CN111575378B - Detection method, detection composition and detection kit for hereditary breast cancer and ovarian cancer syndrome - Google Patents
Detection method, detection composition and detection kit for hereditary breast cancer and ovarian cancer syndrome Download PDFInfo
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Abstract
The invention relates to a detection method, a detection composition and a detection kit for hereditary breast cancer and ovarian cancer syndrome, wherein a coding region of a BRCA1 gene and a plurality of SNP loci on the upstream and downstream of the BRCA1 gene are obtained by screening the hereditary breast cancer and ovarian cancer syndrome, and a method for genetic detection before embryo implantation with strong universality, high diagnosis rate and low cost can be established on the basis of the coding region and the plurality of SNP loci on the upstream and downstream of the coding region on a second generation sequencing platform. And determining the detection result of the embryo by detecting the sequence information of the coding region and the upstream and downstream SNP loci of the detected sample and analyzing and comparing the sequence information. Besides the coding region, 157 SNP are selected for analysis at the upstream and downstream of the BRCA1 gene in the scheme, so that the universality is very high, a large number of samples can be analyzed at one time, the reliability of the result is high, and the detection cost is low.
Description
Technical Field
The invention relates to the field of gene detection, in particular to a detection method, a detection composition and a detection kit for hereditary breast cancer and ovarian cancer syndrome.
Background
Breast cancer (BRC) is one of the most common malignant tumors in women. Most breast cancers are sporadic, some are genetically predisposed, exhibit familial aggregations, and are most commonly less than 50 years of age. Mutations in the breast cancer susceptibility genes 1 (Breast cancer susceptibility gene, BRCA 1) and BRCA2 cause about 60% of hereditary breast cancers, and are the only factors currently known for hereditary breast cancer and ovarian cancer syndrome (Hereditary breast and ovarian cancer syndrome, HBOC). The lifelong risk of breast cancer and ovarian cancer in the carriers of BRCA1 and BRCA2 mutations is significantly increased compared to the general population, with a higher risk of either breast cancer or ovarian cancer.
Hereditary breast cancer and ovarian cancer syndrome are an autosomal disease, with carriers at 50% risk of infecting mutations to their offspring. Prenatal diagnosis or pre-embryo implantation genetic testing (Preimplantation genetic testing, PGT) of female carriers is an effective means of blocking breast and ovarian cancer transmission, eliminating high risk pathogenic factors in the family, and reducing the incidence of tumors. PGT refers to the step of performing genetic detection by biopsy of a plurality of cells when an embryo develops in vitro to a blastomeres stage or blastocyst stage, and finally selecting an embryo without risk of illness to implant into the mother uterus, thereby achieving the purpose of growing healthy offspring. PGT can effectively avoid physical and psychological injuries caused by termination of pregnancy due to pregnant genetics disease fetuses, and is becoming the first choice for high-risk fetuses couples with birth genetics disease. Therefore, it is important to predict the BRCA mutation of an embryo more accurately, and thus take corresponding precautions.
Disclosure of Invention
Based on this, it is necessary to provide a method, a composition and a kit for detecting hereditary breast cancer and ovarian cancer syndrome before embryo implantation, which are highly versatile, high in diagnosis rate and low in cost.
A method for detecting hereditary breast cancer and ovarian cancer syndrome prior to embryo implantation, comprising the steps of:
obtaining genome DNA of an embryo;
detecting sequence information of a BRCA1 gene coding region and SNP loci on the upstream and downstream of the BRCA1 gene in the genome DNA, wherein the BRCA1 gene coding region and the SNP loci on the upstream and downstream of the BRCA1 gene comprise chr17: chr17: the cht-r-cht-h-r-h-r-cht-r-h-cht-h, r-cht-r-cht-h, r-cht-r-cht-r-t-r-cht, r-cht, r-t, r-t, r-cht, r, cht, r, t, r, t, cht, t, r, cht, r, cht, chr, r, 17, chr, 17, chchchchchchchchchchchchchchr, 17, chchchchchchchchchchchchchchchchr, 17, chr, 17, chr, by, of, chr17: chr17: the following are chr17, and chr 17.
The sequence information is aligned and analyzed to determine whether the embryo has hereditary breast cancer and ovarian cancer syndrome.
In one embodiment, the step of detecting the sequence information comprises the steps of: and (3) performing multiplex PCR on the genome DNA by using a plurality of pairs of primers capable of specifically amplifying the coding region of the BRCA1 gene and SNP loci on the upstream and downstream of the BRCA1 gene respectively to obtain amplified products, and sequencing the amplified products to obtain the sequence information.
In one embodiment, the primer is selected from the group consisting of SEQ ID NO: 1-SEQ ID NO:338, at least one of which is a metal foil.
In one embodiment, the step of comparing the analysis comprises the steps of: and (3) comparing the sequence information with the DNA sequences of the parent two parties, and analyzing the haplotype of the embryo.
In one embodiment, the step of comparing the analysis further comprises the steps of: and comparing the sequence information with a human genome reference sequence, and analyzing SNP coverage fold and genotype.
In one embodiment, the step of obtaining the genomic DNA comprises the steps of: trophoblast cells are collected from embryos that develop to the blastomeres or blastocysts stage and the DNA in the trophoblast cells is subjected to whole-genome amplification.
A detection composition for detecting hereditary breast cancer and ovarian cancer syndrome comprises a plurality of detection agents capable of detecting sequence information of the coding region of the BRCA1 gene and SNP loci upstream and downstream of the BRCA1 gene, respectively.
In one embodiment, the detection agent is a PCR primer and the detection agent is selected from the group consisting of SEQ ID NOs: 1-SEQ ID NO:338, at least one of which is a metal foil.
A detection kit for detecting hereditary breast cancer and ovarian cancer syndrome comprises the detection composition and a reaction reagent for library construction.
In one embodiment, the reactant is selected from one or more of multiplex PCR polymerase, DNA ligase, end repair enzyme, dNTPs, and PCR buffer.
The invention screens and obtains a coding region of the BRCA1 gene and a plurality of SNP loci on the upstream and downstream of the BRCA1 gene aiming at hereditary breast cancer and ovarian cancer syndrome, and a method for genetic detection before embryo implantation with strong universality, high diagnosis rate and low cost can be established on the basis of the coding region and the plurality of SNP loci on the upstream and downstream. And determining the hereditary breast cancer and ovarian cancer syndrome detection result of the embryo by detecting the sequence information of the coding region and the upstream and downstream SNP loci of the detected sample and analyzing and comparing. Besides the coding region, 157 SNP are selected for analysis at the upstream and downstream of the BRCA1 gene in the scheme, so that the universality is very high, the pre-experiment at the cell level is omitted, a large number of samples can be analyzed at one time based on a high-throughput sequencing technology, the average sequencing depth can reach more than 100X, and the result reliability is high and the detection cost is low.
Detailed Description
The present invention will be described more fully hereinafter in order to facilitate an understanding of the present invention, and preferred embodiments of the present invention are set forth. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.
In the present invention, reads refer to sequence fragments obtained by sequencing; single nucleotide polymorphism (single nucleotide polymorphism, SNP) refers mainly to DNA sequence polymorphism caused by variation of a single nucleotide at the genomic level; haplotype (Haplotype) refers to a group of interrelated single nucleotide polymorphisms located in a particular region of a chromosome and that tend to inherit as a whole to offspring, also known as haplotypes or haplotypes; sequencing depth, i.e., for example, in one embodiment, a sequencing depth of 1000X, represents that the strip of specific PCR amplification product is sequenced 1000 times.
The method for detecting hereditary breast cancer and ovarian cancer syndrome before embryo implantation in an embodiment of the invention comprises the following steps S1 to S3:
s1, obtaining genome DNA of an embryo.
S2, detecting sequence information of SNP loci on the upper and lower streams of a BRCA1 gene coding region and a BRCA1 gene in genome DNA, wherein the SNP loci on the upper and lower streams of the BRCA1 gene coding region and the BRCA1 gene comprise chr17: chr17: the cht-r-cht-r-h-r-cht-r-cht-h, r-cht-r-cht-r-t, r-r, r-t, r-cht, r, t, r, r, 17, chchchchchchchchchchchchchchchchchchchchchchchchchchchchchchchchchchchchchchchchchchchchchchchchchchchchchch, 17, of, chr17: chr17: the composition comprises chr17, and chr 17.
S3, comparing and analyzing the sequence information to determine whether the embryo has hereditary breast cancer and ovarian cancer syndrome.
According to the European Society of Human Reproduction and Embryo (ESHRE) "for PGD practice guidelines based on amplification" (2011 edition), pre-implantation genetic testing of monogenic genetic disorders has been suggested using a strategy of mutation detection combined with haplotype analysis to ensure accuracy of embryo detection results. Common methods for gene detection at the embryo level are multiplex nested PCR and Karyoming techniques. The multiplex nested PCR method requires that a hybrid Short Tandem Repeat (STR) is screened for BRCA1 female carriers in advance, then a multiplex PCR system containing mutation sites and hybrid STR sites is established at lymphocyte level, and the amplification efficiency and allele release rate of each site are evaluated at cell level, so that the method can be applied to embryo detection after qualification. Because the STR number is limited and the heterozygosity frequency in the crowd is different, the design of the multiple nest PCR method is more personalized and the universality is lower. The high individuation also means that the workload is large and the detection period is long.
Compared with STR loci, the number of single nucleotide polymorphism loci (SNP) is widely distributed, the occurrence frequency in the crowd exceeds 1%, the total number of the SNP is 300 ten thousand, 1/3 Kb is averaged, and the SNP is easy to realize automatic analysis on a high-throughput sequencing platform. The Karyoming technique indirectly eliminates gene defects through SNP linkage analysis. The chip has 30 ten thousand SNP probes for linkage analysis of whole genome, and is a comprehensive universal single gene disease PGD technology. However, karyomopping is not able to detect gene mutations, so that either a pre-prover sample or a suitable family member sample is necessary. If there are not enough pedigree samples, additional mutation detection is required. Therefore, there is a great need in clinic to develop a method for detecting all coding regions of BRCA1 gene and covering enough SNP loci in the gene and upstream and downstream, which is used for judging embryo genotypes.
The BRCA1 gene is located at 17q21 and has a total length of 100kb and contains 22 coding exons, 2 non-coding exons and 22 introns, exon 11 is larger and encodes 61% of amino acid residues, the 22 exons transcribe 7.8kb mRNA, and the gene product is a protein containing 1863 amino acid residues and has a molecular weight of 220KD and contains up to 41.5% Alu repeat sequences and 4.8% other repeat sequences. The N-terminal sequence of the BRCA1 encoded protein contains a cyclic domain (ringdomain) capable of forming a loop 2 cyclic heterodimer with the BRCA 1-related cyclic protein (BRCA 1 associated RING domain protein, BARD 1). The invention screens and obtains a coding region of the BRCA1 gene and a plurality of SNP loci on the upstream and downstream of the BRCA1 gene aiming at hereditary breast cancer and ovarian cancer syndrome, and a method for genetic detection before embryo implantation with strong universality, high diagnosis rate and low cost can be established on the basis of the coding region and the plurality of SNP loci on the upstream and downstream. And determining the hereditary breast cancer and ovarian cancer syndrome detection result of the embryo by detecting the sequence information of the coding region and the upstream and downstream SNP loci of the detected sample and analyzing and comparing. Besides the coding region, 157 SNP are selected for analysis at the upstream and downstream of the BRCA1 gene in the scheme, so that the universality is very high, the pre-experiment at the cell level is omitted, a large number of samples can be analyzed at one time based on a high-throughput sequencing technology, the average sequencing depth can reach more than 100X, and the result reliability is high and the detection cost is low.
It is understood that the detection target of this detection method is an embryo not implanted in the uterus, and is not a living human body or animal body, and the detection result does not relate to the disease diagnosis result of both the parents, and therefore, it is not a disease diagnosis and treatment method.
The SNP locus is finally determined based on the following screening principle and combined with long-term test optimization:
(1) High frequency SNP selection from a thousand-person genome project database
(http://www.ncbi.nlm.nih.gov/variation/tools/1000genomes/)；
(2) High frequency SNP sites with minimum allele frequencies greater than 0.2;
(3) Removing SNP sites with GC content of >70% in the polynucleotide (polyN) and 50bp sequences upstream and downstream of the sites;
(4) Removing SNP sites with a plurality of positions from the upstream and downstream 50bp sequences (namely removing SNP sites with high homology) to human genome hg 19;
(5) SNP selection range: SNP sites within the gene and within 1Mb upstream and downstream are preferentially selected, and if the number of SNPs within 1Mb is small, the range can be appropriately widened, for example, 2Mb upstream and downstream.
In a specific example, the step of detecting the above sequence information includes the steps of: and (3) performing multiplex PCR on the genome DNA by using a plurality of pairs of primers capable of specifically amplifying the coding region of the BRCA1 gene and SNP loci on the upstream and downstream of the BRCA1 gene respectively to obtain amplified products, and sequencing the amplified products to obtain sequence information. It is understood that the method of detecting the above sequence information is not limited thereto, and those skilled in the art may select as needed.
In a specific example, the above primer is selected from the group consisting of SEQ ID NO: 1-SEQ ID NO:338, at least one of which is a metal foil. Preferably, the above primer comprises SEQ ID NO: 1-SEQ ID NO:338. the primers designed and screened by the invention have high specificity and similar annealing temperature, and the sizes of PCR product fragments are all in the range of 125 bp-275 bp. The positions of the coding region and the upstream and downstream SNP loci on chr17 and the sequences of the corresponding 169 pairs of primers are shown in Table 1, wherein, the sequence numbers 1-89 are SNP loci and primer pairs within 2M of the downstream of the BRCA1 gene, the sequence numbers 90-101 are SNP loci and primer pairs of the coding region of the BRCA1 gene, and the sequence numbers 102-169 are SNP loci and primer pairs within 2M of the upstream of the BRCA1 gene. The 169 pairs of primers are mixed into a PCR reaction tube to carry out 169-fold reaction, and the BRCA1 gene coding region and upstream and downstream SNP loci of the tested sample are amplified.
TABLE 1 SNP loci and primer sequence listing
In a specific example, the step of obtaining genomic DNA includes the steps of: trophoblast cells are collected from embryos that develop to the blastomeres or blastocysts stage and the DNA in the trophoblast cells is subjected to whole-genome amplification.
In a specific example, the step of comparing analysis includes the steps of: the sequence information was aligned with the DNA sequences of both parents, and the embryo haplotypes were analyzed.
In a specific example, the step of comparing and analyzing further includes the steps of: the sequence information is compared with a human genome reference sequence, and SNP coverage fold and genotype are analyzed. It will be appreciated that the human genome reference sequences may be from a public database. Specifically, the human genomic reference sequence may be a human genomic reference sequence in NCBI or UCSC databases, such as hg19, hg38, etc.
It will be appreciated that the DNA sequences of both parents may also be obtained by sequencing DNA samples of both parents. Alternatively, the DNA sample of both parents is selected from one or more of peripheral blood genomic DNA, semen DNA, oral mucosa cell DNA, and cell whole genome amplification products. Preferably, the DNA content in each DNA sample is greater than 500ng.
In a specific example, the sequencing method is Ion Torrent PGM or Illumina Miseq, and library building is performed according to the standard library building flow corresponding to the method. Sequence alignment for sequencing analysis the position of the reads on the reference genome may be obtained by any sequence alignment procedure, for example alignment using BWA (Burrow-Wheeler-Aligner) available to the person skilled in the art. In the data analysis, the original data generated by a sequencer such as an Illumina sequencer can be removed by using trimomatic software, BWA software is compared with a human reference genome, and finally haplotype SNP locus coverage fold and genotype are analyzed.
The detection composition for detecting hereditary breast cancer and ovarian cancer syndrome according to an embodiment of the present invention includes a plurality of detection agents capable of detecting sequence information of the coding region of the BRCA1 gene and SNP loci upstream and downstream of the BRCA1 gene, respectively.
Alternatively, the detection agent is a PCR primer and is selected from the group consisting of SEQ ID NO: 1-SEQ ID NO:338, at least one of which is a metal foil. It is to be understood that the detection agent is not limited thereto, and may be, for example, a probe or the like.
The detection kit for detecting hereditary breast cancer and ovarian cancer syndrome comprises the detection composition and a reaction reagent for library construction.
In a specific example, the reagents used in library construction are selected from one or more of multiplex PCR polymerase, DNA ligase, end repair enzyme, dNTPs, and PCR buffer. It is to be understood that the kit is not limited thereto, and various reagents may be added or removed as needed.
In a specific example, the detection kit further comprises a tag sequence, and when the DNA molecule to be detected is derived from a plurality of samples to be detected, each sample may be tagged with a different tag sequence (barcode) for distinguishing between the samples during sequencing, thereby allowing sequencing of a plurality of samples simultaneously.
The following are specific examples.
Example 1
A female and its mother have a heterozygous mutation in the BRCA1 gene (NM-007294.3) of c.4065-4068 delTCAA, and their mother has been removed by ovarian cancer. The female application performs pre-embryo implantation genetic testing (PGT) for the pathogenic mutations described above, giving rise to healthy offspring.
The female is assisted by PGT to obtain 7 embryos (P917-JJJ-1 to P917-JJ-7), the embryos develop to the blastula stage to carry out trophoblast cell biopsy, and the biopsy cells are subjected to whole genome amplification to further detect known variations and haplotypes of BRCA1 genes.
Of the 7 embryos, 5 were normal and 2 were maternal mutation carriers. The pair of couples transplanted a normal embryo, the ultrasonic diagnosis and the genetic diagnosis before 16 weeks of pregnancy indicate that the fetus is normal in development, the genetic detection indicates that the fetal BRCA1 gene does not have the c.4065_4068delTCAA mutation, and the result is consistent with the result of PGT, and the pair of couples succeeds in gestation after the pair of couples transplant and a healthy child which does not carry the BRCA1 gene c.4065_4068delTCAA is bred.
The following is a preimplantation genetic testing procedure:
1. library construction and sequencing
The PCR primers (SEQ ID NO: 1-SEQ ID NO: 338) are used for constructing libraries of amplified products of the whole genome of the biopsy cells according to the Illumina standard library construction flow, and a Miseq sequencer is used for sequencing.
2. Alignment and statistics
The original data generated by the Illumina sequencer was stripped of the linker sequence using trimmabic software, aligned to the human hg19 reference genome using BWA software, and finally analyzed for haplotype SNP coverage and genotype.
3. Analysis of results
The detection results are shown in Table 2, wherein F0 and F1 represent normal male chromosomes; m0 represents a female risk chromosome, and M1 represents a female normal chromosome. The detection results indicate that the P917-JJJ-2, P917-JJJ-3, P917-JJ-5, P917-JJ-6 and P917-JJ-7 embryos do not inherit the risk chromosome of women.
TABLE 2 detection results
The quality control results are shown in Table 3, and the average sequencing depth is 100×or more, the 30×coverage is 70% or more, the 100×coverage is 50% or more, and the quality control is qualified.
TABLE 3 high throughput sequencing quality control
| Numbering device | Name of name | Average sequencing depth of target region (X) | Coverage degree | 30X coverage | Whether or not to pass |
| K05053 | Female party | 600.28 | 99.25 | 90.00 | Qualified product |
| K05226 | Male prescription | 602.99 | 99.01 | 90.04 | Qualified product |
| K05054 | Female father | 512.53 | 99.06 | 87.03 | Qualified product |
| P917-JJJ-1 | JJJ-1 | 433.22 | 99.58 | 89.49 | Qualified product |
| P917-JJJ-2 | JJJ-2 | 479.66 | 99.41 | 89.63 | Qualified product |
| P917-JJJ-3 | JJJ-3 | 487.35 | 99.40 | 90.71 | Qualified product |
| P917-JJJ-4 | JJJ-4 | 500.65 | 99.29 | 86.45 | Qualified product |
| P917-JJJ-5 | JJJ-5 | 578.49 | 99.16 | 90.07 | Qualified product |
| P917-JJJ-6 | JJJ-6 | 647.77 | 99.34 | 89.83 | Qualified product |
| P917-JJJ-7 | JJJ-7 | 881.98 | 99.53 | 92.48 | Qualified product |
The mutation detection results are shown in Table 4, wherein Hom indicates that the mutation site is homozygous, het indicates that the mutation site is heterozygous, heri indicates that the mutation site is hemizygous, and Normal indicates that no mutation is detected at the site. Since the design scheme covers the coding region of the BRCA1 gene, the mutation of the gene in the coding region can be effectively detected, and the sequencing depth is more than 100×.
TABLE 4 high throughput sequencing mutation detection
Effective SNP locus statistics are shown in Table 5, and more than 2 heterozygous SNP loci are respectively located within 1M on both sides of the BRCA1 gene of the male and female, so that the requirement of European Society of Human Reproduction and Embryo (ESHRE) on PGT practice guidelines (2011 edition) based on amplification is met.
TABLE 5 SNP site statistics of target genes
SNP haplotypes (the result of selecting embryo parts SNP) are shown in Table 6, wherein F0 and F1 represent male normal chromosomes; m0 represents a female risk chromosome, and M1 represents a female normal chromosome.
TABLE 6 SNP haplotypes
Example 2
The same detection method is used for detecting 24 BRCA1 carriers, and the mutation site covers 22 exons of BRCA1 pathogenic genes. The detection method provided by the invention has higher universality and accuracy through standard setting that more than 3 effective SNP are arranged on both sides of a mutation site, and the passing rate of 24 testees is 100%.
The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.
The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
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<213> Artificial sequence (Artificial Sequence)
<400> 29
aatacgggga gtgttttggt 20
<210> 30
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 30
gaaagtcatc tcctccaggg 20
<210> 31
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 31
tttgcagaaa ggcaggaaac 20
<210> 32
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 32
tcagtccctc tgctctcag 19
<210> 33
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 33
aagacagatt cagcaggagc 20
<210> 34
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 34
tgtgagagga tgagaggagt 20
<210> 35
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 35
ccacgcacct ctccataaaa 20
<210> 36
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 36
gagatcccag ttcccttctg 20
<210> 37
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 37
ggatgttctc caccgacg 18
<210> 38
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 38
cacttactcc ccagtgacg 19
<210> 39
<211> 17
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 39
ctgtcctcgc ctgacac 17
<210> 40
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 40
aagaggtagc ccagaggac 19
<210> 41
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 41
agcttgctag tgcttcagag 20
<210> 42
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 42
gggggttgtg agataaggag 20
<210> 43
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 43
aagggaggta accattctgc 20
<210> 44
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 44
tttccctgat taagccctgg 20
<210> 45
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 45
agacaatagg gtcctcccag aa 22
<210> 46
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 46
gggcagagtt tatgttgtga ttgg 24
<210> 47
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 47
gccaaaaaca caaccttcct gt 22
<210> 48
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 48
cgaagagaca gaaagtgtgt gtgt 24
<210> 49
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 49
accgtgaggt gatgagattc 20
<210> 50
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 50
acctctttag gagaagcagc 20
<210> 51
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 51
ccatagtctt ctgcaaggga 20
<210> 52
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 52
aaagttgact atccccaggc 20
<210> 53
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 53
atcacccacc tctttcccta 20
<210> 54
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 54
aaagttgact atccccaggc 20
<210> 55
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 55
tcagtacaga gacctccctt 20
<210> 56
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 56
ctgagttaag tgggactgcc 20
<210> 57
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 57
agattctcca catccaccac 20
<210> 58
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 58
gtgtgtatct tgcttggctg 20
<210> 59
<211> 15
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 59
ctctgcgctg ggagc 15
<210> 60
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 60
catggtcatt gtcctcctga 20
<210> 61
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 61
ggggtggagt ttggattttt 20
<210> 62
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 62
cctaggcttt ccctctcttg 20
<210> 63
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 63
tcttaaacct agacttgggg g 21
<210> 64
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 64
accaatctca gttaaggaag gt 22
<210> 65
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 65
ccacaggaag atgacccttt 20
<210> 66
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 66
ttgtggtgtg gaagtctagg 20
<210> 67
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 67
ggcactgttg cagatgaaaa 20
<210> 68
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 68
tttaagccct caacttcccc 20
<210> 69
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 69
tcctcaaggg acactactga 20
<210> 70
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 70
gatggaacta gatgtgggca 20
<210> 71
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 71
ccctgcagct actacttgag tc 22
<210> 72
<211> 26
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 72
actgagatac agactgggat actgag 26
<210> 73
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 73
ctatgagcat cgtgcagatg ga 22
<210> 74
<211> 28
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 74
tgtgtataaa tacttgccga aggtcaag 28
<210> 75
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 75
ctcctgcatc cagagtcac 19
<210> 76
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 76
ttctggatgt tttcttgggc 20
<210> 77
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 77
gtcgggagga aaagcaaacg tc 22
<210> 78
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 78
ccatggtgga agcaaatgat tcc 23
<210> 79
<211> 26
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 79
cccgatcaat aattatcctg ggtttg 26
<210> 80
<211> 30
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 80
ctgtttattc tttcttctct agagagagca 30
<210> 81
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 81
ctgggacgtg ttgagttcta 20
<210> 82
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 82
tcagaaggag tcatttgcca 20
<210> 83
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 83
accctgtttg gaatactcta gtgga 25
<210> 84
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 84
ctcctcaaat gtctggttga tctga 25
<210> 85
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 85
tgaactgaat gaagacgcca ttaca 25
<210> 86
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 86
tgcttacaac cttgactccc ttt 23
<210> 87
<211> 26
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 87
cccaatccct taaaacaatg aactgc 26
<210> 88
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 88
acctcattgg tttccttgtc cttg 24
<210> 89
<211> 27
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 89
ggaagtaaag cttttaaaag ccacgta 27
<210> 90
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 90
cctgctgaag ggaatggaaa gaa 23
<210> 91
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 91
ggaaccggag gttattggat 20
<210> 92
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 92
tcacctgctt catttcctgt 20
<210> 93
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 93
ggaaaaccag agagagcttc tgt 23
<210> 94
<211> 30
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 94
ggaaaacatt cacatctgga tttaactgaa 30
<210> 95
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 95
ggacttccct gtgagacatt 20
<210> 96
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 96
gtttccaaac ggggaacttt 20
<210> 97
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 97
tagggccaga aaagcttagg 20
<210> 98
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 98
tatgcagttc tgtctgctgt 20
<210> 99
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 99
taacaagcaa acagaggtgc 20
<210> 100
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 100
ccgagcagtg tgtttatgc 19
<210> 101
<211> 17
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 101
ctcctggtgc tgacgac 17
<210> 102
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 102
agcatagagg tgcgtgatg 19
<210> 103
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 103
cctttcctct ttaccccagg 20
<210> 104
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 104
ttacatatgg aggccgaagc 20
<210> 105
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 105
ccaacacagg taggcagtaa 20
<210> 106
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 106
gagaaggcag atggacagaa 20
<210> 107
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 107
catcaaggtg aacagggagg 20
<210> 108
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 108
ctcacaaact caatggggtg 20
<210> 109
<211> 26
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 109
aaataaaacc acccgttttc agatgg 26
<210> 110
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 110
gctggctaca gagagagatt gaaga 25
<210> 111
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 111
gacgccaagg aacgatagg 19
<210> 112
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 112
atttatttca ggctctgtcc ca 22
<210> 113
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 113
gtggaaaagt ttgttagccc a 21
<210> 114
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 114
tagcacctta ctccttcact g 21
<210> 115
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 115
acaccattca gcatcagtct 20
<210> 116
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 116
gggaaggaag gagagcaaaa 20
<210> 117
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 117
ctgtcactgt ggttgtccat 20
<210> 118
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 118
ggcttagagc cagattcaga 20
<210> 119
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 119
ttacctatca atcctcaccc c 21
<210> 120
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 120
ggctagactt cagtgtccgt 20
<210> 121
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 121
ccttaagtat cttactagat tggtg 25
<210> 122
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 122
gtgtgtttgt gttcacgata 20
<210> 123
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 123
ctcttccaca tttctaagcg aa 22
<210> 124
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 124
ccccagaaac ttggatagtc t 21
<210> 125
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 125
atcagttgcc agtgtcttca 20
<210> 126
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 126
gaaggaaagg aggactgagg 20
<210> 127
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 127
agaaaggtac taggtcccca 20
<210> 128
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 128
gtaggtgatc ctcttgagcc 20
<210> 129
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 129
ctcaagaagc ccagaggatt 20
<210> 130
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 130
gtctgtcagt ctgtccctg 19
<210> 131
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 131
tggataaccc agaccgaga 19
<210> 132
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 132
tttccagaac tcgaacccaa 20
<210> 133
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 133
gtaactgcca gcttccaatg 20
<210> 134
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 134
ggaaccccat aacgcagag 19
<210> 135
<211> 17
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 135
gtatgcacgc tccctgg 17
<210> 136
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 136
agctttaacc ggctatccc 19
<210> 137
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 137
gatgttttca ggaaggggga 20
<210> 138
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 138
caagggaagg agccttactg 20
<210> 139
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 139
ctggtgttcg attcaacaac gtg 23
<210> 140
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 140
ggtagtcttg ggaggtcagc tt 22
<210> 141
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 141
aatgaataca aaccacggcg 20
<210> 142
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 142
ctgcctctta cggtttggta 20
<210> 143
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 143
aggtctttga acactaggag g 21
<210> 144
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 144
ctagaaaagt ctgtccgtgt g 21
<210> 145
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 145
acgaaggtca ccagaaactg 20
<210> 146
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 146
ttgcgatcag aggacaaagt 20
<210> 147
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 147
cgcttctcct ccctggtttt g 21
<210> 148
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 148
cggaaccggg atctatttcg g 21
<210> 149
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 149
gcatcgttaa cccagattcc ctt 23
<210> 150
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 150
gccagggcaa agatggtaat ga 22
<210> 151
<211> 28
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 151
gtttacctgt tatggatgaa actgacct 28
<210> 152
<211> 29
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 152
ctgtaactaa taaaacgtag gcacagaga 29
<210> 153
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 153
gtgtgtttat ggggtttggg 20
<210> 154
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 154
gatggagcag aatgagacca 20
<210> 155
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 155
tgatcttcca gcagacagtg 20
<210> 156
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 156
ttgcagcaaa aagtcttcgt 20
<210> 157
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 157
tcaatatctt gcactctggg ttc 23
<210> 158
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 158
aggaaacgag tagattggca c 21
<210> 159
<211> 26
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 159
caactccagg tcagttaact aagtcc 26
<210> 160
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 160
ccttggagtc ctgagtggaa ac 22
<210> 161
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 161
tctgcagaaa tgggcaagtt cat 23
<210> 162
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 162
ctgaaaggca gtgggagcac at 22
<210> 163
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 163
ctgtggatat ccccttggac 20
<210> 164
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 164
aggacaccaa tgaggaaagg 20
<210> 165
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 165
gctgctggac aagctagaga tc 22
<210> 166
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 166
cttacctcag ccttctggat ctc 23
<210> 167
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 167
tcccaggtct ctcaaaaagc 20
<210> 168
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 168
ccacttcaga aaccccagt 19
<210> 169
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 169
actggggttt ctgaagtgg 19
<210> 170
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 170
gggtcatccg atctttgtct 20
<210> 171
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 171
gcggttggtt ctttctcttc 20
<210> 172
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 172
ggtgacatca tagctttccg 20
<210> 173
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 173
ggagagagta tgggaaaggc 20
<210> 174
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 174
atgctaggct tggtttgaga 20
<210> 175
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 175
caaggatcga gccaaaagc 19
<210> 176
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 176
gaaacgatga tacccttctg c 21
<210> 177
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 177
tattcctgaa aaggcaggct 20
<210> 178
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 178
tgagttgatg agtctcggtg 20
<210> 179
<211> 30
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 179
cattaatttg ctaaattgct ggctaagaca 30
<210> 180
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 180
ggctgatggg aaagagcaac at 22
<210> 181
<211> 26
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 181
gggaatggag agaaggaaaa tctagt 26
<210> 182
<211> 30
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 182
gcctcttatt aaacatacag aaggaccttt 30
<210> 183
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 183
gcctcgcctc atgtggtttt at 22
<210> 184
<211> 26
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 184
aaatagttcc aggacacgtg tagaac 26
<210> 185
<211> 30
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 185
cggccatgca attattttta ttatgaagtg 30
<210> 186
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 186
cccttgtctc acatgggtga at 22
<210> 187
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 187
gttgctcctc cacatcaaca ac 22
<210> 188
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 188
cctgctttta aacagctggg agat 24
<210> 189
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 189
gcagagggaa ggctcagata ca 22
<210> 190
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 190
cttaacttgt ttacagcgat gccaa 25
<210> 191
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 191
atgttggagc taggtcctta ctct 24
<210> 192
<211> 28
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 192
actaggtgat ttcaattcct gtgctaaa 28
<210> 193
<211> 30
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 193
ccctaatcta agcatagcat tcaattttgg 30
<210> 194
<211> 30
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 194
ggactcatta ctccaaataa acatggactt 30
<210> 195
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 195
attcctcttc tgcatttcct ggatt 25
<210> 196
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 196
ggtactgatt atggcactca ggaaa 25
<210> 197
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 197
tggttctgtt tttgccttcc 20
<210> 198
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 198
aatgctgaag accccaaaga 20
<210> 199
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 199
cttccctaga gtgctaactt ccag 24
<210> 200
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 200
atggaaggta aagaacctgc aact 24
<210> 201
<211> 27
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 201
cttgggatat tcaacactta cactcca 27
<210> 202
<211> 27
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 202
gcaatgcatt atatctgctg tggattt 27
<210> 203
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 203
gcgcttgtac ttgtcaacag tt 22
<210> 204
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 204
agaagattgg ctcttaccac ttgtc 25
<210> 205
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 205
gtggagggta ctttcccag 19
<210> 206
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 206
ttctggctga aggtggaag 19
<210> 207
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 207
agccttgaag gagatgagtg 20
<210> 208
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 208
tggatggaac taacaaggac a 21
<210> 209
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 209
ttttagagtg acattgcact gatg 24
<210> 210
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 210
tgttgaactg ggaggcatat ta 22
<210> 211
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 211
tttacctccc tggctttctg 20
<210> 212
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 212
gtccgttcag gggaacataa 20
<210> 213
<211> 27
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 213
ccctccttgt tttattgact cttcagt 27
<210> 214
<211> 27
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 214
cacatttctg atccagatca catgtct 27
<210> 215
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 215
taattggcac cgttgctttc 20
<210> 216
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 216
tgctcaatag gtgctggtag 20
<210> 217
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 217
tgtaggcaga ttggtctctt tt 22
<210> 218
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 218
aagcttgtta gcactgacgc 20
<210> 219
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 219
ccgtgggtgt aatatggtga 20
<210> 220
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 220
aaagaacgca cacactaagc 20
<210> 221
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 221
aacccggatt taagcgtgta 20
<210> 222
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 222
cgaggtgtag ttttcgaagc 20
<210> 223
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 223
aacccggatt taagcgtgta 20
<210> 224
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 224
cgaggtgtag ttttcgaagc 20
<210> 225
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 225
gtaaacgcaa cacacaacct 20
<210> 226
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 226
ccttgatcgc tggaaggatt 20
<210> 227
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 227
cgttgacaat ctcccgttag 20
<210> 228
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 228
gtggccgatc gttaacattt 20
<210> 229
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 229
aaatggccca atgcaaacac 20
<210> 230
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 230
agtacagtgg ccgatcgtta 20
<210> 231
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 231
tgcaaacacg ttgacaatct 20
<210> 232
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 232
gtacagtggc cgatcgtta 19
<210> 233
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 233
tgcaaacacg ttgacaatct 20
<210> 234
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 234
gtacagtggc cgatcgtta 19
<210> 235
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 235
ggatagtgaa acacatgcgg gaaa 24
<210> 236
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 236
caaggtggaa tgggagcttc tt 22
<210> 237
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 237
gcacgtttga caatcctgtg tct 23
<210> 238
<211> 28
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 238
atctcctgtg ctacatgcaa atatacaa 28
<210> 239
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 239
ctgggcaaca caccactgga at 22
<210> 240
<211> 26
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 240
cgggtagttt aggatagttg gtaggt 26
<210> 241
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 241
gccctccaag taaagtcgaa 20
<210> 242
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 242
tctcctggga tacaacctgt 20
<210> 243
<211> 29
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 243
acaacaacac aaaaaccaag agaaatctt 29
<210> 244
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 244
ccactggtga cgacgtaaag at 22
<210> 245
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 245
gggatgcgat ggtagtgaga at 22
<210> 246
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 246
gcctagggtt cggccttaaa aa 22
<210> 247
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 247
aagcagcata gcagcagaga tt 22
<210> 248
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 248
gctctaattg aggcctgtca caa 23
<210> 249
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 249
ccagtctctg ctggctcttt ag 22
<210> 250
<211> 30
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 250
gactttctat aaatccggtt ttctcatcca 30
<210> 251
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 251
caagcctcag gcttgtatgt ttg 23
<210> 252
<211> 28
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 252
gaggataatt aactctggaa catcaggt 28
<210> 253
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 253
aattatgagg ggccacagag 20
<210> 254
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 254
cagaactgtc tctgtccctg 20
<210> 255
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 255
cagttatgac aacaggccat gaac 24
<210> 256
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 256
gcccagatgc agagaggtta ga 22
<210> 257
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 257
ggcttcaggg taaagctatg 20
<210> 258
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 258
tctgtgagac aaactcagca 20
<210> 259
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 259
cggaactggc ttctctcttt 20
<210> 260
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 260
acctagtttc cagaaccacc 20
<210> 261
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 261
caggggagca gtaacaactt 20
<210> 262
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 262
ctaccatacc agtggacacc 20
<210> 263
<211> 29
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 263
aaacaataat tgcggaaagt gatgaaagt 29
<210> 264
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 264
tggagggaag tagcctgaag tt 22
<210> 265
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 265
gacagataga gaccttcagt gc 22
<210> 266
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 266
ggacctaagc ctagggagat a 21
<210> 267
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 267
cctctaggct cctggacaca ta 22
<210> 268
<211> 27
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 268
tgagagttct aggattctct ggtgatg 27
<210> 269
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 269
atcccagaaa cagagagcag 20
<210> 270
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 270
ctgagcctgc ctgtattcaa 20
<210> 271
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 271
tgacctggga atctgcatct ttt 23
<210> 272
<211> 28
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 272
aacctattta ccagaaatgg ctttgttc 28
<210> 273
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 273
tgactagcag atactgggga 20
<210> 274
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 274
tggcttgcag ggatacatag 20
<210> 275
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 275
accttctgac ctcaaagtgg 20
<210> 276
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 276
ctattgtgct ctgatgctgc 20
<210> 277
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 277
ctgacctcaa agtggctctg at 22
<210> 278
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 278
actggctatt gtgacctcca aag 23
<210> 279
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 279
aggccaatac aagaggtagc 20
<210> 280
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 280
gccaacttct ttacaagggc 20
<210> 281
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 281
cttccccacc cctagaaatc 20
<210> 282
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 282
acctggctaa gaacatggag 20
<210> 283
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 283
tctagattgt aagccccgtg 20
<210> 284
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 284
ggtccatatg tgattaacgc tg 22
<210> 285
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 285
gatctgttct gggactcctg tt 22
<210> 286
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 286
agtgaggcag ggatctgagt ac 22
<210> 287
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 287
agggctatcc cagcgtta 18
<210> 288
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 288
ctttaggcct tggttaggag a 21
<210> 289
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 289
cagtctttgc ttgggctatg 20
<210> 290
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 290
taatttgctc tgtggagggg 20
<210> 291
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 291
gctgaggaca gatgtcccta ct 22
<210> 292
<211> 26
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 292
agtaacttgg tagtgagaag agctga 26
<210> 293
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 293
gggcaggatt ctggttacaa 20
<210> 294
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 294
ttctcacaca cacagactgg 20
<210> 295
<211> 30
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 295
aaagaaatgg attacattct ggaaacttgc 30
<210> 296
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 296
ctgtcaaagc tatctgggtt cgat 24
<210> 297
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 297
ccaggtctac gacactcaag 20
<210> 298
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 298
gtttgcaacc ttaggagcac 20
<210> 299
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 299
ttccccaaca cagagaagac 20
<210> 300
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 300
tgacaagagt gttggaggag 20
<210> 301
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 301
cctaacctcc tagagttcac agtct 25
<210> 302
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 302
ggctgaacac caccatagga at 22
<210> 303
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 303
ggagactagg accacatcag gt 22
<210> 304
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 304
ggactcccag gtggtgagaa ta 22
<210> 305
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 305
cctgtctcta cttccagcag 20
<210> 306
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 306
tgcttgccag gactttatca 20
<210> 307
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 307
gtctccttac ctgtttggct 20
<210> 308
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 308
tccctatcca ccctgaagaa 20
<210> 309
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 309
ctgggtgttt tccctgagag 20
<210> 310
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 310
accaggccca atatcctcta 20
<210> 311
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 311
gtccttcagg agtgcagatt 20
<210> 312
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 312
tagggtaggc atcctcatca 20
<210> 313
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 313
tggccacttc cttaaacact 20
<210> 314
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 314
tcacttggaa tccctcccta 20
<210> 315
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 315
ctcaggctgt aagcaagaga 20
<210> 316
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 316
ttcagtcaca gacactgacc 20
<210> 317
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 317
gagactgtct gccttccttt 20
<210> 318
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 318
attaaattgg ggcttggtgc 20
<210> 319
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 319
atctgcagct gctgtgtg 18
<210> 320
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 320
tccctagact tccaacgaga 20
<210> 321
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 321
ttttccagga agaggtgagg 20
<210> 322
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 322
gtgccaatct ttctgaccac 20
<210> 323
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 323
cactccccct cttcatcatc 20
<210> 324
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 324
tttgtgagta cagtgggctt 20
<210> 325
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 325
ctattaccac ctcccacacc 20
<210> 326
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 326
gtctgtcgta attgttgccc 20
<210> 327
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 327
agaatgttag gcctgggaag 20
<210> 328
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 328
gtggttagct tgaggcaatc 20
<210> 329
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 329
acatgtatga cgggagcctg 20
<210> 330
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 330
atgtaaattg aatgtggctg tca 23
<210> 331
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 331
tggacggcct attatcatca 20
<210> 332
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 332
gaaaaggttg ccttcgctat 20
<210> 333
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 333
cctaggaaag gctgagttgt 20
<210> 334
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 334
acagaagcta aggttcaggg 20
<210> 335
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 335
gaatggggta taggagcctg 20
<210> 336
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 336
gcaagcccat tttctctcag 20
<210> 337
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 337
atcccactcg ggagagttc 19
<210> 338
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 338
ctcaaggggg atgtgacc 18
Claims (7)
1. Use of a detection composition for the preparation of a kit for detecting hereditary breast cancer and ovarian cancer syndrome, characterized in that said detection composition consists of SEQ ID NO: 1-SEQ ID NO:338, and a PCR primer composition shown in the specification;
the use method of the detection composition comprises the following steps:
obtaining genomic DNA of an embryo at a pre-implantation cleavage stage or blastocyst stage;
detecting sequence information of a BRCA1 gene coding region and SNP loci on the upstream and downstream of the BRCA1 gene in the genome DNA, wherein the step of detecting the sequence information comprises the following steps: multiplex PCR is carried out on the genome DNA by using the detection composition to obtain an amplification product, and sequencing is carried out on the amplification product to obtain the sequence information;
the sequence information is aligned and analyzed to determine whether the embryo has hereditary breast cancer and ovarian cancer syndrome.
2. The use according to claim 1, wherein the step of aligning analysis comprises the steps of: and (3) comparing the sequence information with the DNA sequences of the parent two parties, and analyzing the haplotype of the embryo.
3. The use according to claim 2, wherein the step of aligning analysis further comprises the steps of: and comparing the sequence information with a human genome reference sequence, and analyzing SNP coverage fold and genotype.
4. Use according to any one of claims 1 to 3, wherein the step of obtaining said genomic DNA comprises the steps of: trophoblast cells are collected from embryos that develop to the blastomeres or blastocysts stage and the DNA in the trophoblast cells is subjected to whole-genome amplification.
5. A detection composition for detecting hereditary breast cancer and ovarian cancer syndrome, comprising a plurality of detection agents capable of detecting sequence information of a BRCA1 gene coding region and SNP sites upstream and downstream of BRCA1 gene, respectively, said plurality of detection agents consisting of SEQ ID NO: 1-SEQ ID NO:338.
6. A test kit for the detection of hereditary breast cancer and ovarian cancer syndromes comprising the test composition of claim 5 and a reagent for library construction.
7. The test kit of claim 6, wherein the reagents are selected from one or more of multiplex PCR polymerase, DNA ligase, end repair enzymes, dNTPs, and PCR buffers.
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