps

The specific steps are shown in example 1, and the difference from example 1 is that the input DNA is plasma DNA, part of DNA is input into a sample with a small amount, and the number of cycles of the second-step amplification is increased by 1-2.

Second, build the experimental data of the storehouse

To determine whether the amplification primers of the present application can use plasma DNA as a template, this example uses a genomic DNA as a control, and makes three libraries of plasma DNA with gradient input, 1ng, 2.5ng and 5ng, respectively, and the corresponding amplification in the second step selects different amplification cycle numbers as shown in the following table 10:

table 10:

third, sequencing data analysis

By analyzing the sequencing data of the library shown in table 10, it was found that the amplicon of the present application can detect not only genomic DNA but also plasma DNA very well. The data balance of plasma DNA, although not as good as that of genomic DNA, is well covered (see FIG. 5), except that the data deviation of individual sites is slightly larger, which also conforms to the characteristics of plasma DNA, and the major peak of the fragment of plasma is near 167 bp.

In addition, as can be seen from the analysis results of the sequencing data, the amplicon of the present application has another outstanding feature, as shown in fig. 6, that is, the hit rate is extremely high, especially compared with the ordinary amplicon, the hit rate of the amplicon of the present application is about 99%, and primer dimer and non-specific amplification are few, while the ordinary amplicon is designed with the same length as the amplicon of the present application, so that the non-specific amplification and primer dimer are generally higher, especially the dimer is more when plasma DNA is used as a template. Therefore, the target rate is obviously superior when the amplification primer of the application is used for amplification by taking the plasma DNA as a template.

It should be noted that, by using the multiplex PCR amplification method, the present application can detect dozens or even hundreds of microsatellite loci at one time, and is not limited by the detection sample, and can detect both FFPE sample and plasma DNA sample, thus having more clinical popularization value than the existing detection method.

From the above description, it can be seen that the above-described embodiments of the present application achieve the following technical effects: in the improved primer, 4 or 5 bases behind the RNA base represented by R can be well identified and cut by RNase H2, the last three bases are connected through thio, the thio connection can effectively prevent the sequence of a closed region at the tail end of the primer from being cut off by enzyme in the reaction, the tail end structure of the IDT is only suitable for common amplification enzyme and is not suitable for high-fidelity enzyme amplification, and the method is shown in figure 7 (a primer with the last base M in the table 2 is adopted to carry out amplification library building comparison with an ITD primer). The amplicon applied by the sample can be amplified by high-fidelity amplification enzyme without worrying about the problem that the closed part is cut off, so the amplification primer can ensure the amplification accuracy, does not generate non-specific amplification and primer dimer, avoids the waste of sequencing data and simultaneously improves the detection accuracy of the amplicon.

The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.

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tctgcttttt ttttttttgg tcc 23

<210>29

<211>25

<212>DNA

<213> Intelligent (Homo sapiens)

<400>29

gagcattttt tttttttttt ggtct 25

<210>30

<211>27

<212>DNA

<213> Intelligent (Homo sapiens)

<400>30

cgggcttttt tttttttttt ttcttaa 27

<210>31

<211>25

<212>DNA

<213> Intelligent (Homo sapiens)

<400>31

tctacttttt tttttttttt ccttg 25

<210>32

<211>55

<212>DNA

<213> Intelligent (Homo sapiens)

<400>32

atttatcctc ctcctcctcc tcctcctcct cctcctcctc ctcctcctcc tctcc 55

<210>33

<211>23

<212>DNA

<213> Intelligent (Homo sapiens)

<400>33

tattgttttt ttttttttag aag 23

<210>34

<211>27

<212>DNA

<213> Intelligent (Homo sapiens)

<400>34

taatgaaaaa aaaaaaaaaa aagtaat 27

<210>35

<211>28

<212>DNA

<213> Intelligent (Homo sapiens)

<400>35

agaccaaaaa aaaaaaaaaa aaagactt 28

<210>36

<211>40

<212>DNA

<213> Intelligent (Homo sapiens)

<400>36

cgcgcacaca cacacacaca cacacacaca cacaccccgg 40

<210>37

<211>27

<212>DNA

<213> Intelligent (Homo sapiens)

<400>37

gtgttaaaaa aaaaaaaaaa aaggctt 27

<210>38

<211>22

<212>DNA

<213> Intelligent (Homo sapiens)

<400>38

tgtagttttt tttttttctg tc 22

<210>39

<211>27

<212>DNA

<213> Intelligent (Homo sapiens)

<400>39

ataggaaaaa aaaaaaaaaa aacctag 27

<210>40

<211>25

<212>DNA

<213> Intelligent (Homo sapiens)

<400>40

tattgaaaaa aaaaaaaaaa cttaa 25

<210>41

<211>72

<212>DNA

<213> Intelligent (Homo sapiens)

<400>41

tcggcgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg 60

tgtgtgtaat tt 72

<210>42

<211>27

<212>DNA

<213> Intelligent (Homo sapiens)

<400>42

ctaggaaaaa aaaaaaaaaa aaccgct 27

<210>43

<211>46

<212>DNA

<213> Intelligent (Homo sapiens)

<400>43

tgtatacaca cacacacaca cacacacaca cacacacaca ctctct 46

<210>44

<211>25

<212>DNA

<213> Intelligent (Homo sapiens)

<400>44

atgtgttttt tttttttttt cccat 25

<210>45

<211>22

<212>DNA

<213> Intelligent (Homo sapiens)

<400>45

ctgtgaaaaa aaaaaaagcc aa 22

<210>46

<211>26

<212>DNA

<213> Intelligent (Homo sapiens)

<400>46

cttccttttt tttttttttt tcttct 26

<210>47

<211>28

<212>DNA

<213> Intelligent (Homo sapiens)

<400>47

tgcagaaaaa aaaaaaaaaa aaagcctc 28

<210>48

<211>25

<212>DNA

<213> Intelligent (Homo sapiens)

<400>48

tagagttttt tttttttttt aaatg 25

<210>49

<211>25

<212>DNA

<213> Intelligent (Homo sapiens)

<400>49

actgcaaaaa aaaaaaaaaa tagcc 25

<210>50

<211>27

<212>DNA

<213> Intelligent (Homo sapiens)

<400>50

gctgattttt tttttttttt ttacctg 27

<210>51

<211>24

<212>DNA

<213> Intelligent (Homo sapiens)

<400>51

aaggcttttt tttttttttc tgat 24

<210>52

<211>48

<212>DNA

<213> Intelligent (Homo sapiens)

<400>52

ttgtaacaca cacacacaca cacacacaca cacacacaca cactaacc 48

<210>53

<211>25

<212>DNA

<213> Intelligent (Homo sapiens)

<400>53

gctttaaaaa aaaaaaaaaa gaatt 25

<210>54

<211>29

<212>DNA

<213> Intelligent (Homo sapiens)

<400>54

aaaccaaaaa aaaaaaaaaa aaaacacct 29

<210>55

<211>27

<212>DNA

<213> Intelligent (Homo sapiens)

<400>55

atttgttttt tttttttttt ttacata 27

<210>56

<211>47

<212>DNA

<213> Intelligent (Homo sapiens)

<400>56

ttttcttttt tttttttttt tttttttttt tttttttttt ttgagac 47

<210>57

<211>38

<212>DNA

<213> Intelligent (Homo sapiens)

<400>57

tccagttttt tttttttttt tttttttttt tttgagac 38

<210>58

<211>37

<212>DNA

<213> Intelligent (Homo sapiens)

<400>58

caggtaaaaa aaaaaaaaaa aaaaaaaaaa aagggtt 37

<210>59

<211>52

<212>DNA

<213> Intelligent (Homo sapiens)

<400>59

gtgatacaca cacacacaca cacacacaca cacacacaca cacacacata tt 52

<210>60

<211>33

<212>DNA

<213> Intelligent (Homo sapiens)

<400>60

tcctattttt tttttttttt ttttttttgt gag 33

<210>61

<211>20

<212>DNA

<213> Intelligent (Homo sapiens)

<400>61

gaaggaaaaa aaaaagcctg 20

<210>62

<211>35

<212>DNA

<213> Intelligent (Homo sapiens)

<400>62

tttgattttt tttttttttt tttttttttt gagaa 35

<210>63

<211>50

<212>DNA

<213> Intelligent (Homo sapiens)

<400>63

tactctgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtaaat 50

<210>64

<211>36

<212>DNA

<213> Intelligent (Homo sapiens)

<400>64

ctggtaaaaa aaaaaaaaaa aaaaaaaaaa agccac 36

<210>65

<211>31

<212>DNA

<213> Intelligent (Homo sapiens)

<400>65

gaagattttt tttttttttt ttttttaata t 31

<210>66

<211>31

<212>DNA

<213> Intelligent (Homo sapiens)

<400>66

ttgctaaaaa aaaaaaaaaa aaaaaaggcc a 31

<210>67

<211>56

<212>DNA

<213> Intelligent (Homo sapiens)

<400>67

gcatggtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tatggg 56

<210>68

<211>44

<212>DNA

<213> Intelligent (Homo sapiens)

<400>68

cctttacaca cacacacaca cacacacaca cacacacacg gctc 44

<210>69

<211>62

<212>DNA

<213> Intelligent (Homo sapiens)

<400>69

aagaatgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtct 60

aa 62

<210>70

<211>31

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(27)..(31)

<223> terminal blocking sequence, C is ribonucleotide, GAG are connected by-S-bond, and terminal base G3' end has C3 spacer arm modification.

<400>70

aggcaaggat ataaatgttg aattcccaga g 31

<210>71

<211>33

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(29)..(33)

<223> terminal blocking sequence, the 5 th reciprocal position is RNA residue, the three reciprocal positions are connected by-S-, and the 3' end at the terminal position is blocked and modified by a C3 spacer arm.

<400>71

gcatctatat ggtcatattg ttgaaatgat tct 33

<210>72

<211>26

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(22)..(26)

<400>72

acttcctgtg aactaaggtt gctgca 26

<210>73

<211>24

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(21)..(24)

<223> terminal blocking sequence, the 4 th reciprocal position is RNA residue, the three terminal positions are connected by-S-, and the 3' end of the terminal position is blocked and modified by a C3 spacer arm.

<400>73

tcgctgactt tttggagtag gatg 24

<210>74

<211>27

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(23)..(27)

<223> terminal blocking sequence, the 5 th reciprocal position is RNA residue, the three terminal positions are connected by-S-, and the 3' end of the terminal position is blocked and modified by a C3 spacer arm.

<400>74

cagattgtgg tgcagtacat ttaagaa 27

<210>75

<211>26

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(23)..(26)

<400>75

ttgctcacaa gaggatttca atgttc 26

<210>76

<211>28

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(24)..(28)

<400>76

ggcattggaa acttcatatc ctggacat 28

<210>77

<211>28

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(24)..(28)

<400>77

gaattttgca tttctcaagt gctcctgt 28

<210>78

<211>29

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(25)..(29)

<400>78

gcacatcaag tttggtagga tattggtaa 29

<210>79

<211>30

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(26)..(30)

<400>79

tagctaaaat ccttttggag gtatgccttg 30

<210>80

<211>33

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(29)..(33)

<400>80

acaataaaga tctcatggtt aaagactaac gga 33

<210>81

<211>34

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(30)..(34)

<400>81

gtcttttgta actccataat ttaacatcag cttc 34

<210>82

<211>32

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(28)..(32)

<400>82

agatttagag tataacctga tgcaaagcct ca 32

<210>83

<211>31

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(27)..(31)

<400>83

ccttcactgg cttttctaaa ataaaaccac a 31

<210>84

<211>25

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(21)..(25)

<400>84

gatgacattc acagccttca catac 25

<210>85

<211>26

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(22)..(26)

<400>85

agctgtactc tgaggaatac acaaac 26

<210>86

<211>28

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(24)..(28)

<400>86

aaaagctgct ggacatactc taagttac 28

<210>87

<211>27

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(23)..(27)

<400>87

agaggcattg cttattgagg ttcaaaa 27

<210>88

<211>30

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(26)..(30)

<400>88

gtcatttaag ttttctaagg agccacaaac 30

<210>89

<211>30

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(26)..(30)

<400>89

ggtagattct ccctacttct acttccctta 30

<210>90

<211>25

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(21)..(25)

<400>90

ttcctgcttt cccagatgac cagaa 25

<210>91

<211>24

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(20)..(24)

<400>91

atcttggaat gctgcacctc tctt 24

<210>92

<211>29

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(25)..(29)

<400>92

ccttcattca attaagcatg tgtccttgg 29

<210>93

<211>28

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(24)..(28)

<400>93

ctgtgtgaag ctttcaattt accagaag 28

<210>94

<211>26

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(22)..(26)

<400>94

agctaaaaca gaagtctcac ccagga 26

<210>95

<211>25

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(21)..(25)

<400>95

gtaaacacct ttgactggct gattt 25

<210>96

<211>25

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(21)..(25)

<400>96

aaggatggct agaatgaggg cagag 25

<210>97

<211>26

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(22)..(26)

<400>97

cctacctcag aaagatagca ccctag 26

<210>98

<211>28

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(24)..(28)

<400>98

cagaaatggt taaatgcagg caagaaaa 28

<210>99

<211>28

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(24)..(28)

<400>99

acattagcat atggtttgat ccgggcat 28

<210>100

<211>26

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(22)..(26)

<400>100

gctcactaaa tgaatgggtg actttc 26

<210>101

<211>26

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(22)..(26)

<400>101

ctcaaggtct ctgcagagtt aggtta 26

<210>102

<211>24

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(20)..(24)

<400>102

tctctcggct tcccctaaac acac 24

<210>103

<211>25

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(21)..(25)

<400>103

caacagacag gaaagtgcag gagag 25

<210>104

<211>26

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(22)..(26)

<400>104

ttgacgatgc ttatgcggtt tctgaa 26

<210>105

<211>25

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(21)..(25)

<400>105

cccaggtgac taagaaatgc ctttc 25

<210>106

<211>25

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(21)..(25)

<400>106

gtctgtgctg tcgtgaaatt ccaag 25

<210>107

<211>25

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(21)..(25)

<400>107

tgaatcaagt cggtgaactg cccct 25

<210>108

<211>25

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(21)..(25)

<400>108

ccgaggtgat tgattggttt ggtca 25

<210>109

<211>25

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(21)..(25)

<400>109

ctgtaactat tcagccagca cttgc 25

<210>110

<211>30

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(26)..(30)

<400>110

caggtaagtg gattgatgtg gttatcttac 30

<210>111

<211>31

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(27)..(31)

<400>111

ccttgattat tttgcttggt tctcaactga g 31

<210>112

<211>22

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(18)..(22)

<400>112

ctctggaccc gagacagctg ct 22

<210>113

<211>23

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(19)..(23)

<400>113

aggttgagaa gccagtggcc gag 23

<210>114

<211>24

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(20)..(24)

<400>114

taggcagggt ccccaaatac atca 24

<210>115

<211>24

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(20)..(24)

<400>115

gctgagtaga caccatgcca aaaa 24

<210>116

<211>28

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(24)..(28)

<400>116

agggttacta ctttttagcc tgtcttaa 28

<210>117

<211>27

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(23)..(27)

<400>117

tctccgacaa caacaacaaa accacac 27

<210>118

<211>25

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(21)..(25)

<400>118

tttggtgaca ggtctcacat gagct 25

<210>119

<211>26

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(22)..(26)

<400>119

ggtatttctg tcacccacat ccctca 26

<210>120

<211>31

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(27)..(31)

<400>120

ctgtcatact tagctactac cttgatcctt t 31

<210>121

<211>32

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(28)..(32)

<400>121

tgctattgtc gttattaaag tccagatctg at 32

<210>122

<211>27

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(23)..(27)

<400>122

tccatattcc atagtgtcct cccactt 27

<210>123

<211>28

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(24)..(28)

<400>123

agccgttcac taaagataac aatgaagg 28

<210>124

<211>25

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(21)..(25)

<400>124

cagcagcttc attctttcca gcata 25

<210>125

<211>26

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(22)..(26)

<400>125

tgcctcccag ttcttatcct aggaaa 26

<210>126

<211>27

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(23)..(27)

<400>126

ctcaactctc tcacagtgtt ggggatt 27

<210>127

<211>29

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(25)..(29)

<400>127

gtaaggtgta gatggaaatg ggatatagg 29

<210>128

<211>28

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(24)..(28)

<400>128

ggcacgcatt atttcagaac atactaga 28

<210>129

<211>28

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(24)..(28)

<400>129

gtctcctaat gttatcctct cctcactt 28

<210>130

<211>31

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(27)..(31)

<400>130

ggtagtataa tcacgagcta atggtcccaa a 31

<210>131

<211>32

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(28)..(32)

<400>131

catttccatc tgattgttga cgatattgac aa 32

<210>132

<211>34

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(30)..(34)

<400>132

gaactattat atgttttgat ttgagggctg atag 34

<210>133

<211>33

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(29)..(33)

<400>133

gggtacaatt gtttttatca gagaaacagc ata 33

<210>134

<211>28

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(24)..(28)

<400>134

gaaacctacc agagtccttg atacagtt 28

<210>135

<211>27

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(23)..(27)

<400>135

ttggtgtctt taaccctcaa aagagga 27

<210>136

<211>26

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(22)..(26)

<400>136

ccctgtttcc ttgtcttgat tgtctc 26

<210>137

<211>26

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(22)..(26)

<400>137

gaggaatggg aagaagaagt ggcaga 26

<210>138

<211>27

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(23)..(27)

<400>138

tgtatgcaca cacaccttta aacacag 27

<210>139

<211>28

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(24)..(28)

<400>139

gggcctgata tatggtagga taacctta 28

<210>140

<211>28

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(24)..(28)

<400>140

atcaggatac caaaaattac cccccgat 28

<210>141

<211>28

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(24)..(28)

<400>141

tgagtgttgg aatcctagtg aaagaaag 28

<210>142

<211>28

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(24)..(28)

<400>142

gttctgtaac ataacccaac tcaccaat 28

<210>143

<211>27

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(23)..(27)

<400>143

cttgtcaaca cgaggttatg tcaactt 27

<210>144

<211>26

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(22)..(26)

<400>144

tgcattgatg aggtcttgtt gctagc 26

<210>145

<211>26

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(22)..(26)

<400>145

acagcatccc agacattatg actctg 26

<210>146

<211>33

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(29)..(33)

<400>146

gagtaggaga gaaatttgac ataatcaagt aaa 33

<210>147

<211>32

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(28)..(42)

<400>147

cttcatcaac atgtttctgt ttaactacag tg 32

<210>148

<211>31

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(27)..(31)

<400>148

tcaagtagat ttgctttgaa ataggaggct a 31

<210>149

<211>30

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(26)..(30)

<400>149

ttcctatgtg atgtgttttc aaaggcaagg 30

<210>150

<211>28

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(24)..(28)

<400>150

tgccttttat gtatattcgc tgtggagt 28

<210>151

<211>29

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(25)..(29)

<400>151

gatgagtaat gctatataca ccgccactg 29

<210>152

<211>23

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(21)..(25)

<400>152

agagactaca gctggctcct tca 23

<210>153

<211>25

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(21)..(25)

<400>153

agagtgttct cctaatccca gaacc 25

<210>154

<211>30

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(26)..(30)

<400>154

caactatgta acttgtcagg aacatgattg 30

<210>155

<211>29

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(25)..(29)

<400>155

gacactgcaa aagcatgaaa attcccaca 29

<210>156

<211>33

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(29)..(33)

<400>156

cgatcattca ataaatgcag aaaaagtagc aca 33

<210>157

<211>33

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(29)..(33)

<400>157

gaagcaattt ctaacatcat ttcatgagcc tca 33

<210>158

<211>30

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(26)..(30)

<400>158

catttctcta ccctctttca aacttccttc 30

<210>159

<211>31

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(27)..(31)

<400>159

caaagttgat ctgactcttt taatgacttg c 31

<210>160

<211>27

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(23)..(27)

<400>160

ccatggtctt gtaaataggc atcatcc 27

<210>161

<211>23

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(19)..(23)

<400>161

attgtaggca tggcactgcg aag 23

<210>162

<211>30

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(26)..(30)

<400>162

agatgaacta ttcctagttt ccttcctgcc 30

<210>163

<211>30

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(26)..(30)

<400>163

gaataaaaca gcattttaaa ccccagacca 30

<210>164

<211>32

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(28)..(32)

<400>164

ctgcttcaag ttaatgccat atttaaagaa cc 32

<210>165

<211>32

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(28)..(32)

<400>165

agaataagaa acataatcta gcccaaaccc ac 32

<210>166

<211>24

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(20)..(24)

<400>166

tatcatttcc caccctgacc accc 24

<210>167

<211>24

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(20)..(24)

<400>167

caccttgagt taggctgagc caca 24

<210>168

<211>26

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(22)..(26)

<400>168

attaggaaag gctctctcct gctgac 26

<210>169

<211>26

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(22)..(26)

<400>169

cctccatgct cagaaaagag agaaga 26

<210>170

<211>27

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(23)..(27)

<400>170

cctgggactg tagaaatttc accgttg 27

<210>171

<211>28

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(24)..(28)

<400>171

actacaaagt atctagctgg cttcagag 28

<210>172

<211>24

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(20)..(24)

<400>172

acagctggac agttaaacgg caga 24

<210>173

<211>25

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(21)..(25)

<400>173

aaggacgctc taggaaagta attgc 25

<210>174

<211>28

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(24)..(28)

<400>174

ttgtgacatc tgaaaagcta cagggaga 28

<210>175

<211>29

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(25)..(29)

<400>175

tgtgttttca aataggatgc tcttgtatc 29

<210>176

<211>26

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(22)..(26)

<400>176

aacaacacca tctgctcatt tcacac 26

<210>177

<211>26

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(22)..(26)

<400>177

cccttgcaat gtcatgtttt gggaaa 26

<210>178

<211>23

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(19)..(23)

<400>178

ctcaggtttc ctccagctca aga 23

<210>179

<211>23

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(19)..(23)

<400>179

catcagagtc accaacccca atg 23

<210>180

<211>24

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(20)..(24)

<400>180

ccacaaccct gcttttgttc cttt 24

<210>181

<211>23

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(19)..(23)

<400>181

ctagcctggg tggtagagca aga 23

<210>182

<211>34

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(30)..(34)

<400>182

gtttagttag attaataccc accttagaac ttgc 34

<210>183

<211>33

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(29)..(33)

<400>183

gagatagttt aggagaaagt gatacaaggg atg 33

<210>184

<211>31

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(27)..(31)

<400>184

ccagtatatg aaattggata ttgcagcagt c 31

<210>185

<211>30

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(26)..(30)

<400>185

gctcctttat aagcttcttc agtatatgtc 30

<210>186

<211>31

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(27)..(31)

<400>186

ctagtttttc agggaattga gagttacagg t 31

<210>187

<211>32

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(28)..(32)

<400>187

gcatatgaat accaggatag ctttataaag ca 32

<210>188

<211>25

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(21)..(25)

<400>188

ttcaaccctc actcatgttc ctggc 25

<210>189

<211>28

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(24)..(28)

<400>189

cccaagactt cagaaaattc tctctggc 28

<210>190

<211>28

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(24)..(28)

<400>190

cattgaagtc tgcagttgaa aagcccaa 28

<210>191

<211>28

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(24)..(28)

<400>191

ctagcaatga ccaataagca agtcactg 28

<210>192

<211>31

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(27)..(31)

<400>192

gagaacacga aaaatattcc tactccgcat t 31

<210>193

<211>32

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(28)..(32)

<400>193

cgctggcaca gttctatttt tatatttaaa tg 32

<210>194

<211>26

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(22)..(26)

<400>194

ccccattgct gaattttacc tcctga 26

<210>195

<211>25

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(21)..(25)

<400>195

gagattgtgc cattgcattc caacc 25

<210>196

<211>27

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(23)..(28)

<400>196

agttctggcc agagaaatta gacacag 27

<210>197

<211>28

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(24)..(28)

<400>197

gggatagttt tacttctccc tttctgac 28

<210>198

<211>25

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(21)..(25)

<400>198

cataaatgtc acgtccagct ctgat 25

<210>199

<211>26

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(22)..(26)

<400>199

ctgatgccat ccagttttgt tcttac 26

<210>200

<211>30

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(26)..(30)

<400>200

tgggagtgat tctctaaaga gttttgtgtt 30

<210>201

<211>31

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(27)..(31)

<400>201

aactaaattt cctctgcttt tggttaccac a 31

<210>202

<211>29

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(25)..(29)

<400>202

gaatgacgag aacataacac tagagacag 29

<210>203

<211>28

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(24)..(28)

<400>203

ctacaggaac acatgaagaa agtctcac 28

<210>204

<211>25

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(21)..(25)

<400>204

aaacactgtt tggactccac ctagg 25

<210>205

<211>26

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(22)..(26)

<400>205

ctgttggatt ctggaaacct actcct 26

<210>206

<211>28

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(24)..(28)

<400>206

aggcatgaat tactactgtc ctactgtg 28

<210>207

<211>25

<212>DNA/RNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(21)..(25)

<400>207

agtgagctgt gattgcacta cactc 25

<210>208

<211>32

<212>DNA

<213> Intelligent (Homo sapiens)

<400>208

ttgtcttcct aagaccgctt ggcctccgac tt 32

<210>209

<211>25

<212>DNA

<213> Intelligent (Homo sapiens)

<400>209

gaacgacatg gctacgatcc gactt 25

<210>210

<211>33

<212>DNA

<213> Intelligent (Homo sapiens)

<400>210

acactctttc cctacacgac gctcttccga tct 33

<210>211

<211>34

<212>DNA

<213> Intelligent (Homo sapiens)

<400>211

gtgactggag ttcagacgtg tgctcttccg atct 34

Claims

1. A primer combination for PCR amplification is characterized in that the primer combination is a combination of primers shown in SEQ ID NO. 70 to SEQ ID NO. 207.

2. A kit, comprising: the primer combination of claim 1.

3. The kit of claim 2, further comprising a sequencing platform adapter universal primer.

4. The kit according to claim 3, wherein the universal primer is a universal primer of MGI sequencing platform shown in SEQ ID NO:208 and SEQ ID NO:209, or the universal primer is a universal primer of Illumina sequencing platform shown in SEQ ID NO:210 and SEQ ID NO: 211.

5. A method for detecting MSI status for non-diagnostic purposes, said method comprising the step of performing PCR amplification detection of a target MSI site, characterized in that amplification is performed using a primer combination according to claim 1; or using the kit of claim 3 or 4 for amplification.

6. Use of the primer combination of claim 1 for the preparation of a kit for detecting MSI status.

7. The use of claim 6, wherein the kit is an amplicon library construction kit.