CN114058689A - Gene mutation detection kit and application thereof - Google Patents

Gene mutation detection kit and application thereof Download PDF

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CN114058689A
CN114058689A CN202010754298.0A CN202010754298A CN114058689A CN 114058689 A CN114058689 A CN 114058689A CN 202010754298 A CN202010754298 A CN 202010754298A CN 114058689 A CN114058689 A CN 114058689A
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许争峰
胡平
谭建新
姜正文
余锋
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GENESKY DIAGNOSTICS (SUZHOU) Inc
Nanjing Maternity and Child Healthcare Hospital
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Nanjing Maternity and Child Healthcare Hospital
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Abstract

The invention provides an ATP7B, PAH, PTS, MUT, MMACHC gene mutation detection kit and application thereof. Specifically, the invention utilizes the SNaPshot technology to detect 224 hot point mutations of ATP7B, PAH, PTS, MUT and MMACHC genes. 51 pairs of PCR primers were designed to amplify 51 fragments of 100-500bp comprising 224 sites, and 224 extension primers immediately adjacent to the target site were also designed for single base extension. The kit has very high accuracy, can quickly carry out gene diagnosis on related diseases, and provides effective reference for clinicians.

Description

Gene mutation detection kit and application thereof
Technical Field
The invention relates to the field of genetics, in particular to a gene mutation detection kit and application thereof.
Background
Genetic metabolic diseases are diseases in which gene mutations lead to the deficiency of certain enzymes, receptors or vectors necessary for maintaining normal metabolism of the body or to abnormal occurrence of proteins, resulting in corresponding pathological changes and clinical manifestations. Although the incidence of the single genetic metabolic disease is low, the overall incidence can reach 1/500 of live babies. At present, genetic diagnosis of inherited metabolic diseases is mainly based on a secondary sequencing technology. However, the second-generation sequencing sample is complex to prepare, the generated data volume is large, the requirements on splicing of data in the later period and bioinformatics analysis are high, and the method is not suitable for large-scale popularization. In addition, despite the great development of the second generation sequencing technology, the price thereof is still relatively high. Therefore, a rapid gene detection method for genetic metabolic diseases, which is simple and convenient to operate, easy to interpret and low in price, is urgently needed clinically.
Disclosure of Invention
The invention aims to provide a gene mutation detection kit and application thereof.
The invention aims to establish a rapid detection method for 224 mutations of pathogenic genes MUT and MMACHC, which are easy to operate, easy to interpret and low in price and comprise hepatolenticular degeneration pathogenic genes ATP7B, phenylketonuria pathogenic genes PAH and PTS and methylmalonic acidemia pathogenic genes.
In a first aspect of the present invention, there is provided a gene mutation detection kit comprising a nucleic acid detection reagent that specifically detects a mutation site of ATP7B gene; and, the mutation site of the ATP7B gene is selected from the group consisting of:
group A
Figure BDA0002611031590000011
Figure BDA0002611031590000021
In another preferred embodiment, the mutation site of ATP7B gene is selected from the group consisting of:
group B
Figure BDA0002611031590000022
Figure BDA0002611031590000031
In another preferred example, the kit further comprises a nucleic acid detecting reagent that specifically detects a mutation site of the MMACHC gene, and the mutation site of the MMACHC gene is selected from the group consisting of:
group C
Figure BDA0002611031590000032
Figure BDA0002611031590000041
In another preferred example, the kit further comprises a nucleic acid detecting reagent specifically detecting a mutation site of the MUT gene, and the mutation site of the MUT gene is selected from the group consisting of D:
group D
Figure BDA0002611031590000042
Figure BDA0002611031590000051
In another preferred example, the kit further comprises a nucleic acid detecting reagent specifically detecting the mutation sites of the PAH gene and the PTS gene, and the mutation sites of the PAH gene and the PTS gene are selected from the group consisting of E:
group E
Figure BDA0002611031590000052
Figure BDA0002611031590000061
In another preferred embodiment, the mutation sites of the PAH gene and PTS gene are selected from the group F:
group F
Figure BDA0002611031590000062
Figure BDA0002611031590000071
In another preferred embodiment, the mutation sites of the PAH gene and PTS gene are selected from the group G:
group G
Figure BDA0002611031590000072
Figure BDA0002611031590000081
In another preferred embodiment, the nucleic acid detection reagents contained in the kit for specifically detecting the mutation sites of group a, group B, group C, group D, group E, group F, and group G are located in different containers.
In another preferred embodiment, the kit comprises specific multiplex PCR primers for the mutation site.
In another preferred embodiment, the multiplex PCR primer pair can simultaneously obtain amplicons containing different mutation sites through amplification, and each amplicon segment contains at least 1-5 mutation sites.
In another preferred embodiment, the sequences of the multiplex PCR primers are shown in SEQ ID NO. 1-103.
In another preferred embodiment, the kit comprises multiple PCR primers for ATP7B gene (mutation sites of group A and group B), and the sequences of the multiple PCR primers are shown as SEQ ID NO: 1-32.
In another preferred embodiment, the kit comprises multiple PCR primers for MMACHC gene (mutation site of group C), and the sequences of the multiple PCR primers are shown as SEQ ID NO: 33-40.
In another preferred embodiment, the kit comprises multiple PCR primers for MUT gene (mutation site of group D), and the sequences of the multiple PCR primers are shown in SEQ ID NO: 41-62.
In another preferred embodiment, the kit comprises multiplex PCR primers for the PAH gene and PTS genes (mutation sites of group E, group F and group G), and the sequences of the multiplex PCR primers are shown in SEQ ID NOS: 63-103.
In another preferred embodiment, the kit further comprises a single base extension primer for the mutation site.
In another preferred embodiment, the single base extension primer is a SNaPshot single base extension primer.
In another preferred embodiment, the kit comprises a single base extension primer for the mutation site of group A, and the sequence of the single base extension primer is shown in SEQ ID NO: 104-136.
In another preferred embodiment, the kit comprises a single base extension primer for the mutation site of group B, and the sequence of the single base extension primer is shown in SEQ ID NO: 137-171.
In another preferred embodiment, the kit comprises a single base extension primer for the mutation site of group C, and the sequence of the single base extension primer is shown in SEQ ID NO: 172-190.
In another preferred embodiment, the kit comprises a single base extension primer for the mutation site of group D, and the sequence of the single base extension primer is shown in SEQ ID NO. 191-215.
In another preferred embodiment, the kit comprises a single base extension primer for the mutation site of group E, and the sequence of the single base extension primer is shown in SEQ ID NO: 216-251.
In another preferred embodiment, the kit comprises a single base extension primer for the mutation site of group F, and the sequence of the single base extension primer is shown in SEQ ID NO: 252-288.
In another preferred embodiment, the kit comprises a single base extension primer for the mutation site of group G, and the sequence of the single base extension primer is shown in SEQ ID NO: 289-321.
In another preferred embodiment, the kit further comprises standard DNA which is also included in the kit.
In another preferred embodiment, the kit further comprises a sequencing enzyme, preferably the sequencing enzyme comprises a snapshot sequencing enzyme.
In another preferred embodiment, the kit further comprises a fluorescently labeled ddNTP.
In a second aspect of the present invention, there is provided a method of detecting a mutation site from ATP7B, PAH, PTS, MUT and MMACHC genes, comprising the steps of:
(i) providing a sample to be detected;
(ii) providing a kit according to the first aspect of the present invention, performing multiplex PCR reaction and single base extension reaction on a sample to be tested;
(iii) (iii) detecting and typing the sample obtained in step (ii).
In another preferred embodiment, the single base extension reaction is a SNaPshot single base extension reaction.
In another preferred embodiment, the method further comprises performing parallel detection on the control samples.
In another preferred embodiment, the control sample comprises a positive control sample and a negative control sample.
In another preferred embodiment, the negative control sample is ultrapure water.
In another preferred example, in the step (ii), after the sample to be tested is subjected to the multiplex amplification reaction, a purification treatment with Shrimp Alkaline Phosphatase (SAP) is performed before the single base extension reaction is performed.
In another preferred embodiment, the sample obtained in step (ii) is subjected to Shrimp Alkaline Phosphatase (SAP) purification treatment before said step (iii).
In another preferred embodiment, in the step (iii), the detection is performed by capillary electrophoresis.
In another preferred embodiment, in said step (iii), the detection is performed by mass spectrometry.
In another preferred embodiment, the method is non-diagnostic and non-therapeutic.
In another preferred embodiment, the sample is a human DNA-containing sample.
In a third aspect of the invention there is provided the use of a kit according to the first aspect of the invention for detecting mutation sites from ATP7B, PAH, PTS, MUT and MMACHC genes.
In another preferred embodiment, the kit is used for genetic diagnosis of a disease selected from the group consisting of:
hepatolenticular degeneration, phenylketonuria, tetrahydrobiopterin deficiency, methylmalonic acidemia, or a combination thereof.
In the fourth aspect of the invention, the use of a nucleic acid detection reagent for preparing a gene mutation detection kit is provided, wherein the nucleic acid detection reagent comprises a mutation site nucleic acid detection reagent for specifically detecting the ATP7B gene; and, the mutation site of the ATP7B gene is selected from group a.
In another preferred embodiment, the mutation site of the ATP7B gene is selected from group B.
In another preferred embodiment, the nucleic acid detecting reagent comprises a nucleic acid detecting reagent that specifically detects a mutation site of MMACHC gene, and the mutation site of MMACHC gene is selected from group C.
In another preferred embodiment, the nucleic acid detecting reagent comprises a nucleic acid detecting reagent that specifically detects a mutation site of a MUT gene, and the mutation site of the MUT gene is selected from group D.
In another preferred example, the nucleic acid detecting reagent comprises a nucleic acid detecting reagent that specifically detects a mutation site of the PAH gene and the PTS gene, and the mutation site of the PAH gene and the PTS gene is selected from group E.
In another preferred embodiment, the mutation site of the PAH gene and PTS gene is selected from the group F below.
In another preferred embodiment, the mutation site of the PAH gene and PTS gene is selected from the group G below.
In another preferred embodiment, the gene mutation detection kit is used for detecting mutations in ATP7B, PAH, PTS, MUT and/or MMACHC genes.
In another preferred embodiment, the gene mutation detection kit is used for gene diagnosis of a disease selected from the group consisting of:
hepatolenticular degeneration, phenylketonuria, tetrahydrobiopterin deficiency, methylmalonic acidemia, or a combination thereof.
It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.
Drawings
FIG. 1 shows the working principle of the detection method of the present invention.
FIG. 2 shows a capillary electrophoresis peak profile of ATP7B-Panel1, which contains 33 hot spot mutations of ATP7B gene, as a negative sample.
FIG. 3 shows a capillary electrophoresis peak profile of ATP7B-Panel2, a negative sample, which contains 37 hot-spot mutations of ATP7B gene.
FIG. 4 shows a diagram of the capillary electrophoresis peak shape of a negative sample PKU-Panel1, which contains 34 hot spot mutations of the PAH gene and 4 hot spot mutations of the PTS gene.
FIG. 5 shows a diagram of the capillary electrophoresis peak shape of a negative sample PKU-Panel2, which contains 32 hot spot mutations of the PAH gene and 6 hot spot mutations of the PTS gene.
FIG. 6 shows a diagram of the capillary electrophoresis peak shape of a negative sample PKU-Panel3, which contains 31 hot spot mutations of the PAH gene and 3 hot spot mutations of the PTS gene.
FIG. 7 shows a capillary electrophoresis peak profile of a negative sample MUT, containing 25 hot spot mutations of the MUT gene.
FIG. 8 shows a capillary electrophoresis peak profile of MMACHC in a negative sample, including 19 hot spot mutations of MMACHC gene.
FIG. 9 shows the capillary electrophoresis peak shape of ATP7B: c.2621C > T heterozygous mutation (the upper panel is heterozygous mutant sample, the lower panel is negative sample).
FIG. 10 shows a first generation sequencing validation graph of ATP7B: c.2621C > T heterozygous mutations.
FIG. 11 shows capillary electrophoresis peak patterns of MMACHC: c.217C > T heterozygous mutations (heterozygous mutant samples in the upper panel and negative samples in the lower panel).
FIG. 12 shows a primary sequencing validation graph of MMACHC: c.217C > T heterozygous mutations.
FIG. 13 shows capillary electrophoresis peak profiles of MUT c.729-730 insTT heterozygous mutations (heterozygous mutant samples in the upper panel and negative samples in the lower panel).
FIG. 14 shows a primary sequencing validation of MUT c.729_730insTT heterozygous mutation.
FIG. 15 shows the capillary electrophoresis peak pattern of the PAH c.1033G > A heterozygous mutation (the upper panel is heterozygous mutant sample, the lower panel is negative sample).
FIG. 16 shows a first-generation sequencing validation of C.1033G > A heterozygous mutations for PAH.
Detailed Description
The inventor of the invention has extensively and deeply studied and unexpectedly found a kit for detecting the gene mutation of ATP7B, PAH, PTS, MUT and MMACHC for the first time and application thereof. Specifically, the invention utilizes the SNaPshot technology to detect 224 hot point mutations of ATP7B, PAH, PTS, MUT and MMACHC genes. 51 pairs of PCR primers were designed to amplify 51 fragments of 100-500bp comprising 224 sites, and 224 extension primers immediately adjacent to the target site were also designed for single base extension. The PCR product was obtained by Multiplex PCR using HotStarTaq from Qiagen, and the PCR product was purified by shrimp alkaline enzyme (SAP) (from Promega) and exonuclease I (EXO I) (from epicentre) and then subjected to extension reaction using the SNaPshot Multiplex kit from ABI. The extension product was purified with shrimp alkaline enzyme (SAP) (from Promega) and loaded on ABI3730 xl. SNP typing was analyzed by GeneMapper4.0(applied biosystems).
The main working principle of the invention is shown in figure 1, in a reaction system containing sequencing enzyme, four kinds of fluorescence labeled ddNTP, extension primers with different lengths and close to the 5' end of a polymorphic site and a PCR template, one base of primer extension is terminated, after capillary electrophoresis of an ABI sequencer, the type of the incorporated base can be known according to the color of the peak, thereby determining the genotype of the sample, and determining the mutation site corresponding to the extension product according to the position of peak movement on the peak map.
The main advantages of the invention include:
(a) the kit has a breakthrough in the screening of hereditary diseases.
(b) The kit detects 224 hot point mutations of related genes ATP7B, PAH, PTS, MUT and MMACHC in hepatolenticular degeneration, phenylketonuria, tetrahydrobiopterin deficiency and methyl malonic acidemia, can quickly perform gene diagnosis of related diseases, and provides effective reference for clinicians.
(c) The technology adopts the SNaPshot technology with high accuracy, and has very high accuracy.
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are percentages and parts by weight.
Universal material
The DNA samples in the examples were obtained from the maternal care facility, Nanjing and isolated from human peripheral blood.
The mutation site information and corresponding primers and groupings referred to in the examples are shown in tables 1-4.
TABLE 1 mutation site information
Figure BDA0002611031590000121
Figure BDA0002611031590000131
Figure BDA0002611031590000141
Figure BDA0002611031590000151
Table 2: PCR primer sequence Listing
Figure BDA0002611031590000152
Figure BDA0002611031590000161
Figure BDA0002611031590000171
Figure BDA0002611031590000181
TABLE 3 correspondence between PCR primers and respective mutation sites
Figure BDA0002611031590000182
Figure BDA0002611031590000191
TABLE 4 extension primer information
Figure BDA0002611031590000192
Figure BDA0002611031590000201
Figure BDA0002611031590000211
Figure BDA0002611031590000221
Figure BDA0002611031590000231
Figure BDA0002611031590000241
Figure BDA0002611031590000251
Figure BDA0002611031590000261
Figure BDA0002611031590000271
Figure BDA0002611031590000281
Figure BDA0002611031590000291
EXAMPLE 1 detection of mutation sites
The method adopts the SNaPshot technology to detect 224 hot spot mutations of hepatolenticular degeneration, phenylketonuria, tetrahydrobiopterin deficiency and related genes of methyl malonic acidemia ATP7B, PAH, PTS, MUT and MMACHC, and comprises the following specific experimental operation steps:
1) DNA samples 1. mu.l of 1% agarose gel electrophoresis was used for quality inspection and concentration estimation, and the samples were diluted to a working concentration of 5-10 ng/. mu.l depending on the estimated concentration.
2) PCR reaction
Reaction system (10. mu.l):
Figure BDA0002611031590000292
PCR cycling program:
5min at 95 ℃; 11 × (94 ℃ for 20s,65 ℃ -0.5 ℃/cycle for 40s,72 ℃ for 1.5 min); 24 × (94 ℃ C. for 20s,59 ℃ C. for 30s,72 ℃ C. for 1.5 min); 2min at 72 ℃; keeping the temperature at 4 ℃.
3) PCR product purification
To 10. mu.l of the PCR product, 1U of SAP enzyme and 1U of Exonaclease I enzyme were added, incubated at 37 ℃ for 1 hour, and then inactivated at 75 ℃ for 15 minutes.
4) Snapshot multiplex single base extension reaction
Extension reaction system (10. mu.l):
Figure BDA0002611031590000293
PCR cycling program:
1min at 96 ℃; 28 × (96 ℃ C. for 10s,50 ℃ C. for 5s,60 ℃ C. for 30 s); keeping the temperature at 4 DEG C
5) Purification of extension products
Mu.l of extension product was added with 1U of SAP enzyme, incubated for 1 hour at 37 ℃ and then inactivated for 15 minutes at 75 ℃.
6) ABI3730XL sequencer on extension products
0.5. mu.l of the purified extension product was mixed with 0.5. mu.l of Liz120 SIZE STANDARD and 9. mu.l of Hi-Di, denatured at 95 ℃ for 5 minutes and applied to ABI3730XL sequencer
7) Raw data collected on the ABI3730XL sequencer was analyzed using GeneMapper4.0(applied biosystems co., ltd., USA).
In this example, 5375 samples were tested, among which 119 samples positive for ATP7B gene, 61 samples positive for MMACHC gene, 14 samples positive for MUT gene, 97 samples positive for PAH gene, and 27 samples positive for PTS gene. Subsequently, the result of the positive detection sample is verified by SANGER sequencing, and the accuracy reaches 100%.
The detection results of the partial positive mutant samples and their negative control and verification results are shown in FIGS. 9-16.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
Sequence listing
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<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 29
ccttttgcca acactaggca ttg 23
<210> 30
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 30
cagctggagc acagtgggta aga 23
<210> 31
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 31
cctcctctgt gtctgtggtg ct 22
<210> 32
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 32
attgcctgct catggtgctg at 22
<210> 33
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 33
taacggccca attgtccttg ag 22
<210> 34
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 34
aggaacccag gaggatcaga gg 22
<210> 35
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 35
cctaccgctg ccaggaccta 20
<210> 36
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 36
ttgggagctg ggggaaaact 20
<210> 37
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 37
aaactagggc tccctcggac aa 22
<210> 38
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 38
aaggctgagg aatgtgggac ct 22
<210> 39
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 39
gggacctcca tgaccttgct t 21
<210> 40
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 40
gttgggcagg tggagtggag 20
<210> 41
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 41
agccattcac acgtggacca ta 22
<210> 42
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 42
cgtgcatgtg ttgttttctt tca 23
<210> 43
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 43
gctggtcagc agggattatc ag 22
<210> 44
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 44
tctctttagg tacaccttgt tcttctcca 29
<210> 45
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 45
tggtaccatc caaaatgata tactaaagga 30
<210> 46
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 46
gcccagattc ctgcaagtaa cg 22
<210> 47
<211> 35
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 47
aaatatagac cttgattttc tagtgtgtga tacaa 35
<210> 48
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 48
tacctgctca gtaagtgacc atcca 25
<210> 49
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 49
tgcagcagtt gctgtttaat catgt 25
<210> 50
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 50
tctgaaaaca aagttgcaaa gtgga 25
<210> 51
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 51
tcctctcaca cccccttctc ag 22
<210> 52
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 52
ttcaatctgc ctgtttcgca ct 22
<210> 53
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 53
tcaaatccag cagggatcaa gc 22
<210> 54
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 54
tttcttttgg gctcacatgg ttt 23
<210> 55
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 55
ggtcgcagac ctcgtcttct tg 22
<210> 56
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 56
ttgccaagtc agtggctaca tacc 24
<210> 57
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 57
accctcgctg ctggtcataa aa 22
<210> 58
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 58
atcacccctc cacacatgac aa 22
<210> 59
<211> 34
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 59
gtttatgctt agaaagtgga tttaatagga cctt 34
<210> 60
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 60
tctgctaaag tataggccag ctcca 25
<210> 61
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 61
tgctgtttat ccatttgtgt taagtggtg 29
<210> 62
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 62
tggcctaaga aaccttacat attacctctt 30
<210> 63
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 63
gggcacagag ctaatatttg gaca 24
<210> 64
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 64
caaaggtcag aatttttgcc aatc 24
<210> 65
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 65
taggcacttc cgggactctc ag 22
<210> 66
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 66
ttgcaaacct gcctgtacct ga 22
<210> 67
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 67
gaaaccagga agcaccagca gt 22
<210> 68
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 68
gcttgctttg tccatggagg tt 22
<210> 69
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 69
catggaagtt tgctacgaca ttatcc 26
<210> 70
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 70
ggcctgcgtt agttccagtg ac 22
<210> 71
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 71
tgctgttatt ttatgaagac agtgtgg 27
<210> 72
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 72
cctggttccc aagaaccatt ca 22
<210> 73
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 73
cccagccctc gtgtaaatag ga 22
<210> 74
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 74
gcccccattc aaagcattca ta 22
<210> 75
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 75
ttctctcttc ccctcaacaa gcaa 24
<210> 76
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 76
gtccccgact ccctctgcta ac 22
<210> 77
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 77
ccctctcctc tgcctcaatc ct 22
<210> 78
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 78
gcctatgtcc ctgggcagtt atg 23
<210> 79
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 79
gtggaccagc cagcaatgaa c 21
<210> 80
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 80
tctggcttgg cttaaacctc ctc 23
<210> 81
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 81
cacccatttc aggtgggatc at 22
<210> 82
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 82
gtgggctgtt ctgaaggcat ct 22
<210> 83
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 83
aacctggctt ccaggggagt ag 22
<210> 84
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 84
ccaatcccct ccagaaacac c 21
<210> 85
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 85
tccttggttc ctgtgaaggt ca 22
<210> 86
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 86
ggaatcgggg tgagatgaga ga 22
<210> 87
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 87
cccagagcta gtggctcacc tt 22
<210> 88
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 88
ctccaaatgg tgcccttcac tc 22
<210> 89
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 89
acccatggct tacatggagg tg 22
<210> 90
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 90
cgtcgctgcc aggcacaagt 20
<210> 91
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 91
ccgctgtacc tgtgtgcaca cc 22
<210> 92
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 92
ggaacagaga agggggtttg aa 22
<210> 93
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 93
catccttatt gaagggctga aatg 24
<210> 94
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 94
agatagtaag ccactttgcg gatca 25
<210> 95
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 95
ccccttgttc cacagcaact g 21
<210> 96
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 96
tgtgcttgga tgttgatctg ttga 24
<210> 97
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 97
gctgaaagag agcccagcaa tc 22
<210> 98
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 98
tgcaaatgtg ggcacagtct ct 22
<210> 99
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 99
agcacccaac atgccattac ct 22
<210> 100
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 100
tggaacaatt tggaatttga gtcgta 26
<210> 101
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 101
atacagtgcc acccacctca cc 22
<210> 102
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 102
actttgattg ttgtgtgatt tctgaagtt 29
<210> 103
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 103
ctgggctttg tgcaatgcta ac 22
<210> 104
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 104
ttttgtctgc ctgggccaag gcc 23
<210> 105
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 105
ttttcacgga cttccaggca gtgc 24
<210> 106
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 106
tgcctcagcc actcacggtt t 21
<210> 107
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 107
tatcccaacc aggttataaa tcagtg 26
<210> 108
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 108
ttttttttag accacgagat gaaaggacag 30
<210> 109
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 109
ttttttctgt gtctgtggtg ctctcatccc 30
<210> 110
<211> 35
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 110
tttttttttt tttcagtgac gctatgacag accac 35
<210> 111
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 111
tttttttttg gggacgccat gggtaatggt g 31
<210> 112
<211> 36
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 112
tttttttttt tctgttacct ttgccaagtg ttccag 36
<210> 113
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 113
ttttttttct tggtgtttta ttttcatagg cacccatt 38
<210> 114
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 114
tttttttttt ttttttgcat tgccttcctt ttgtcttag 39
<210> 115
<211> 43
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 115
tttttttttt tttttttttt tttttttctt cgacacgccc ccc 43
<210> 116
<211> 43
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 116
tttttttttt tttttttttc atgtcattta catggtccct gag 43
<210> 117
<211> 46
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 117
tttttttttt tttttttttt tttttttctg ttgcaggcac acaacc 46
<210> 118
<211> 43
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 118
tttttttttt tttttttttt ttttcggttt gctttccaga cgt 43
<210> 119
<211> 48
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 119
tttttttttt tttttttttt ttttttttgg aggccagcag tgaacacc 48
<210> 120
<211> 49
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 120
tttttttttt tttttttttt cgtttgttgc agggaggagc aagtcccca 49
<210> 121
<211> 51
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 121
tttttttttt tttttttttt tttttttttt tccaatcagc acagagaagg t 51
<210> 122
<211> 54
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 122
tttttttttt tttttttttt ttttttcact aagaaacccr gaagcactgt aatt 54
<210> 123
<211> 54
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 123
tttttttttt tttttttttt ttttttttca ggacgcgcag gtcatccctg tgat 54
<210> 124
<211> 55
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 124
tttttttttt tttttttttt tttttttttt tctgagggga ctcaccactt gagct 55
<210> 125
<211> 57
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 125
tttttttttt tttttttttt tttttttttt ttttttttgc agagcatggg tgtggac 57
<210> 126
<211> 57
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 126
tttttttttt tttttttttt ttttcattcc aggactgtcc attctaaatc tcatctt 57
<210> 127
<211> 59
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 127
tttttttttt tttttttttt ttttttttgt cccatttatc atcatcatgt caactttga 59
<210> 128
<211> 61
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 128
tttttttttt tttttttttt tttttttttt ttttttttct cccagacaga ggtgatcatc 60
c 61
<210> 129
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 129
tttttttttt tttttttttt tttttttttt tttttttttt aggcaagccc ctggagatgg 60
<210> 130
<211> 61
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 130
tttttttttt tttttttttt tttttttttt tttggttgtt tttggcagat aaagactgtg 60
a 61
<210> 131
<211> 65
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 131
tttttttttt tttttttttt tttttttttt gatgatgata aatgggacaa aatatccact 60
aaacc 65
<210> 132
<211> 66
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 132
tttttttttt tttttttttt tttttttttt tttttttttt tttttttgcc tgcccctgct 60
ccctgg 66
<210> 133
<211> 65
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 133
tttttttttt tttttttttt tttttttttt ttttttttgc acatggctct gtgctcatta 60
aagct 65
<210> 134
<211> 72
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 134
tttttttttt tttttttttt tttttttttt tttttttttt ttttttttta ccggttgcgc 60
ctcagccact ca 72
<210> 135
<211> 72
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 135
tttttttttt tttttttttt tttttttttt tttttttttt ttttttcagg accaggttga 60
tgcgtatcct tc 72
<210> 136
<211> 75
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 136
tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt gagaggagcc 60
ctgtgacatt cttcg 75
<210> 137
<211> 76
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 137
tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttgt 60
gttcattgcc ctgggc 76
<210> 138
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 138
ctgtggaatt gggtgcaaa 19
<210> 139
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 139
tgctttccag acgtccatca 20
<210> 140
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 140
ttttcggtgc ccaccatgac agccgtg 27
<210> 141
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 141
tttttcaagg tctctgttcc aagttcc 27
<210> 142
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 142
tttttttttt tgccaaggcc ggggagtca 29
<210> 143
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 143
ttttttcaga gccatgtgca tttatagacc cc 32
<210> 144
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 144
tttttaagtt tgacccggaa attatcggtc ca 32
<210> 145
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 145
tttttttttt tcagacgtcc atcacggtgc tgt 33
<210> 146
<211> 34
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 146
ttttttttgt gtgtctaaca tagaaaggaa tctg 34
<210> 147
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 147
tttttttttt tgaagaaagt cgccatggtg ggg 33
<210> 148
<211> 35
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 148
tttttttttt ttttcccaga ccttctctgt gctga 35
<210> 149
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 149
tttttttttt tttactgcaa ggagtagtga gagtcaaa 38
<210> 150
<211> 41
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 150
tttttttttg tatctctgaa caacaccaaa atcgataaaa c 41
<210> 151
<211> 42
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 151
tttttttttt tttttttcca gagcagtggc accgcagaag tg 42
<210> 152
<211> 43
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 152
tttttttttt tttttttttt tcgcagaacg gcatcctcat caa 43
<210> 153
<211> 47
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 153
tttttttttt tttttcaaaa tcgataaaac cgattacaat ccatacc 47
<210> 154
<211> 46
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 154
tttttttttt tttttttttt ttttttacct gctgcaatgg gtatcc 46
<210> 155
<211> 47
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 155
tttttttttt tttttttttt ttttttttag gtgctgcctt cgcacaa 47
<210> 156
<211> 49
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 156
tttttttttt tttttttttt tgtggatatt ttgtcccatt tatcatcat 49
<210> 157
<211> 52
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 157
tttttttttt tttttttttt tttttttgac cacaacatca ttccaggact gt 52
<210> 158
<211> 56
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 158
tttttttttt tttttttttt tttttttttt ttttcacatc ccccagcagg agcacc 56
<210> 159
<211> 54
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 159
tttttttttt tttttttttt tttttttttt ttcctgaggg gcagtgtggc caca 54
<210> 160
<211> 57
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 160
tttttttttt tttttttttt ttttttttat ctctgaacaa caccaaaatc gataaaa 57
<210> 161
<211> 56
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 161
tttttttttt tttttttttt ttttttttga tctcccaact ggaaggggtg cagcaa 56
<210> 162
<211> 59
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 162
tttttttttt tttttttttt tttttttttt tttgctctgt gctcattaaa gctacccac 59
<210> 163
<211> 59
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 163
tttttttttt tttttttttt tttttttttt ttttgcctga acgtagaagt accacccac 59
<210> 164
<211> 62
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 164
tttttttttt tttttttttt tttttttttt tttccacgct cactctgata aggacgacgt 60
cg 62
<210> 165
<211> 61
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 165
tttttttttt tttttttttt tttttttttt ttttggtgag gacaatttaa tcatcaggtg 60
a 61
<210> 166
<211> 72
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 166
tttttttttt tttttttttt tttttttttt tttttttttt ttttttagtt caacatgggc 60
gttcatctct ta 72
<210> 167
<211> 63
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 167
tttttttttt tttttttttt tttttttttt ttttttgttg gctgacctgt gtctcagaga 60
ttt 63
<210> 168
<211> 63
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 168
tttttttttt tttttttttt tttttttttt tttttttttt tctgtgatct gtcccactcc 60
ctt 63
<210> 169
<211> 66
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 169
tttttttttt tttttttttt tttttttttt tttttttttt ttttttttgc caagtgttcc 60
agccac 66
<210> 170
<211> 66
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 170
tttttttttt tttttttttt tttttttttt tttttttttt tctgtgggat tcttgccatc 60
ctgtgt 66
<210> 171
<211> 69
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 171
tttttttttt tttttttttt tttttttttt tttttttttt tttttgcatg aaggtttccc 60
tggaacaag 69
<210> 172
<211> 75
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 172
tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttagctcag 60
cgtgtaacgt gcgct 75
<210> 173
<211> 36
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 173
tttttttttt ttttcagcag gagggataaa ctaacc 36
<210> 174
<211> 48
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 174
tttttttttt tttttttttt tttttgcttc gaggtttacc ccttccag 48
<210> 175
<211> 57
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 175
tttttttttt tttttttttt tttttttttt ttttttttag gcgtgctgag taccagg 57
<210> 176
<211> 77
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 176
tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttcac 60
tgggtcagtc agcattc 77
<210> 177
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 177
tttttttttt ctaccatctg ggccgtgtta 30
<210> 178
<211> 51
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 178
tttttttttt tttttttttt tttttttgca gatagaaatc attgctgact a 51
<210> 179
<211> 78
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 179
tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttgac 60
tacgaggtrc accccaac 78
<210> 180
<211> 70
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 180
tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt taaccgaygc 60
cccaagatcc 70
<210> 181
<211> 51
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 181
tttttttttt tttttttttt tttttggtag tagtaagcag ccccagctac a 51
<210> 182
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 182
ttttttttgg tcagcctcca catcttgtc 29
<210> 183
<211> 44
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 183
tttttttttt tttttttttt tttccccaaa tcgggggtgt atgc 44
<210> 184
<211> 63
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 184
tttttttttt tttttttttt tttttttttt ttttttttta gcgcatatca ggtgtgtgca 60
tac 63
<210> 185
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 185
tttttttttt tttggcagca gcactacccc t 31
<210> 186
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 186
tttttttttt tttttttttt tttttttttt tttttttttg tacctacaag agctgaccgt 60
<210> 187
<211> 41
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 187
tttttttttt tttttttttg tgtcacagca tcccggtaag t 41
<210> 188
<211> 56
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 188
tttttttttt tttttttttt ttttttttca atttccactg gcgtgattgg acttac 56
<210> 189
<211> 46
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 189
tttttttttt tttttttttt tttttagtag cgctcctggg gtgtca 46
<210> 190
<211> 72
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 190
tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tgtggagtgg 60
agaagtaggc ct 72
<210> 191
<211> 50
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 191
tttttttttt tttttttttt tttttcacag tactaaaacc agcatactgg 50
<210> 192
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 192
tttttttttc tgaatcatag ccacgatgtg 30
<210> 193
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 193
ttttgaatgg ctggagttgc tattg 25
<210> 194
<211> 62
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 194
tttttttttt tttttttttt tttttttttt ttttttaatt cctttagaaa aaatgtcagt 60
tt 62
<210> 195
<211> 69
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 195
tttttttttt tttttttttt tttttttttt tttttttttt tgcagttatt ccagttcttg 60
caaatttta 69
<210> 196
<211> 62
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 196
tttttttttt tttttttttt tttttttttt tttttgttct ggaggaaaaa tgtatgtatt 60
tc 62
<210> 197
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 197
tttttttttt tttttttttt tttttttttt ttttcctttg ctgtatattc aaatatgtca 60
<210> 198
<211> 70
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 198
tttttttttt tttttttttt tttttttttt tttttttctt attcactgtt acaatttttt 60
tttttatgta 70
<210> 199
<211> 56
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 199
tttttttttt tttttttttt tttttttttt tgaaattgaa ttaaattttg gcatgt 56
<210> 200
<211> 57
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 200
tttttttttt tttttttttt tttttttttt tttttttgct tcctgcatat ggtatcc 57
<210> 201
<211> 67
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 201
tttttttttt tttttttttt tttttttttt tttttagaaa ttcattccaa ttccccagaa 60
gaaagac 67
<210> 202
<211> 51
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 202
tttttttttt tttttttttt tttttttgca aagatgagag ctggtagaag a 51
<210> 203
<211> 71
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 203
tttttttttt tttttttttt tttttttttt tttttttttt tttcttcttc taagagcaca 60
ctgtcagaca t 71
<210> 204
<211> 49
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 204
tttttttttt tttttttttt ttttttcttt gagtttttgt tttttatca 49
<210> 205
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 205
tttgccattg cttctattgc agta 24
<210> 206
<211> 43
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 206
tttttttttt tttttttttg tgagacattc catcatgyaa gaa 43
<210> 207
<211> 52
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 207
tttttttttt tttttttttt ttttttgtct cacaaatgat gtttatgatg ct 52
<210> 208
<211> 49
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 208
tttttttttt tttttttttt ttttaaaatt tttctttggg aaattacca 49
<210> 209
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 209
tttttttttc aagagccagg atatttccat ct 32
<210> 210
<211> 40
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 210
tttttttttt ttttcatctg tgatttctcc cactgtacat 40
<210> 211
<211> 34
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 211
tttttttttt ttcgatatgc tccactcacc attc 34
<210> 212
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 212
aaaatgggac aagatggcc 19
<210> 213
<211> 35
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 213
tttttttttt agtttttagt acctggaaaa gaggg 35
<210> 214
<211> 54
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 214
tttttttttt tttttttttt tttttttttt tccctagttc ctgaactcat caaa 54
<210> 215
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 215
tttttttttt tttttttttt ttacacatga caagaatatc tggcc 45
<210> 216
<211> 41
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 216
tttttttttt ttttttttca tcccagcttg cactggtttc c 41
<210> 217
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 217
ttttttacag gaaattggcc ttgcct 26
<210> 218
<211> 69
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 218
tttttttttt tttttttttt tttttttttt tttttttttt ttttttttta ctgtgtgcag 60
tggaagact 69
<210> 219
<211> 68
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 219
tttttttttt tttttttttt tttttttttt tttttttttt tttttcttgt ataaaaccca 60
tgcttgct 68
<210> 220
<211> 77
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 220
tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttatc 60
ctgtgtaccg tgcaaga 77
<210> 221
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 221
ttttttttgg cccttctcag ttcgctac 28
<210> 222
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 222
tttttcccct caacaagcaa ggcagactta 30
<210> 223
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 223
tttttttgta ctgtgtgcag tggaagactc 30
<210> 224
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 224
ttttcttagg aactttgctg ccacaatacc t 31
<210> 225
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 225
tttctttgag ttttctttct tcttttcatc cca 33
<210> 226
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 226
tttttttctg trgctggcct gctttcctct 30
<210> 227
<211> 77
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 227
tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttcggagt 60
tccagcccct stattac 77
<210> 228
<211> 79
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 228
tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttagctg 60
gaggacagta ctcacggtt 79
<210> 229
<211> 35
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 229
tttttttttt tttttttgtg tcccgccgca tctcc 35
<210> 230
<211> 35
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 230
tttttttttt tctctgtgct ttctgtcttt cagtg 35
<210> 231
<211> 79
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 231
tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttggaga 60
ctccataaag gcatatggt 79
<210> 232
<211> 74
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 232
tttttttttt tttttttttt tttttttttt tttttttttt tttttttaaa ccacacaaat 60
attttctgca cata 74
<210> 233
<211> 85
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 233
tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 60
aactggtagc tggaggacag tactc 85
<210> 234
<211> 43
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 234
tttttttttt tttttttttt cccaggcttg ggcaggaaac tct 43
<210> 235
<211> 75
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 235
tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tcgcatctcc 60
ttcagcgcga gccac 75
<210> 236
<211> 44
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 236
tttttttttt tttttttgag ttttaatgat gccaaggaga aagt 44
<210> 237
<211> 83
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 237
tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 60
taactggatg ctgaccaccc tgt 83
<210> 238
<211> 47
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 238
tttttttttt tttttttttt tccgagtggc ctcgtaaggt gtaaatt 47
<210> 239
<211> 51
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 239
tttttttttt tttttttttt tttttttgga actttgctgc cacaatacct c 51
<210> 240
<211> 50
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 240
tttttttttt tttttttttt ttttttttca tggatccaag cccatgtata 50
<210> 241
<211> 51
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 241
tttttttttt tttttttttt ttttttagtg gcctcgtaag gtgtaaatta c 51
<210> 242
<211> 53
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 242
tttttttttt tttttttttt tttttttttt ttcttcctcc atgtattcca ctc 53
<210> 243
<211> 54
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 243
tttttttttt tttttttttt ttttgcaaag tatggcacat ccatatccag attc 54
<210> 244
<211> 55
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 244
tttttttttt tttttttttt cctctaattc ttacctgtgt ctttcttctt atctc 55
<210> 245
<211> 56
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 245
tttttttttt tttttttttt tttttttttt ttttttttca cttggggcct acagta 56
<210> 246
<211> 58
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 246
tttttttttt tttttttttt ttttgtaagg tgtaaattac ttactgttaa tggaatca 58
<210> 247
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 247
tttttttttt tttttttttt tttttttttt ttttttttgg cagacttact ggcggtagtt 60
<210> 248
<211> 61
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 248
tttttttttt tttttttttt tttttttttt tttttgccta cagtactgct tatcagagaa 60
g 61
<210> 249
<211> 61
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 249
tttttttttt tttttttttt tttttttttt tttttttttt cctctcggga tttcttgggt 60
g 61
<210> 250
<211> 65
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 250
tttttttttt tttttttttt tttttttttt tttttttttc tagggtttta aagatcctgt 60
gtacc 65
<210> 251
<211> 66
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 251
tttttttttt tttttttttt tttttttttt tttttttttt ttttctgctt tcctctcggg 60
atttct 66
<210> 252
<211> 62
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 252
tttttttttt tttttttttt tttttttttt tttttttttt tttactggat gctgaccacc 60
ct 62
<210> 253
<211> 89
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 253
tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 60
ttttttgcaa acaaggagac tccataaag 89
<210> 254
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 254
cttgcactgg tttccgcctc 20
<210> 255
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 255
ttcatcccag cttgcactgg tt 22
<210> 256
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 256
tttaaaaatc aggtgtctct tttctccta 29
<210> 257
<211> 72
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 257
tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt cccagcttgc 60
actggtttcc gc 72
<210> 258
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 258
tttttttttt tagccagatt cataaccatt ccc 33
<210> 259
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 259
tttttttttt tgcaatgtca gcaaactgct tc 32
<210> 260
<211> 83
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 260
tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 60
tttctccttg tttgcagagc cca 83
<210> 261
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 261
ttttttggta gctggaggac agtactcacg gt 32
<210> 262
<211> 36
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 262
tttttttccc agatataaac agctacattt tcagtc 36
<210> 263
<211> 80
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 263
tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttgg 60
tagctggagg acagtactca 80
<210> 264
<211> 37
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 264
tttttttttt tttttgtctt tcagtgacat ctgccat 37
<210> 265
<211> 37
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 265
tttttttttt ttttaaatgg tgccatatca ctgatct 37
<210> 266
<211> 80
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 266
tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttcccttg 60
atcataagaa tctggatatg 80
<210> 267
<211> 41
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 267
tttttttttt tttttttgta ttgtccaaga cctcaatcct t 41
<210> 268
<211> 44
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 268
tttttttttt ttttttttac ctgacccaca ttgaatctag acct 44
<210> 269
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 269
tttttttttt tttttttttt ttctcatcct acgggccatg gactc 45
<210> 270
<211> 47
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 270
tttttttttt tttttttttt tttgagccca aactccacag taaacca 47
<210> 271
<211> 86
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 271
tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttg 60
ctctcagatt gactttccat tccaga 86
<210> 272
<211> 49
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 272
tttttttttt tttttttttt cattatccaa gacaarcatg attgtagca 49
<210> 273
<211> 50
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 273
tttttttttt tttttttttt tttttttttt tagacatgga tccaagccca 50
<210> 274
<211> 50
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 274
tttttttttt tttttttttt ttttttggtt ttaaagatcc tgtgtaccgt 50
<210> 275
<211> 51
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 275
tttttttttt tttttttttt tttttggcga gcttttcaat gtattcatca g 51
<210> 276
<211> 76
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 276
tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttcagt 60
tcgctacgac ccatac 76
<210> 277
<211> 54
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 277
tttttttttt tttttttttt ttttttccaa gacaarcatg attgtagcac tgac 54
<210> 278
<211> 70
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 278
tttttttttt tttttttttt tttttttttt tttttttttt tttagctttt caatgtattc 60
atcaggtgca 70
<210> 279
<211> 58
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 279
tttttttttt tttttttttt tttttttttt ttttttaatc caaatggcca tgggcaca 58
<210> 280
<211> 73
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 280
tttttttttt tttttttttt tttttttttt tttttttttt ttttaaaaaa tccattcctt 60
acctgggaaa act 73
<210> 281
<211> 57
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 281
tttttttttt tttttttttt tttttttttc ctccagaaag ttcttcctgt aggagtt 57
<210> 282
<211> 59
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 282
tttttttttt tttttttttt tttttttctg tgtctttctt cttatctcgt gaaagctca 59
<210> 283
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 283
tttttttttt tttttttttt tttttttttt tttttttttt ttgcactggt ttccgcctcc 60
<210> 284
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 284
tttttttttt tttttttttt tttttttttt tttatgattg tagcactgac ctcaaataag 60
<210> 285
<211> 63
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 285
tttttttttt tttttttttt tttttttttt ttttttttta tacctctcca tgtactgtca 60
cca 63
<210> 286
<211> 66
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 286
tttttttttt tttttttttt tttttttttt tttttttttt ttttttcacc tttgtcacca 60
cctcac 66
<210> 287
<211> 68
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 287
tttttttttt tttttttttt tttttttttt tttttttttt tttcaarcat gattgtagca 60
ctgacctc 68
<210> 288
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 288
tttttttttt tttttttttc acttggggcc tacagtact 39
<210> 289
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 289
cagctccagg gggagaagct tt 22
<210> 290
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 290
ttggtagttg taggcaatgt cagca 25
<210> 291
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 291
tttttttttg gccaggccac ccaagaaatc c 31
<210> 292
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 292
ttttttagat tgactttcca ttccagattt act 33
<210> 293
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 293
tttttttttt tcccagctgg aagacgtttc t 31
<210> 294
<211> 35
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 294
tttttttttt ttttaacagg ttctattttc cccca 35
<210> 295
<211> 35
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 295
tttttttttt ttctgataag cagtactgta ggccc 35
<210> 296
<211> 34
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 296
ttttttttgg agaatgatgt aaacctgacc caca 34
<210> 297
<211> 36
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 297
tttttttttt tttggtagct ggaggacagt actcac 36
<210> 298
<211> 37
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 298
ttttttttta agtactgtgg cttccatgaa gataaca 37
<210> 299
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 299
ttttttttta cttttcaaga agtggaaaaa tgtgattg 38
<210> 300
<211> 43
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 300
tttttttttt tttttttttt taaatcccga gaggaaagca ggc 43
<210> 301
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 301
tttttttttt tttttttttt cgatctgaaa acaagggcac atgtc 45
<210> 302
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 302
tttttttttt tttttttttt aatcctttgg gtgtatgggt cgtag 45
<210> 303
<211> 47
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 303
tttttttttt tttttttttt tttgcctgcc tgctctgaca aacrtca 47
<210> 304
<211> 49
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 304
tttttttttt tttttttttt tcaccttact ttctccttgg catcattaa 49
<210> 305
<211> 52
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 305
tttttttttt tttttttttt ttttttttta tcaggtgcac ccagagaggc aa 52
<210> 306
<211> 52
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 306
tttttttttt tttttttttt tttttttttt atctgctcat gggccttcca ct 52
<210> 307
<211> 54
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 307
tttttttttt tttttttttt tttttttggc taaaaaatcc attccttacc tggg 54
<210> 308
<211> 57
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 308
tttttttttt tttttttttt tttttttttt gaagataaca ttccccagct ggaagac 57
<210> 309
<211> 54
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 309
tttttttttt tttttttttt tttttttttc tcctagggtt ttaaagatcc tgtg 54
<210> 310
<211> 57
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 310
tttttttttt tttttttttt tttttttttt tgacagccat csaaaattac actgtca 57
<210> 311
<211> 58
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 311
tttttttttt tttttttttt tttttttttt ttttttgaaa tcccgagagg aaagcagg 58
<210> 312
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 312
tttttttttt tttttttttt tttttttcta ttcatagcat gggaaatcta agaggtataa 60
<210> 313
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 313
tttttttttt tttttttttt tttttttttt ttggtcatac ctgtaattca ccaaaggatg 60
<210> 314
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 314
tttttttttt tttttttttt tttttttttt ttttttcaga cttactggcg gtagttgtag 60
<210> 315
<211> 63
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 315
tttttttttt tttttttttt tttttttttt tttttttttt ttaagatcct gtgtaccgtg 60
caa 63
<210> 316
<211> 66
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 316
tttttttttt tttttttttt tttttttttt tttttttttt ttccatttgg attgttgcat 60
ttccca 66
<210> 317
<211> 67
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 317
tttttttttt tttttttttt tttttttttt tttttttttt tttttcatcc cagcttgcac 60
tggtttc 67
<210> 318
<211> 71
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 318
tttttttttt tttttttttt tttttttttt tttttttttt ttttcctgtg gttttggtct 60
taggaacttt g 71
<210> 319
<211> 74
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 319
tttttttttt tttttttttt tttttttttt tttttttttt tttttttttc ttcttttcat 60
cccagcttgc actg 74
<210> 320
<211> 73
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 320
tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt gtctctagga 60
ggcgattatg cag 73
<210> 321
<211> 76
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 321
tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttgcggaa 60
ctggatgctg accacc 76

Claims (10)

1. A gene mutation detection kit, which is characterized by comprising a nucleic acid detection reagent for specifically detecting a mutation site of the ATP7B gene; and, the mutation site of the ATP7B gene is selected from the group consisting of:
group A
Figure FDA0002611031580000011
Figure FDA0002611031580000021
2. The kit of claim 1, wherein the mutation site of the ATP7B gene is selected from the group consisting of:
group B
Figure FDA0002611031580000022
Figure FDA0002611031580000031
3. The kit of claim 1, wherein the kit further comprises a nucleic acid detection reagent that specifically detects a mutation site of the MMACHC gene, and wherein the mutation site of the MMACHC gene is selected from the group consisting of group C:
group C
Figure FDA0002611031580000032
4. The kit of claim 1, further comprising a nucleic acid detection reagent that specifically detects a mutation site of the MUT gene, and wherein the mutation site of the MUT gene is selected from the group consisting of D:
group D
Figure FDA0002611031580000033
Figure FDA0002611031580000041
5. The kit of claim 1, further comprising a nucleic acid detection reagent that specifically detects the mutation sites of the PAH gene and the PTS gene, and wherein the mutation sites of the PAH gene and the PTS gene are selected from the group consisting of E:
group E
Figure FDA0002611031580000042
Figure FDA0002611031580000051
6. The kit of claim 1, wherein the kit comprises specific multiplex PCR primers for the mutation site, preferably wherein the sequences of the multiplex PCR primers are shown in SEQ ID NOs 1-103.
7. The kit of claim 1, further comprising a single base extension primer for the mutation site selected from the group consisting of:
a single base extension primer for the mutation site of group A, and the sequence of the single base extension primer is shown in SEQ ID NO:104-136,
a single base extension primer for the mutation site of group B, and the sequence of the single base extension primer is shown in SEQ ID NO:137-171,
a single base extension primer for the mutation site of group C, and the sequence of the single base extension primer is shown as SEQ ID NO:172-190,
a single base extension primer for the mutation site of group D, and the sequence of the single base extension primer is shown as SEQ ID NO. 191-215,
a single base extension primer for the mutation site of group E, and the sequence of the single base extension primer is shown in SEQ ID NO:216-251,
a single-base extension primer directed to the mutation site of group F and having a sequence shown by SEQ ID NO:252-288, and
a single base extension primer for the mutation site of group G, and the sequence of the single base extension primer is shown in SEQ ID NO: 289-321.
8. The use of a nucleic acid detection reagent for preparing a gene mutation detection kit, wherein the nucleic acid detection reagent comprises a mutation site nucleic acid detection reagent for specifically detecting an ATP7B gene; and, the mutation site of the ATP7B gene is selected from group a.
9. The use according to claim 8, wherein the gene mutation detection kit is for detecting mutations in the ATP7B, PAH, PTS, MUT and/or MMACHC genes.
10. The use according to claim 8, wherein the gene mutation detection kit is used for genetic diagnosis of a disease selected from the group consisting of:
hepatolenticular degeneration, phenylketonuria, tetrahydrobiopterin deficiency, methylmalonic acidemia, or a combination thereof.
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