CN111139297A

CN111139297A - Kit for preimplantation embryo genetic diagnosis and prenatal diagnosis of DMD

Info

Publication number: CN111139297A
Application number: CN202010255720.8A
Authority: CN
Inventors: 乔杰; 闫丽盈; 陈伟; 朱晓辉
Original assignee: Peking University Third Hospital Peking University Third Clinical Medical College
Current assignee: Peking University Third Hospital Peking University Third Clinical Medical College
Priority date: 2020-04-02
Filing date: 2020-04-02
Publication date: 2020-05-12
Anticipated expiration: 2040-04-02
Also published as: CN111139297B

Abstract

The invention discloses a kit for preimplantation embryo genetic diagnosis and prenatal diagnosis of DMD. The kit comprises a DMD gene-linked SNP site capture probe set, and the DMD gene-linked SNP site refers to DMD There are more than 199 SNPs with high mutation frequency in the population within 2M of each gene upstream and downstream. The probe set in the kit of the invention can simultaneously capture DMD gene and DMD gene-linked SNP quickly and accurately, and can improve the detection specificity and accuracy of preimplantation genetic diagnosis of DMD.

Description

Kit for preimplantation embryo genetic diagnosis and prenatal diagnosis of DMD

Technical Field

The invention belongs to the field of gene detection and molecular genetics, and particularly relates to a kit for preimplantation embryonic genetics diagnosis and prenatal diagnosis of DMD.

Background

Pseudohypertrophic muscular dystrophy (DMD), or progressive Duchenne muscular dystrophy, is one of the most common lethal X-linked recessive neuromuscular genetic diseases. The incidence of DMD is 1/3500 for live born male infants, with about 65% of all cases genetically related. The clinical manifestations of the disease are mainly progressive skeletal muscle atrophy and weakness at the proximal end of limbs and gradual loss of the activity, and 90% of the children patients have pseudohypertrophy of muscles, with gastrocnemius muscle being the most obvious; most children are accompanied by myocardial damage, and some patients are also accompanied by intellectual disability. Patients get ill from 3-5 years old, lose walking ability around 12 years old, and die due to respiratory tract infection and heart failure and exhaustion at 20-30 years old; becker Muscular Dystrophy (BMD) is a benign subtype of DMD, has the morbidity of 1/30000, and has similar clinical manifestations to DMD but a light disease condition, a late onset age, a slow progression rate and normal intelligence, and the survival period of more than 50 years. The DMD/BMD patients are mostly without obvious abnormality at birth and lack effective treatment means at present, so that genetic diagnosis is carried out on probands and carriers, and on the basis, genetic diagnosis (PGD) before embryo transplantation or prenatal diagnosis is carried out, so that the prevention of the birth of the children patients is a key measure for avoiding and reducing the occurrence of pseudohypertrophic muscular dystrophy in offspring and lightening the burden of families and society caused by the occurrence.

DMD/BMD is caused by the mutation of the DMD gene in the Xp21.2 region of the X chromosome, and the Dystrophin protein (Dystrophin protein) encoded by the DMD gene can protect and stabilize muscle fibers. The DMD gene is the largest human gene known at present and contains 79 exons. The mutation has high incidence and various mutation types, and the statistics of various mutations can reach more than 4000. Wherein, the deletion mutation of the exon accounts for about 60-70% of all mutations of the DMD, the repetitive amplification mutation of the exon accounts for about 5-10%, and in addition, 25-35% of the deletion mutation of the exon is related to the micro mutation of the DMD gene.

The detection of different mutation types of DMD is usually carried out by combining qPCR method with Sanger sequencing, Multiple Ligation Probe Amplification (MLPA), multiple PCR or microarray comparative genomic hybridization (a-CGH) and other technologies. However, the qPCR method can only verify known deletion mutation and cannot detect deletion of an unknown region; the MLPA technology has lower flux, and a PCR method is still needed to be used for further verification experiments for single exon deletion; the detection precision of a-CGH is relatively limited, and the precise site and fragment size of exon deletion repeat cannot be determined; the DMD gene probe capture technology is combined with the low-depth NGS, so that the flux is improved, and meanwhile, various DMD mutation types and mutation positions can be accurately detected. However, single detection of mutations in the DMD gene itself still leads to misdiagnosis when performing pre-implantation embryonic genetic diagnosis due to the presence of Allelic Dropout (ADO) in a small number of cellular genomic assays. In addition to detecting the target gene clinically, SNP or STR genetic markers around the target gene are often additionally detected, and linkage analysis is performed by combining family genetic linkage relation to improve the accuracy of carrying diagnosis of the mutation of the embryo to be implanted. In addition, evaluation of chromosomal ploidy Variation (CNV) is an important component in genetic diagnosis before embryo implantation, and only embryos with normal chromosomal ploidy can be used for subsequent transplantation.

Therefore, the existing detection technology has certain defects in DMD mutation detection and DMD gene genetic diagnosis before embryo implantation, and the method capable of simultaneously and accurately detecting multiple mutation types of genomes CNV and DMD and linked genetic markers thereof is needed in the field, so that the method can simultaneously meet the requirements of gene diagnosis, prenatal diagnosis and prenatal genetic diagnosis and is convenient for clinical application and popularization.

No existing kit can simultaneously meet the detection requirements of gene diagnosis, prenatal diagnosis and pre-implantation genetic diagnosis in one detection; and also meets the requirements of low cost. Therefore, there is a need in the industry for: one kit is suitable for family verification, PGD and amniotic fluid detection.

Prior art CN110541025A (published 2019, 12/06/l) also discloses a DMD genetic defect detection method, but it uses 1246 primer set. The detection precision is often reduced due to potential mutual interference between primers in the PCR process. Secondly, since the patent document provides a way to reconstruct a library based on PCR amplification, the amplified product is not suitable for the detection of CNV in an embryo sample. Furthermore, the patent document describes the determination of linked SNPs around the DMD gene based solely on public databases, which are well known in the art, but does not take into account the amplification bias of genomic regions during amplification of a small number of cellular genomes, which results in difficult or insufficient detection of SNPs located in low amplification regions and increases the possibility of misreading in downstream analysis. Therefore, the technical problem to be solved by those skilled in the art is how to select the capture probe set of the DMD gene and its linked SNP sites to improve the detection accuracy and provide a kit suitable for embryo sample detection.

Disclosure of Invention

The invention aims to provide a capture probe group and a kit for pre-implantation embryonic genetic diagnosis and prenatal diagnosis of DMD, which can simultaneously meet the detection of genetic diagnosis, prenatal diagnosis and prenatal genetic diagnosis. The invention combines the DMD gene capture sequencing technology, and can simultaneously detect the genome ploidy of multiple samples, the DMD gene and the adjacent SNP sites thereof. By optimizing the chain SNP capture probe, the efficiency and the stability of capturing the amplification product of the embryo sample are improved. The kit is suitable for prenatal diagnosis, can meet the detection requirement of trace samples in the genetic diagnosis of the embryo before implantation, and has stronger universality clinically.

To achieve the above objects, the present invention first relates to a kit for prenatal and prenatal diagnosis of DMD, the kit comprising a DMD gene-linked SNP site capture probe set, which comprises oligonucleotide probes having base sequences shown in SEQ ID Nos. 1-199, and does not comprise other oligonucleotide probes.

In a preferred embodiment of the present invention, the DMD gene-linked SNP site capture probe set comprises 199 oligonucleotide probes, wherein the 199 oligonucleotide probes have the sequences shown in SEQ ID nos. 1 to 199.

In a preferred embodiment of the invention, the oligonucleotide probes are all single-stranded DNA.

In a preferred embodiment of the invention, the oligonucleotide probes are each labeled with biotin.

In a preferred embodiment of the invention, the kit further comprises linked SNP site capture probes located in a small number of cell genome amplification preferential regions in the upstream and downstream 2M internal regions of the DMD gene, an enrichment buffer BL, an enrichment buffer HY, a library enrichment binding buffer, a washing buffer WB1, a washing buffer WB3, a library enrichment eluent, a buffer NE, a PCR reaction solution, and a product purification eluent.

In another aspect, the invention also relates to the use of the above-mentioned kit for the preparation of a kit for the prenatal and prenatal genetic diagnosis of DMD. Preferably, the kit is applied to family verification, embryo genetic diagnosis before implantation and amniotic fluid detection at the same time.

The operation process for prenatal diagnosis of the DMD gene provided by the invention comprises the following steps:

1) designing capture probe set for DMD gene and SNP (single nucleotide polymorphism) linked with DMD gene

2) Extraction, fragmentation and library construction of genomic DNA

3) Capture enrichment of fragments containing a region of interest

4) Gene sequencing: high throughput sequencing of captured library fragments

5) And (4) biological information analysis.

The method comprises the following steps: firstly, extracting 3-5 ug of DNA from tissue cells such as amniotic fluid, peripheral blood or muscle and the like, and randomly breaking the DNA into fragments with the size of 200-300bp to construct a whole genome DNA library; and then carrying out hybridization incubation on the DMD gene carrying the biotin marker and the capture probe containing the DMD-linked SNP site developed by the user and the constructed library of the sample to be detected, wherein the library fragment containing the DMD gene and the library fragment containing the DMD-linked SNP site can be specifically hybridized with the capture probe. And then, through the combination of biotin on the probe and streptavidin on the magnetic beads, further washing to remove library fragments without DMD linkage SNP sites, and then eluting by using an enrichment eluent to realize the enrichment of the target fragments. Different individual sample libraries are distinguished by different sequence tags, so that multiple samples can be captured simultaneously. And performing high-throughput sequencing on the libraries before and after enrichment by using a sequencer of a second-generation sequencing platform such as Illumina HiseqX Ten, NovaSeq6000 and the like, wherein the sequencing average depth of each sample is 1X times of the genome at the lowest, and the total sequencing data amount of two libraries of each sample is 3G base. Comparing the obtained off-machine data to a reference genome after quality control, and performing DMD gene mutation, deletion, amplification analysis process and SNPs analysis.

The invention provides a method for performing high-throughput sequencing after capturing DMD genes and DMD gene linkage SNP (single nucleotide polymorphism) of a family sample carrying DMD defective genes simultaneously and performing genetic diagnosis before implantation of embryos of the DMD genes by combining haplotype SNP linkage analysis, which comprises the following steps:

(1) obtaining of embryonic cell sample: obtaining a fertilized egg through single sperm injection, culturing until the blastocyst stage, and separating the outer trophoblast cells to obtain a sample containing 3-10 cells;

(2) whole genome amplification: adding lysis solution into a sample, putting the sample into a PCR instrument for lysis and inactivation of protease, carrying out whole genome amplification on the obtained cell lysis sample, and realizing by adopting a multiple annealing circular amplification technology (MALBAC); specifically, pre-amplification mixed liquor is added into a cell lysis sample to carry out a first round of linear amplification, then amplification mixed liquor is added to carry out a second round of exponential amplification, and an amplification product is purified; the products after MALBAC amplification are between 300-2000 bp.

Wherein, the conditions of the first round of linear amplification are as follows:

(1) reacting at 94 ℃ for 3 min;

(2) reacting at 20 ℃ for 40 s;

(3) reacting at 30 ℃ for 40 s;

(4) reacting at 40 ℃ for 30 s;

(5) reacting at 50 ℃ for 30 s;

(6) reacting at 60 ℃ for 30 s;

(7) reacting at 70 ℃ for 4 min;

(8) reacting at 95 ℃ for 20 s;

(9) reacting at 58 ℃ for 10 s;

(10) and (5) repeating the steps (2) to (9) for 8 cycles.

The conditions for the second round of exponential amplification were:

(1) reacting at 94 ℃ for 30 s;

(2) reacting at 94 ℃ for 20 s;

(3) reacting at 58 ℃ for 30 s;

(4) reacting at 72 ℃ for 3 min;

(5) repeating the steps (2) to (4) for 17 cycles;

(6) the amplified product was stored at 4 ℃.

(3) Obtaining a family genome sample: extracting peripheral blood of members of the family members of the group, and extracting genome DNA of the members; extracting a genomic DNA sample of a tissue related to the president (if any);

(4) constructing a library before enrichment: 2ug of whole genome amplification product or family member genome DNA is taken as initial amount to construct NGS library which is a library before enrichment;

(5) hybridization enrichment of fragments containing the DMD gene and the SNP sites linked with the DMD: after the capture probe and the library are hybridized and incubated, the capture probe can specifically hybridize with the DMD gene and a related sequence containing the DMD-linked SNP site, biotin on the probe can be combined with streptavidin on a magnetic bead, and library fragments without the DMD-linked SNP site can be removed by further washing, so that library fragments containing the DMD gene and the DMD-linked SNP site are enriched. The enriched library is generally amplified again to meet the requirements of the subsequent order machine;

(6) sequencing the library before and after sample enrichment by adopting a high-throughput sequencing platform, wherein the sequencing platform can be a sequencer of a second-generation sequencing platform such as Illumina HiseqX Ten, NovaSeq6000 and the like;

(7) bioinformatics analysis: after the machine is off, the data are compared to a reference genome after quality control, and DMD gene mutation, deletion, amplification analysis process and SNPs analysis are carried out. For accurate single gene pathogenic site, SNP linkage information analysis, the average sequencing depth of each specimen is at least 1X of the genome, namely the total sequencing data amount of two libraries of each sample is 3G base.

The specific operation of carrying out haplotype SNP linkage analysis on the embryo sample by combining family genetic information is as follows:

(1) obtaining the SNPs of the target region by using software, wherein the software comprises at least one of, but is not limited to, samtools mpileup, GATK, FreeBayes and VarScan;

(2) SNP filtration: the coverage of specific SNP sites in a single sample is at least 20, the heterozygosity of the sites is 0-5% and 95-100% when the sites are determined to be pure, the heterozygosity of the sites is 30-70% when the sites are heterozygotes, and potential error SNPs are removed;

(3) screening for differential SNP, and constructing male parent and female parent SNP-haplotype: in the same SNP locus, 1 base in 4 allelic bases of a male parent and a female parent is different from other 3 bases, and the discrimination can be realized;

(4) analysis of SNP-haplotypes: the number of the embryo SNP-haplotypes is 2, the embryo SNP-haplotypes are respectively inherited from 1 male parent and 1 female parent, and specific SNP-haplotypes are judged which is father source inheritance and which is mother source inheritance according to the distinguishing type SNP and Mendelian inheritance principle; the minimum number of the differential type SNP is 10, if more than 3 SNP errors exist, the embryo SNP-haplotype data is regarded as insufficient data volume and is difficult to analyze;

(5) and (4) analyzing results: and (4) determining the SNP-haplotypes of the male parent and the female parent according to the step (4), distinguishing the haplotype linked with the pathogenic locus, comparing the specific alleles of the SNP where the embryo is located, and judging whether the embryo carries the pathogenic locus or not according to the Mendel inheritance principle.

The invention has the following beneficial effects:

the method has high sensitivity, and because the method is used for capturing and sequencing in a DNA library mode, partial degradation of the sample DNA hardly influences the final result. And the excessive probes ensure the complete capture of the target fragments, so that certain low-abundance variant DNA can be captured and detected, and the sensitivity is much higher than that of the traditional Sanger sequencing. This provides a better platform for comprehensive detection and accurate prenatal diagnosis of the DMD gene or genetic diagnosis before embryo implantation.

The probe set can be used for quickly and accurately capturing the DMD gene and the SNP linked with the DMD gene at the same time, and can improve the detection specificity and accuracy of genetic diagnosis before implantation of the DMD embryo.

The invention optimizes the selection of the DMD gene linkage SNP capture probe by utilizing the amplification preference characteristics of a small amount of cell genomes, and improves the efficiency and the stability of the linkage SNP capture probe for capturing the amplification products of the embryo samples.

The invention realizes that DMD genetic variation and SNP locus linkage analysis are simultaneously carried out on a plurality of samples in one capturing period by introducing different barcode to different samples for distinguishing during library construction. The method has the advantages of low sequencing depth, high detection flux, no need of additional special primer or probe design for different mutations of the DMD gene, simple operation, suitability for prenatal diagnosis besides meeting the detection requirements of trace samples in PGD, and strong universality in clinic.

Drawings

The invention is further described with reference to the accompanying drawings and the embodiments.

FIG. 1 is a genetic map of pedigree 1 in example 3 according to the present invention.

FIG. 2 is an electrophoretogram of PCR products of DMD exon deletion regions of pedigree 1 in example 3 according to the present invention.

FIG. 3 is a diagram of the genomic CNV of each embryo sample in example 3 according to the present invention.

FIG. 4 shows the result of DMD gene mutation analysis by whole gene capture sequencing of target gene in example 3 of the present invention.

FIGS. 5 (A) to (C) are electrophoretograms of PCR products of exon deleted region of DMD gene of each embryo in example 3 of the present invention.

FIG. 6 is a genetic map of pedigree 2 in example 4 according to the present invention.

FIG. 7 is an electrophoretogram of PCR products of DMD exon deletion regions of pedigree 2 in example 4 according to the present invention.

FIG. 8 is a diagram of the genomic CNV of each embryo sample in example 4 according to the present invention.

FIG. 9 is an electrophoresis diagram of PCR products of DMD exon deletion regions of pedigree 2 and amniotic fluid samples in example 4 of the present invention.

FIG. 10 is the electrophoresis chart of the PCR product of the deleted region of the DMD gene exon from each embryo in example 4 of the present invention.

Detailed Description

The invention is further described in the following detailed description in conjunction with specific examples, which are intended to be illustrative rather than limiting, and that the methods and reagents used in the invention, as well as related reagents, can be varied and substituted to achieve the same technical results.

The experimental procedures, in which specific conditions are not specified, in the following examples were carried out according to the routine procedures in the art or according to the conditions suggested by the manufacturers.

Example 1: design and preparation of Probe sets of the invention

1. Screening of SNP sites linked to DMD gene

Based on low-depth sequencing data of MALBAC amplification products of sequenced healthy embryo cells and genetically abnormal embryos, 199 linked SNP sites in a small number of cell genome amplification preference areas in upstream and downstream 2M areas of the DMD gene are screened out according to the following steps. The specific screening process and criteria were:

1) for each existing sequencing sample, homogenizing the reads coverage of the coverage sites according to the sequencing depth of the sample, and selecting the sites with the coverage exceeding an ideal threshold (namely the coverage times are at least more than 3);

2) selecting sites with the frequency of more than 80% in all samples (in the example, selecting MALBAC post-amplification and pre-sequencing data of 24 embryos in total), and merging adjacent sites, wherein the obtained region is determined as a small amount of cell genome amplification preference region;

3) an SNV information recording file (dbSNP 144) provided by 1000 genome project was selected (reference: 1. auton, A., Abecasis, G., Altsuhler, D. et al. A global reference for human genetics. Nature 526, 68-74 (2015). https:// doi.org/10.1038/Nature15393 or database of Single Nucleotide Polymorphisms (dbSNP). Bethesda (MD). NationallCenter for Biotechnology Information, Nationalllibrary of medicine: { build 144}) Available: http:// www.ncbi.nlm.nih.gov/SNP) determining SNPs located in a small number of cell genome amplification preference regions as amplification preference regions SNPs;

4) selecting an SNV information recording file provided by a 1000 genome project, determining SNPs positioned in a repeat region of a genome and 100bp regions at the upstream and downstream of the repeat region, and determining the SNPs after filtering and removing the sites from the SNPs in the amplification preference region as potential probe SNPs sites;

5) 199 potential probe SNPs with highest variation frequency in the population within 2M of the upper and lower reaches of the DMD gene are selected to be determined as the SNP sites linked with the DMD gene.

6) According to the selected SNP sites of the small amount of cell genome amplification preferential regions positioned within 2M of the upper stream and the lower stream of the DMD gene, a probe sequence can be designed and synthesized by a known and general method by a person skilled in the art, and the length of the probe sequence is 120 bp. In this example, the synthesized probe sequence was labeled with biotin, specifically (see also the method in patent WO 2013/003585): a large amount of designed probes (MyGenosics. US) are synthesized by adopting an in-situ synthesis technology, and a large amount of probes with biotin labels are amplified by utilizing a PCR method to form a DMD gene-linked SNP capture probe set.

The PCR method specifically comprises the following steps:

the synthesized probe was uniformly mixed in dH of a total volume of 1.2ml₂In O, 15. mu.l of the total amount was subjected to PCR amplification in three tubes using a universal PCR primer (5 '-end sequence GACTACATGGGACAT, 3' -end sequence GGAACCTACGACGTA, primer GACTACATGGGACAT being a biotin-labeled primer).

PCR amplification System: substrate DNA, 5. mu.l; forward primer (25 μ M), 2 μ l; reverse primer (25. mu.M), 2. mu.l; MgCl2(50mM), 4. mu.l; 10X Platinum Taq buffer (from Life Technologies), 5. mu.l; dNTPs (10 mM each), 4. mu.l; platinum Taq (5U/. mu.l, from Life Technologies), 1. mu.l; H2O, 27 μ l; the total volume was 50. mu.l.

Amplification conditions: 30s at 98 ℃; (98 ℃, 30s, 60 ℃, 25s, 72 ℃,45 s)35 cycles; 72 ℃ for 5 min.

After the PCR product was purified using MinElute PCR purification kit (from Life Technologies), 500ng of PCR product was bound using MyOne avidin magnetic beads (from Invitrogen). Then adding alkaline NaOH to denature and elute the complementary strand without biotin; the whole magnetic beads were then washed with a 100 ℃ formamide liquid to separate the probes from the magnetic beads. And precipitating with ethanol to obtain the biotin-labeled probe set of the embodiment, wherein the base sequence of the probe set is shown as SEQ ID No. 1-199.

Example 2: kit composition, preparation and use of the invention

The detection kit for the preimplantation genetic diagnosis and the prenatal diagnosis of the embryo of the DMD gene is a kit for performing molecular genetic detection by simultaneously detecting the mutation of the DMD gene and the SNP site linked with the DMD gene.

The kit comprises the following components: the probe set obtained in example 1, DMD whole gene capture probe set (cf. CN 106834507 a), buffer HY, buffer BL, library enrichment binding buffer, washing buffer WB1, washing buffer WB3, buffer NE, PCR reaction solution, and product purification eluent. The specific composition is shown in table 1. TABLE 1

The using method of the kit comprises the following steps:

sample library preparation:

a) ultrasonic fragmentation: the starting amount (product of MALBAC amplification of cellular genomic DNA) was 3. mu.g, diluted to 30 ng/. mu.L with 1 Xlow TEBuffer. Ultrasonic fragmentation is carried out by adopting a Covaris S2 ultrasonic instrument, the value of a Covaris system is set according to the standard, 6 times of circulation is multiplied by 60S, the temperature of a water bath is 5 ℃, the duty ratio is 20%, the intensity is 5, and the mode is Frequency shocking.

b) Filling the tail end in a plain mode: mu.L of fragmented DNA, 8. mu.L of dNTPs, 2. mu.L of End Polishingase I (10U/. mu.L, Agilent), 16. mu.L of End Polishingase II (5U/. mu.L, Agilent) were each added with water to a total volume of 200. mu.L. Incubate at 25 ℃ for 30 min. The DNA was purified using the PureLink PCR purification kit (Invitrogen).

c) Connecting the P1 and P2 linkers: illumina linker 1(P1eA) 50. mu. mol/L and linker 2(P2eA) 50. mu. mol/L each 26. mu.L of (Illumina), blunt-ended DNA 48. mu.L, T4 DNA ligase 10. mu.L (50U), water to a total volume of 200. mu.L, and incubation at room temperature for 15 min.

d) And (3) recovering the DNA fragments: using a pre-made 2% SizeSelect Gel (Applied Biosystems), it was placed on the E-Gel iBase. And adding the connected and purified DNA fragments into the sample loading holes in 20 mu L portions respectively in 3 portions, filling the molecular weight control with 0.2 mu g of 50bpladder, filling the non-sample holes and the recovery holes with 20 mu L of water and 25 mu L of water respectively, performing electrophoresis for about 12min, and sucking out the DNA fragments between 150 and 200bp entering the sample recovery holes.

e) Notch translation: the recovered purified fragments required a balanced linear amplification of the amount of library template using nick translation. For each 100. mu.L of recovered sample, 400. mu.L of master mix (Agilent) was added and the reaction was programmed: 20min at 72 ℃ and 5min at 95 ℃; then 10 to 12 cycles are carried out at 95 ℃ for 15s, 54 ℃ for 15s and 70 ℃ for 1 min; re-extension at 70 deg.C for 5 min; storing at 4 ℃. The product was purified and quantified, and 1. mu.L of the sample product was subjected to Flash Gel (2.2%, Lonza) electrophoresis for about 10 min.

2. Sample enrichment and hybridization:

(1) preparing a hybridization solution: mu.g of the constructed DNA gene library, 13ul of BL buffer, 5. mu.l of GenCapTM prepared probe were mixed well and placed at 95 ℃ for 7 minutes, 65 ℃ for 2 minutes, and 23. mu.l of preheated hybridization buffer HY (preheated at 65 ℃ and shaken well before use to resuspend the pellet) was added. Hybridization was carried out for 22 hours at 65 ℃ on a PCR instrument.

The volume of the buffer HY was adjusted according to the volume of the library prepared, and the adjustment ratio was HY buffer volume to total volume/1.8

(2) And (3) purification: the buffer used in this step was left at room temperature, except for the washing buffer WB 3.

1) A 1.5ml centrifuge tube was added with 50ul MyOne magnetic beads, shaken vigorously for at least 5 seconds, centrifuged briefly to collect the liquid on the tube wall, and the tube placed in a magnetic rack (for example: DynaMag) for 1 minute;

2) keeping the centrifugal tube on the magnetic frame, and discarding the clear liquid;

3) taking down the centrifugal tube, adding 50ul of 1X Binding buffer solution, violently shaking for at least 5 seconds, centrifuging for a short time to collect liquid on the tube wall, placing the centrifugal tube on a magnetic frame for 1 minute, keeping the centrifugal tube on the magnetic frame, and discarding clear liquid; three repetitions (three washes total);

4) resuspending the precipitate with 50ul 1 Xbinding buffer, shaking vigorously for at least 5 seconds, and collecting the liquid on the tube wall in a gentle spin manner;

5) adding an equal volume of Binding buffer to the hybridization solution, and transferring into the solution obtained in step 4). (final total volume about 200 ul);

6) violently shaking the mixed solution for at least 5 seconds, and placing the mixed solution on a rotary blending instrument for one hour at room temperature;

7) violently shaking the mixed solution for at least 5 seconds, centrifuging for a short time, and placing the mixed solution on a magnetic frame for 1 minute;

8) keeping the centrifugal tube on a magnetic frame, and discarding clear liquid;

9) adding 0.5ml of washing buffer WB1, violently shaking and mixing uniformly for at least 5 seconds, and placing on a rotary mixer for 15 minutes at room temperature;

10) taking down the centrifugal tube, violently shaking for at least 5 seconds, centrifuging for a short time, and placing on a magnetic frame for one minute;

11) keeping the centrifugal tube on a magnetic frame, and discarding clear liquid;

12) adding 0.5ml of washing buffer WB3, and shaking vigorously for at least 5 seconds;

13) the tube was placed in a thermostatic mixer (65 ℃, 850rpm) for 15 minutes;

14) repeat step 10-13 four times (5 washes of WB3 buffer total);

15) violently shaking for 5 seconds, centrifuging for a short time, and placing on a magnetic frame for one minute;

16) keeping the centrifugal tube on a magnetic frame, and completely discarding clear liquid;

18) adding 50 μ l of Elute buffer solution to resuspend the precipitate, shaking vigorously for at least 5 seconds, and placing on a rotary mixer for at least 10 minutes;

19) taking down the centrifugal tube, shaking and uniformly mixing for 5 seconds, centrifuging for a short time, and placing the centrifugal tube on a magnetic frame for 1 minute;

20) transferring the clear solution to a 1.5ml centrifuge tube with 70ul of NE buffer solution;

21) the resulting solution was purified using QIAquick MinElute kit to obtain a final volume of 20. mu.l template DNA.

3. Sample enrichment

(1) And (3) PCR reaction system: primer A is Illumina forward primer PCR PE 1.0, primer B is lluminar primer PCR PE 2.0, and enzyme is Hotstart Phusion enzyme (New England Biolabs);

(2) PCR cycling conditions: pre-denaturation at 98 ℃ for 30 seconds; 15 cycles: 25 seconds at 98 ℃ and 30 seconds at 65 ℃ and 30 seconds at 72 ℃ for 30 seconds; 5 minutes at 72 ℃; 4-1 h;

(3) the PCR product was purified by NucleoSpin purification kit, and the final product was eluted twice with 17.5. mu.l of 65 ℃ preheated Elute buffer, finally obtaining 35ul of enriched DNA;

(4) the product is subjected to capillary electrophoresis and the concentration measurement by the Qubit, and then quantitative PCR is carried out.

4. Sequencing the obtained DNA by using a secondary high-throughput sequencing platform such as Illumina HiSeq X Ten to obtain sequencing data.

Example 3: verification of Using Effect of the kit of the present invention

The present invention will now be described more fully hereinafter with reference to specific embodiments thereof. The exemplary embodiments of the present invention and the description thereof are provided to explain the present invention and not to limit the present invention.

The child in pedigree 1 suffered from Duchenne muscular dystrophy (DMD, Exon31-43 loss), X-linked recessive inheritance. Female is the carrier (DMD, Exon31-43 heterozygous deletion) and male is normal. The specific genetic map is shown in FIG. 1

FIG. 2 is an electrophoretic map showing exon deletion in the DMD gene of an example pedigree. The exon loading sequence is: MALBAC1, MALBAC2, MALBAC3, mother (carrier), father, president (DMD Exon31-43 deletion) showed that president carried DMD gene Exon31-43 homozygous deletion mutations.

1. Taking human embryo single cells:

1.1 fertilization of sperm and egg

Injecting single sperm (ICSI) into the ovum in MII stage, placing the ovum into (G-MOPS) operating solution, transferring to the platform of micromanipulator, and performing micromanipulation.

1.2 in vitro culture of embryos

Culturing the spermatogonium in G1 culture solution (vitrolite) or Gm culture solution (Global) for about 72 hours to 5-8 cell stage, perforating on a transparent tape by laser, transferring to balanced G2 culture solution (vitrolite) or Gm culture solution (Global), continuously culturing to the blastocyst, and separating the incubated ectotrophoblast cells into a small cluster containing 3-10 cells by laser.

2. MALBAC amplification and product purification

A single cell whole genome amplification kit (MALBAC single cell whole genome amplification kit (KT 110700150/YK 001B)) produced by Yikang company is used for carrying out whole genome DNA amplification of a small amount of embryonic cells.

2.1 lysis of cells

The embryonic cells were transferred to a PCR tube containing 5ul of lysate, lysed in a PCR instrument at 50 ℃ for 50 minutes.

2.2 inactivation of proteases

The PCR tube was inactivated in a PCR instrument at 80 ℃ for 10 minutes.

2.3 first round Linear amplification

mu.L of the pre-amplification mix was added to 5. mu.L of the cell lysis sample.

Temperature process: 3 minutes at 94 ℃, 8 × (20 ℃ C. -40 seconds, 30 ℃ C. -40 seconds, 40 ℃ C. -30 seconds, 50 ℃ C. -30 seconds, 60 ℃ C. -30 seconds, 70 ℃ C. -4 minutes, 95 ℃ C. -20 seconds, 58 ℃ C. -10 seconds).

2.4 second round exponential amplification

Mu.l of the amplification mixture was added to 35. mu.l of the pre-amplification mixture.

Temperature process: 94 ℃ for-30 seconds, 17 × (94 ℃ for-20 seconds, 58 ℃ for-30 seconds, 72 ℃ for-3 minutes), stored at 4 ℃.

2.5 purification of genomic DNA amplification products from embryo samples

Single cell or equivalent DNA may be amplified by single cell amplification reaction to obtain 2-4. mu.g of amplification product ranging between 250 and 2000bp per 65. mu.L of reaction system, and purification of the MALBAC product is performed using a commercial purification kit such as DNA Clean & concentrate-5 (VISTECH cat No. DC2005).

3. Family member genome DNA extraction

Genomic DNA from peripheral Blood of family members or tissues of president infants was extracted using a commercial genomic DNA extraction Kit such as QIAamp DNA Blood Mini Kit (250) (Cat No. 51106).

4. As shown in example 2, using the kit of the invention: the detection kit for embryo preimplantation genetic diagnosis and prenatal diagnosis of the DMD gene is used for performing library construction, hybridization capture and enrichment on an embryo genome product and family member genome DNA after amplification and purification of the MALBAC.

5. High throughput sequencing

And sequencing the sample by adopting a high-throughput sequencing platform. The sequencing platform is not particularly limited, and second generation sequencing platforms include, but are not limited to, GA, GAII, GAIIx, HiSeq1000/2000/2500/3000/4000, X Ten, XFIve, NextSeq500/550, MiSeq, SOLID by Applied Biosystems, 454FLX by Roche, Ion Torrent, Ion PGM, Ion Proton I/II by thermo Fisher scientific (Life technologies); the third generation of single molecule sequencing platform: including but not limited to the HeliScope system from Helicos BioSciences, the SMRT system from Pacific BioSciences, Gridios, MinION from Oxford Nanopore Technologies. The type of sequencing can be Single ended (Single End) sequencing or double ended (Paired End) sequencing. Each specimen was sequenced at an average depth of 1X.

6. Data analysis

6.1 reference sequence alignment

And (4) removing the joint and low-quality data from the sequencing result, and aligning to a reference genome. The reference genome may be a whole genome, an arbitrary chromosome, a part of a chromosome, a gene. Reference genome usually the gene sequence for which it is generally accepted that the sequence can be determined as NCBI or UCSC is selected as the reference sequence (e.g. human Hg38), or any chromosome. The Alignment software may be any free or commercial software, such as BWA (Burrows-Wheeler Alignment tool), SOAPaligner/soap2(ShortOligonucleotide Analysis Package), Bowtie/Bowtie 2.

6.2 genomic CNV analysis

And (3) comparing the off-line data with a reference sequence (such as human Hg38) after quality control, analyzing the data of the same batch of embryos together according to the coverage degree, and determining the genome CNV condition of each embryo.

FIG. 3 is a genomic CNV map (10 Mb resolution at a single point) of an example pedigree of each embryo sample. The results showed abnormalities in the genome of embryo 1, 47-XX (triploid for chromosome 7); embryo 2 genome normal, 46-XY; embryonic 3 genomic abnormalities, 45-XX (1 chromosome 15 deletion); embryo 4 genome normal, 46-XX, embryo 5 genome abnormal, 45-XX (chromosome 15 deletion of 1), embryo 6 genome abnormal, 47-XX (chromosome 8 is triploid), embryo 7 genome abnormal, 45-XX, +8 (x 3, mos), -9q (q21.11 → qter, 67M, x 1, mos), -16 (x 1), embryo 8 genome normal-46, XX.

6.3 SNP-haplotype linkage analysis

6.3.1 SNP trapping and SNP filtration

The SNPs of the target region are obtained using software including, but not limited to, at least one of GATK, samtools mpileup, FreeBayes, VarScan.

SNP filtration: the reads coverage of SNP sites is at least 20, the heterozygosity of allele is 0-5% to 95% -100%, the heterozygosity of the heterozygote of allele is 30% -70%, and the rest sites are regarded as sites which cannot be judged, so that potential error SNP is removed.

6.3.2 screening for differential SNP and construction of paternal and maternal SNP-haplotypes

The SNP of the differential type is that because DMD gene is deleted and mutated into X-linked monogenic recessive inheritance, the SNP-haplotype of heterozygous maternal genotype can be deduced from the same SNP locus according to the genotype of male president. As shown in table 2, at SNP locus rs4829199, the premalignant genotype was AA and the maternal allele genotype was GA. The base A of the site in the female parent is linked with the pathogenic site in the same haplotype; this locus base G is linked to the normal allele within the same haplotype as the parent. This SNP site is the discrimination SNP, and the rest of the sites are analogized in the same way, as shown in Table 2.

TABLE 2 SNP genotypes upstream and downstream of the DMD Gene, 2M or more regions of differentiation

Judging the embryo sex according to the number of the 199 SNP loci in the heterozygous loci of the embryo

1. In Table 2, "-" indicates that no corresponding SNP data (no data coverage or low depth) was obtained;

2. in this example, there were 19 discrimination SNPs in total among 199 SNP sites; wherein the number of sites: embryo 1: 19, the number of the channels is 19; embryo: 18 of the total number of the cells; 3:19 embryos; 4:19 embryos, 5:18 embryos; 6, 15 embryos; 7 embryos are added; 8:18 embryos

3. The SNP sites marked in bold are positioned within 1M of the upstream and downstream of the SMN1 gene

"" identifies sites at which allelic shedding (ADO) may occur; "" indicates the site where the allelic shedding phenomenon (ADO) is present

5. Light grey indicates no mutant gene, grey indicates single mutant gene, dark grey indicates diseased embryo

6. The column "mutation" in Table 2 indicates the carrier of the causative mutation.

6.3.3 analysis of embryonic SNP-haplotypes

The number of the embryo SNP-haplotypes is 2, the embryo SNP-haplotypes are respectively inherited from 1 male parent and 1 female parent, and specific SNP-haplotypes are judged which is father source inheritance and which is mother source inheritance according to the distinguishing type SNP and the Mendelian inheritance principle. In the embryo SNP-haplotype analysis, the minimum number of discrimination SNPs is 6, and if more than 3 SNPs are wrong, the analysis of the embryo SNP-haplotype is not judged.

6.3.4 analysis of results

And determining SNP-haplotypes of the male parent according to 6.3.2, distinguishing the haplotypes linked with pathogenic sites, and comparing specific alleles of the SNP where the embryo is located. And judging whether the embryo carries a pathogenic gene locus or not according to the Mendelian genetic principle. For example, the SNP locus rs4829199, and the alleles of

embryos

2,3,4,5,6 all have a base A, which indicates that the 5 embryos carry maternal pathogenic gene mutation; the alleles of

embryos

1, 7 and 8 contain a base G, which indicates that the embryo does not carry maternal pathogenic gene mutations.

6.4 DMD Gene mutation analysis

The off-line data is compared on a reference sequence (such as human Hg38) after quality control, mutation carrying information of a DMD gene region is analyzed, and FIG. 4 shows the mutation conditions of the DMD gene in parents, presbyopes, transplanted embryos (E8) and amniotic fluid, which shows that the kit can accurately detect the mutation of the DMD gene in families, amniotic fluid and transplanted embryos.

7. Method feasibility verification

7.1 multiple PCR-based Re-validation of SNP site linkage analysis results

Specific primers are designed for target gene linkage SNP loci, and multiple PCR is utilized to carry out SNP locus linkage analysis for verifying the accuracy of the high-throughput sequencing SNP-haplotype analysis method disclosed by the invention, which is specifically shown in Table 3. As can be seen from Table 3, the SNP site linkage analysis results of the remaining 7 embryos were consistent with the SNP-haplotype analysis results of Table 2, except that it was difficult to conclude that the detection of embryo 7 was abnormal.

TABLE 3

7.2 embryo DMD Gene exon deletion electropherogram validation

Designing a specific primer for the exon region of the target gene, performing common PCR amplification and gel electrophoresis, and comparing with a high-throughput sequencing result to verify the feasibility of the detection method for the mutation region of the target gene in the patent, wherein the specific results are shown in FIGS. 5 (A) to 5 (C): the loading order of each exon was: fig. 5 (a): MALBAC normal control, MALBAC-1, embryo 2, embryo 3, X (amplification negative control), Y (amplification positive control), blank control; fig. 5 (B): normal gDNA, embryo 4-first polar body, embryo 4-second polar body, embryo 5-first polar body, embryo 5-second polar body, embryo 6-first polar body, embryo 6-second polar body, embryo 7-first polar body, embryo 7-second polar body, MALBAC amplification negative control, MALBAC amplification positive control, blank control; fig. 5 (C): normal gDNA, MALBAC-1-polar body, embryo 8-first polar body. Individual unlanded samples may be the result of amplification failure, not necessarily deletion. As can be seen from FIGS. 5 (A) to 5 (C), the results of exon deletion gel electrophoresis were consistent with the results of high-throughput sequencing in Table 2, except for embryo 2 and embryo 3. Electrophoresis results show that the

embryos

2 and 3 do not carry pure and deletion mutations, and are different from SNP linkage analysis results.

Example 4:

the child in pedigree 2 suffered from Duchenne muscular dystrophy (DMD, Exon 49-52 deleted), X-linked recessive inheritance. Female is the carrier (DMD, Exon 49-52 heterozygous deletion) and male is normal. The specific genetic map is shown in FIG. 6

FIG. 7 is an electrophoretic map showing exon deletion in the DMD gene of an exemplary family. The exon loading sequence is: mother (carrier), father, president (DMD Exon 49-52 deletion), MALBAC 1-MALBAC 3 (oral mucosa cell genome amplification sample of president), MALBAC negative control, MALBAC positive control and blank control. The results show that the presymptors carry the homozygous deletion mutation of DMD gene Exon 49-52.

1. Taking human embryo single cells:

1.1 fertilization of sperm and egg

1.2 in vitro culture of embryos

2. MALBAC amplification and product purification

2.1 lysis of cells

2.2 inactivation of proteases

The PCR tube was inactivated in a PCR instrument at 80 ℃ for 10 minutes.

2.3 first round Linear amplification

2.4 second round exponential amplification

2.5 purification of genomic DNA amplification products from embryo samples

5. Family member genome DNA extraction

6. As shown in example 2, using the kit of the invention: the detection kit for embryo preimplantation genetic diagnosis and prenatal diagnosis of the DMD gene is used for performing library construction, hybridization capture and enrichment on an embryo genome product and family member genome DNA after amplification and purification of the MALBAC.

5. High throughput sequencing

And sequencing the sample by adopting a high-throughput sequencing platform. The sequencing platform is not particularly limited, and second generation sequencing platforms include, but are not limited to, GA, GAII, GAIIx, HiSeq1000/2000/2500/3000/4000, X Ten, NextSeq500/550, MiSeq, SOLID by Applied Biosystems, 454FLX by Roche, Ion Torrent, Ion PGM, Ion Proton I/II by ThermoFisher scientific (Life Technologies); the third generation of single molecule sequencing platform: including but not limited to the HeliScope system from Helicos BioSciences, the SMRT system from Pacific BioSciences, Gridios, MinION from Oxford Nanopore Technologies. The type of sequencing can be Single ended (Single End) sequencing or double ended (Paired End) sequencing. Each specimen was sequenced at an average depth of 1X.

6. Data analysis

6.1 reference sequence alignment

6.2 genomic CNV analysis

The offline data, after quality control, were aligned to a reference sequence (e.g., human Hg38), and the same batch of embryo data was analyzed together according to coverage to determine the genomic CNV profile of each embryo fig. 8 is a genomic CNV map of each embryo sample in an exemplary family (single point 10Mb resolution). The results showed that the embryonic chromosome 4 set was abnormal, and the remaining embryonic chromosomes were normal.

6.3 SNP-haplotype linkage analysis

6.3.1 SNP trapping and SNP filtration

The SNP of the differential type is that because DMD gene is deleted and mutated into X-linked monogenic recessive inheritance, the SNP-haplotype of heterozygous maternal genotype can be deduced from the same SNP locus according to the genotype of male president. As shown in table 4, at SNP locus rs5928818, the antecedent genotype was TT and the maternal allele was CT. Indicating that the base T of the site in the female parent is interlocked with the allele with deletion in the same haplotype; this locus base G is linked to the normal allele within the same haplotype as the parent. This SNP site is the discrimination SNP, and the rest of the sites are analogized in the same way, as shown in Table 4.

TABLE 4 DMD Gene-related region-typing SNP genotypes

7. In Table 2, "-" indicates that no corresponding SNP data (no data coverage or low depth) was obtained;

8. in this example, 16 discrimination SNPs were present in total in the 199 SNP sites; wherein the number of sites: embryo 1: 13, the number of the channels is 13; embryo 2: 8, the number of the cells is 8; 3:16 embryos; 4:16 embryos; embryo 5: 9 are provided with

9. The SNP locus of the bold mark is positioned within 1M of the upstream and downstream of the DMD gene

10. "" identifies sites where allelic shedding (ADO) may occur; "" indicates the site where the allelic shedding phenomenon (ADO) is present

11. Light grey indicates no mutant gene carried, grey indicates single mutant gene carried

12. The column "mutation type" in Table 2 indicates the carrier of the causative mutation.

6.3.3 analysis of embryonic SNP-haplotypes

6.3.4 analysis of results

And determining SNP-haplotypes of the male parent according to 6.3.2, distinguishing the haplotypes linked with pathogenic sites, and comparing specific alleles of the SNP where the embryo is located. And judging whether the embryo carries a pathogenic gene locus or not according to the Mendelian genetic principle. For example, base a is present in both alleles of SNP locus rs17329762 and

embryos

4,5, indicating that these 2 embryos carry maternal mutations in the disease-causing gene; all alleles of

embryos

1,2 and 3 are basic groups G, which indicates that the 3 embryos do not carry maternal pathogenic gene mutation, and the rest sites are analogized in sequence for judgment.

7. Method feasibility verification

7.1 PCR electrophoresis based analysis of DMD Gene mutations: FIG. 9 shows a female square; a male party; the mutation conditions of the DMD gene in the proband and the fetal amniotic fluid (from left to right) further show that the kit can be used for accurately predicting the mutation of whether the transplanted embryo carries pure and deleted DMD gene.

7.2 multiple PCR-based Re-validation of SNP site linkage analysis results

Specific primers are designed for target gene linkage SNP loci, and multiple PCR is utilized to carry out SNP locus linkage analysis for verifying the accuracy of the high-throughput sequencing SNP-haplotype analysis method disclosed by the invention, which is specifically shown in Table 5. As can be seen from Table 5, the SNP locus linkage analysis results of

embryos

1,2, and 3 are consistent with the SNP-haplotype analysis results of Table 2, it is indicated that

embryos

4 and 5 do not carry pure mutations, and that the CNV abnormality is not judged due to abnormal amplification, which indicates that the SNP detection is abnormal, and it is difficult to draw a conclusion; embryo 5 previously found that recombination may occur without making a decision as to whether the embryo carries a mutant gene, where the SNP points to the embryo carrying the mutation.

TABLE 5

7.3 embryo DMD Gene exon deletion electropherograms

Designing specific primers for target gene exon regions, performing common PCR amplification and gel electrophoresis, and comparing with a high-throughput sequencing result to verify the feasibility of the target gene mutation region detection method in the patent, wherein the specific result is shown in FIG. 10: the loading order of each exon was: normal gDNA, embryo 1, embryo 2, embryo 3, embryo 4, embryo 5, MALBAC negative control, MALBAC positive control, blank control. Individual unlanded samples may be the result of amplification failure, not necessarily deletion. As can be seen in fig. 10, the gel electrophoresis results show that all 5 embryos do not carry homozygous deletions, which is consistent with the high throughput sequencing results prediction of table 4 and the prediction of conventional SNP multiplex PCR.

In conclusion, the method and the kit of the invention are used for simultaneously carrying out capture sequencing analysis on the DMD gene of two family samples and the SNP linked to the amplification preference area of a small amount of cell genome. Meanwhile, the conventionally adopted SNP site PCR capturing method is used for verification, the two groups of analysis results are consistent, and the method has strong feasibility and accuracy. The verification of the electrophoresis after the deletion exon PCR shows that the SNP linkage analysis result in the embodiment 4 completely accords with the detection result of the mutation region of the gene body; although the amplification results of the

ancestral embryos

2 and 3 in example 3 were different from the SNP results, the results of the remaining 6 embryos were all accurately judged. The amniotic fluid verification of the finally selected embryo 8 also reflects the accuracy of interpretation, and shows that the SNP linkage analysis result of the kit can complement the detection judgment of the mutation region of the gene body, so that the judgment error caused by single utilization of SNP linkage analysis or locus detection can be reduced, and the PGD diagnosis is more accurate. The above examples show that the probe set and the kit for detecting the DMD gene and the linked SNPs provided by the invention can be used for quickly and accurately detecting actual samples, are simple and convenient to operate and have strong universality.

The above detailed description is only a preferred embodiment of the present invention, and should not be taken as limiting the invention, and any modifications, equivalents, improvements, etc. made within the spirit and principle of the present invention should be included in the scope of the present invention. While the foregoing is directed to the preferred embodiment of the present invention, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention as defined in the appended claims.

Sequence listing

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<213> human cells (human cells)

<400>31

gtgccctggg gagaaatagg tagtaaagac atgtttgtgg ctggcagaga atcctgggaa 60

gctgtttcac tggactttgc ctgcaagcaa agcaatttta cttggcatga aggaatccaa 120

ttaagataga agagaaaaaa ggaatgccca gggaggaacc 160

<210>32

<211>160

<212>DNA

<213> human cells (human cells)

<400>32

ccttttctgt tgtgcaaact tttaaagcct tctttcagtc cttttcgtca tagccaaact 60

ttcctagaca atagttttct aattcattac aatgtcttct gatcctgatt ctacatatta 120

aacatatgaa ttttaacaaa aatctcttcc ttagaaaata 160

<210>33

<211>160

<212>DNA

<213> human cells (human cells)

<400>33

cacaaaaaca ttttgctagg aactcttcca gttttcaacg tttaagcttg attccttagt 60

ttcagggtaa atttgtctct atcattaaga ctttaaaggt gcccctatat cttacatcct 120

aagataatgt gccttttctg ttgtgcaaac ttttaaagcc 160

<210>34

<211>160

<212>DNA

<213> human cells (human cells)

<400>34

catttttctt ctcttgaata ttatattatt tatcctgttc tcagagctgt tagattgccc 60

tcttattaat ttgtagtgac tttaaccctg cttgcaatat tcaaatgtta ttactgtcag 120

aggtcctgtt ttctcagtct ttgccctttt gctcaacatt 160

<210>35

<211>160

<212>DNA

<213> human cells (human cells)

<400>35

tgcttttgaa atatttatga aataatcaca gtttaactgt ttaattgcat gagctagatt 60

tggttgagta caagcagaac actgcctatt ctttctcact tcctctttaa atcaagtcat 120

ggtcttgtcc tttcctaccc atgaatgaaa ccgaacgcag 160

<210>36

<211>160

<212>DNA

<213> human cells (human cells)

<400>36

tgccttatga gtgaagaaag ttggatgatt gggtgtaaaa gatgtaaggt taatgggaaa 60

acttccctag tccacaaatc atcttattat ccagtctgta attctttgtt ccatgccaaa 120

aatctatatt tcgtcaagta ataatagatg ctttcaggca 160

<210>37

<211>160

<212>DNA

<213> human cells (human cells)

<400>37

ccagttagtg agcatctttg tttgttaata tctgtacttc atacttaatg gactaatttt 60

ttctctatca aaaaagctgc ctttgttaaa acatttcaat caactaaatt acttaactct 120

aatgcctatt ttgagggaaa aatatactta gagggagaat 160

<210>38

<211>160

<212>DNA

<213> human cells (human cells)

<400>38

tagccattga agactcagaa caatagggac tccaatttgc tacctgttct tgaaaatgac 60

aatcagttac ttaggaaagc aactttgaaa aatacaaagg aaagtcagag agattggaac 120

caaataatga aggtcaagca tcatcaaaaa cacaagttgt 160

<210>39

<211>160

<212>DNA

<213> human cells (human cells)

<400>39

ctatacaact gtatcagaac cctcccatca gcctcattct tctcttgttc cagtgtttac 60

tgggctctgg cagccttctc atatagacca cttgagaatg tctaacttac tgttcatagc 120

ttccccaaat ctaccttctc ctttactctc ttctcactcc 160

<210>40

<211>160

<212>DNA

<213> human cells (human cells)

<400>40

tttttcttgc ttttttaccc tggtcccttg tttctggact tacttggggt ttaaaagatg 60

tatttgctta agaataactt aattacccat tctggataaa aatttaatgg aatgtgttag 120

aaggcttaag tgagatatta aaattaattg aaatcacgac 160

<210>41

<211>160

<212>DNA

<213> human cells (human cells)

<400>41

aaaaattcat tataaattga actacagaaa aagccaaata taaacattta caaggattaa 60

tcaagttata ttttgagact actaagcaac ctcaataaat gtgaaataac atcagcagtt 120

tatgtattct tattttcaag aaaaggagaa tcttacacta 160

<210>42

<211>160

<212>DNA

<213> human cells (human cells)

<400>42

gatgttcttt ctctgaaagg aagggtacaa gccctggatt tctttgatgt tgctctgaga 60

cacttgcgct ggcaacacca ggggctttga tgatgttctt ctagcactct gacatcctga 120

catctgggct tgtattcttc tgaagcagca aattgaaaca 160

<210>43

<211>160

<212>DNA

<213> human cells (human cells)

<400>43

ggacagtgag gcctcattca gaactttttg atgccacttg cctgattaga gagaatgctt 60

ttcctgcgtc ttcaagcagc catcagtttc acattttcat attcatccca agtgtctgtc 120

actaggcacc ctcattatcc caggagatcc ccaaaaagga 160

<210>44

<211>160

<212>DNA

<213> human cells (human cells)

<400>44

catctgggct tgtattcttc tgaagcagca aattgaaaca atagtcaaaa actccagttt 60

aaaagaaaaa ggacagtgag gcctcattca gaactttttg atgccacttg cctgattaga 120

gagaatgctt ttcctgcgtc ttcaagcagc catcagtttc 160

<210>45

<211>160

<212>DNA

<213> human cells (human cells)

<400>45

tcatgaagta ccacaaagcc taccctgcct ttgtatccca gaataaattt ccttctctct 60

ccctcccagt tctcagaata catcctgaaa atgtctctgt atctaactga tgtgtttacc 120

tcctagttac caaggagctt ttctgaccat agactttccc 160

<210>46

<211>160

<212>DNA

<213> human cells (human cells)

<400>46

tttcatgcca taataaaata acctctattg tgaggataat ttttcaaaag gacttagtgt 60

aatgagcata catttttggc taggtcagat ttctaatagt tggcagccca ggtattagca 120

acctcatatt ctacttccct gaataagaag tctctaggag 160

<210>47

<211>160

<212>DNA

<213> human cells (human cells)

<400>47

actgttatgg ctttcatctg ttgctgcctt gaagttgagg agaaaagagc aagcagtggc 60

ctctttgtga ggtccatgcc gactggaact atcatcacac aaattactac taatatttgc 120

tttgttaaaa aaaaacttta ggaacaaaaa aaaaatgagt 160

<210>48

<211>160

<212>DNA

<213> human cells (human cells)

<400>48

ccccatgata cataagatgg gatgaaattt gtatctttcc tggcttgttt ccatccaaga 60

gtaagaagta tattttgaga aatgtatgta gttcacatgg atggatttgt gtagttcata 120

gacttaccct aggatgcaat ttgaaacctc ttcattctgt 160

<210>49

<211>160

<212>DNA

<213> human cells (human cells)

<400>49

tgtggtagtc acttaggtca gcatcatcac cctactttca aaaaattagc aagtgattga 60

ttcacagcag agatgccaca tctactaaca aacatcattt ctaatgagat ggatcatctc 120

atctcctatt atttccaaaa tcactaggat aggctgcacc 160

<210>50

<211>160

<212>DNA

<213> human cells (human cells)

<400>50

ctctaggtaa tgttaaaatt ataatgtact ttattttttg cccttcatgt gaaaatgcac 60

acattttcag ttatctgctt ggcgtatcct cctacgtatg gcatgaaacc ttcaaattat 120

cctctccaaa cttgatccta tgcctatttc tatttcttaa 160

<210>51

<211>160

<212>DNA

<213> human cells (human cells)

<400>51

ttaaagagat ggtagggaag gaaaagggtc aggggaagaa tatgcagaga tcagggagac 60

tgaggaccta gggggactga atgaattatt gaccagagtg tatgaaacac tttggctccc 120

tagtgactaa gtgtcatggc tgttgagact catgcttaaa 160

<210>52

<211>160

<212>DNA

<213> human cells (human cells)

<400>52

aatttcagaa tggggtttca gaaatcgatg ttgcagaatc ttcagtgagc aaaaactttt 60

tttagaaaaa gatatgctct ggacttaaaa gaaagctaaa gaagaaagaa taaccatttc 120

ccatgaaggc acaaactttt ctatggtaag aaatgtgtag 160

<210>53

<211>160

<212>DNA

<213> human cells (human cells)

<400>53

ctctgtggga cagtgtgtcc agcacccaac agacaacctg aaagtttagt gcagccaata 60

tcccactggg cccagaagac aggaccctca ggtgccacaa cacaccaacc atttacttgg 120

gaccttggga cacatctacc tactcagtaa aatggggtgg 160

<210>54

<211>160

<212>DNA

<213> human cells (human cells)

<400>54

taaatagaagcatcaggaag cctggatcca ttagtcttca cttgccacac agctgtgaag 60

acatggggca gggaagggtg ttggtatata ccagctgata cagaatcatt tttacttttt 120

ccctccctcc catcctccct ctttcttttt ttgctgtttc 160

<210>55

<211>160

<212>DNA

<213> human cells (human cells)

<400>55

ctgtgaggct ctagagcact gccctcaaga aaaaaactgc tggaggcagc atttattgga 60

gccaccaacc gttgagttta ccagatgagg ccattcacat cttccctcta tccccaagtt 120

gggcttcact gtcatctagc ttcctgccca cattcctgcc 160

<210>56

<211>160

<212>DNA

<213> human cells (human cells)

<400>56

agtgtgacct tcagtctgca gaggcaagaa tttcagcccc tgccaaacat caaggcaaga 60

cactgagtga tgactgggga accacacact ccagaagatc caaagagccc tgtccctgct 120

atcagatctg gtatctggta gggcatagaa tagctatcag 160

<210>57

<211>160

<212>DNA

<213> human cells (human cells)

<400>57

agtgagagaa tggtgagtac catagtctaa gaagtgtgac cttcagtctg cagaggcaag 60

aatttcagcc cctgccaaac atcaaggcaa gacactgagt gatgactggg gaaccacaca 120

ctccagaaga tccaaagagc cctgtccctg ctatcagatc 160

<210>58

<211>160

<212>DNA

<213> human cells (human cells)

<400>58

taaagaattg agggccttaa ttgtaacttt tcaacctgaa atacctgacc tctgctaacc 60

atacaacatg ttggtgagaa ctggggagtt ccaaaaagga taaaaaaggc agaatagtgt 120

tctcctgaga atagtattaa ggacccaatt gcaaatgatt 160

<210>59

<211>160

<212>DNA

<213> human cells (human cells)

<400>59

agacaggcca ggaatagcat gagtagtgga cgaggacacg tagctggaag gagggagaaa 60

ttgatgtccc aagcttagac ggacttttca tgcttcttga atttttgcac tacagagggt 120

ggatatctgt atttgctggt ctatgtttgc caccagatac 160

<210>60

<211>160

<212>DNA

<213> human cells (human cells)

<400>60

ttattttaag gttttcatac ctcaggcaag gtgatttatt attattggga ctttatataa 60

ctctttattt ttgcaaagtt cttcacagtt atttttctct cctcattgtg taccatgcct 120

ttcctctcct gccactgaaa ttggttttgg gggaggcggc 160

<210>61

<211>160

<212>DNA

<213> human cells (human cells)

<400>61

tcttcatcac ttttgtgtaa acttgagttt accccaatac ccagactctt atctgttcta 60

actactatgt ttgttctaag aagctggtaa ctccacagta actccagagt aactcgagtt 120

tctcatgata tacagagcaa gggactggaa gaaatcatgg 160

<210>62

<211>160

<212>DNA

<213> human cells (human cells)

<400>62

tcttgaggtg agatacgcag aagtcactgg actggaagtc tgccaagggc tgggagatcc 60

agagtccact ccattcaaag aacattttaa ataggttatg ttgaatataa tttggcaact 120

gtaagcatgg cctacatttt tttgtttgag agaatggatg 160

<210>63

<211>160

<212>DNA

<213> human cells (human cells)

<400>63

ccaaagcact aaaagcaggt gtagagaaag aatgtgaaaa tgagaatcaa aaacaactgg 60

gaattgttta taggattcat ttacctaaag tactgctgct cctagcccca gagtgtcttg 120

agaactgact ctgctcagga gctggcacag gtttgcagtc 160

<210>64

<211>160

<212>DNA

<213> human cells (human cells)

<400>64

tttgagaacc cttgtccgag tttctcataa gcaaatttga aacaaacaaa aaaagcaact 60

tcaaaggaat tcctcaactg ttttataaat gcacttacta tggatggtta cctttagaaa 120

atttacaagg aaaaatcttg aaattttatg gggattgttg 160

<210>65

<211>160

<212>DNA

<213> human cells (human cells)

<400>65

agtggatgat ttttaagcag gtctaaatgt aatatagtgt atggttgaca agcgtgtctg 60

gataattacc atgttccaag gacacattgt taggcagctc tattgtaatt agagttttaa 120

aatcaataaa agttattttc tatcttgaaa tataaatgcc 160

<210>66

<211>160

<212>DNA

<213> human cells (human cells)

<400>66

taaatgtaat atagtgtatg gttgacaagc gtgtctggat aattaccatg ttccaaggac 60

acattgttag gcagctctat tgtaattaga gttttaaaat caataaaagt tattttctat 120

cttgaaatat aaatgccatg tattaaatga tgaagcagaa 160

<210>67

<211>160

<212>DNA

<213> human cells (human cells)

<400>67

ttgtacccct taaatttata ctaatggcat atcactagaa ggaacgtcat tgccttcttc 60

tttgttttct ttctctcccc tcctaattca gtttaaccac tgatttttta tatctaatcc 120

ctccttattc caggcatctc aaacacaagt tgtccacaat 160

<210>68

<211>160

<212>DNA

<213> human cells (human cells)

<400>68

gaaagtattc cctcatgata atgagaaaaa tccagaacac aaggcttgat tactcctaca 60

atatttgcct gctttgactc tgactgtgct taagtatatc caggcatgct gtgcaaagta 120

cagaactgaa ataaaataat ccctagtcca atcataggct 160

<210>69

<211>160

<212>DNA

<213> human cells (human cells)

<400>69

cctctagcac atacacaggc aactgtgaag attgagatgt ctcattttat tttcccctag 60

taaacgtaat ataaagtggg tttgtggttg gggtgagagg agaatcacct tttcatatct 120

gaaaacctaa tttattttcc cttccactca aaatgatgta 160

<210>70

<211>160

<212>DNA

<213> human cells (human cells)

<400>70

gtttatggag ttacatgaac tcatctaggc attttcaatg ccttgtgcag gccccgagcc 60

acacttagtc cattcttacg ttctccctcc ctgacctcgt gacctctact cagtcctcta 120

ccaggcagtt ccagcatgcc tgccatagat catcatcctg 160

<210>71

<211>160

<212>DNA

<213> human cells (human cells)

<400>71

cttttcaaga atattaccac tgtcaccagt gattgaatat taaacatttc ctgtgttttt 60

ctctctgtat atttctcccc actcttcctg ctctttactt ctgtactaac ttcttaaaac 120

tatagttaag ggcccacaaa ctcaaatgcc gtcagggatg 160

<210>72

<211>160

<212>DNA

<213> human cells (human cells)

<400>72

gggaagagat taaaggaata aatgaagtaa tcacccagaa attctccaat cagaaaaaaa 60

ttttgtgttg ccttaaactc tctttttaaa gcatatattt atacctttgc ttatgtatta 120

cctatacttg tgtatttgtg acatatataa agttatatat 160

<210>73

<211>160

<212>DNA

<213> human cells (human cells)

<400>73

aggatagcca catcgagttt ttcaacaata gcattgccct cttgaccaac gcatttctta 60

tttctttaat aaaggaagtg tcctgagggc cttcccagag aaacaaatcc gatgtaattt 120

cgctaaaaat taattaaaag gcctgctctg aacctgtgtg 160

<210>74

<211>160

<212>DNA

<213> human cells (human cells)

<400>74

tcatacccaa caactaactg aggtgagatg tgctaaagaa agcaagatac aactaaactc 60

tattgttgcc atggtaacct gtcaggttac ctaacagtaa taagttttat tgagagtaaa 120

ggtggataaa cactcttttt tgagacataa ttcacatacc 160

<210>75

<211>160

<212>DNA

<213> human cells (human cells)

<400>75

aaattgggag tatcagggag aatcaaggag agacattctg caaatttgaa gaggtagggc 60

tacagcaaaa agtttacctg ttacaagatg tgatccaatt tatatcagaa gctagacaag 120

tgggctattc tgcttatcca actaatctat gcaacacatt 160

<210>76

<211>160

<212>DNA

<213> human cells (human cells)

<400>76

tgtgcctttt actgcataga aatcatattt caggaacaat aattttaaaa gaataccata 60

gtatgtgaga ggtggcatgc agtggaggcc tctgttgtcc aggttatgct tttctattac 120

agctgcagct gtgggagaca gtactgacta aagtcagtag 160

<210>77

<211>160

<212>DNA

<213> human cells (human cells)

<400>77

aacatcatgg catcttggcc caccacagcc ctatagcagc cctaatccca cattatgggt 60

catgttatat gcttaccaga gtggcaaagg agggaggaga tgacagagtt atagctttcc 120

tagttcacat gtaactcttc tgaaattagg taataactcc 160

<210>78

<211>160

<212>DNA

<213> human cells (human cells)

<400>78

atccctaaat atattgtatt acacaataag catttaagtt gtgttttcta tatgagggaa 60

taatcatgcg gaataataaa caaacttgtg tcttctaaac agtgttaaaa ccatgatgga 120

cgttttcttc ttggcctaaa catgattttc ccagagtctg 160

<210>79

<211>160

<212>DNA

<213> human cells (human cells)

<400>79

tttaaagaaa gtcaacgggg taatgtgttc agtttctttt ttaaataaaa agtatgcaat 60

attataatga ttagagtccc atgtagtaga aggaagagaa taaaggactg aaattataag 120

actatttgtg cccttgattt taatttctta ccttgagtcc 160

<210>80

<211>160

<212>DNA

<213> human cells (human cells)

<400>80

ctctgtcgcc caggctggaa tgcagtggag ctcaaaactg atcttgttca tcaggaaaat 60

agaatgaaca taattatggg gtatgattca aagaactagt ctttcaagag taaaagtatg 120

gtccttctgg gaatgatgaa tttgaatata gaaagcaaaa 160

<210>81

<211>160

<212>DNA

<213> human cells (human cells)

<400>81

gacatatgcc atttttcatt ctaatcaaaa gtccccaagc acacagaaac cattgaccag 60

aatgtaacca tttgtacatg ccaagataaa aattataggt gtttgttagt ttcacactga 120

tttcttcaaa ttaagcaatt caattatgaa ttaccacaat 160

<210>82

<211>160

<212>DNA

<213> human cells (human cells)

<400>82

attaaagggc tggaagtagg aggaggtgag gcagatgagg atctgggagg caggtgttgg 60

cagactaaat ctggggaggt gtataaatag ggtacaatac agtagatatg tcaacaggtg 120

gatttgtcaa tgtgtgttat tattttgaac tggcgtgaag 160

<210>83

<211>160

<212>DNA

<213> human cells (human cells)

<400>83

cttgccctag ggctgattaa agggctggaa gtaggaggag gtgaggcaga tgaggatctg 60

ggaggcaggt gttggcagac taaatctggg gaggtgtata aatagggtac aatacagtag 120

atatgtcaac aggtggattt gtcaatgtgt gttattattt 160

<210>84

<211>160

<212>DNA

<213> human cells (human cells)

<400>84

tgaggatctg ggaggcaggt gttggcagac taaatctggg gaggtgtata aatagggtac 60

aatacagtag atatgtcaac aggtggattt gtcaatgtgt gttattattt tgaactggcg 120

tgaaggcagc agaacatagc ggtgaacgct tatatcctgg 160

<210>85

<211>160

<212>DNA

<213> human cells (human cells)

<400>85

gatcttgttc atcaggaaaa tagaatgaac ataattatgg ggtatgattc aaagaactag 60

tctttcaaga gtaaaagtat ggtccttctg ggaatgatga atttgaatat agaaagcaaa 120

agaagggttc taagtagtga ggccaaactg tggattaaga 160

<210>86

<211>160

<212>DNA

<213> human cells (human cells)

<400>86

atgcattcta catataaagg cctttacact cacttccatg agataagtta acacaaagta 60

attttatgag gttaattaca atggtgaact ccaaatgagc acaactgtta gaatcgttac 120

gtactcctga gtggcaagtg ctccccttcc cctcctatac 160

<210>87

<211>160

<212>DNA

<213> human cells (human cells)

<400>87

gaagtatcca ggttaggtgc ttacagaatg tgtttatgaa tttccttaat gctaatttgg 60

aattagttat gcattctaca tataaaggcc tttacactca cttccatgag ataagttaac 120

acaaagtaat tttatgaggt taattacaat ggtgaactcc 160

<210>88

<211>160

<212>DNA

<213> human cells (human cells)

<400>88

cttcagtaaa gctaagatgg ggtcttccag ctgaaggtgc tcacagatct cattctccca 60

tctccaggta tactaaccat ctattcttct gcccacattt cttggtttac ccagccatca 120

tgcctcgtgg tcagaagagt aagctccgtg cccgtgagaa 160

<210>89

<211>160

<212>DNA

<213> human cells (human cells)

<400>89

tttcattcta tgtaaaggga attgagggag gaaaaagtaa ttatgtaatc ctcaagaagt 60

ttagtgtgtt tggctacatt gtttatacgt atgtgatcag aatatgtcat ttcttcattg 120

aactttgtta atatctcccc tgtatttctg atatatagtc 160

<210>90

<211>160

<212>DNA

<213> human cells (human cells)

<400>90

tgacaaaaac agtgaacggt ttttcaaggg aggtaaaagc tgccagagct gagcctgatg 60

ttctttctct gaaaggaagg gtacaagccc tggatttctt tgatgttgct ctgagacact 120

tgcgctggca acaccagggg ctttgatgat gttcttctag 160

<210>91

<211>160

<212>DNA

<213> human cells (human cells)

<400>91

cccaattatc ctccttgttg agacataatt taatgctact gttttgtcac atactcctcc 60

aattcctgat tcagagttct ggattgttgg agaagggccc atgagcttta aacctagaag 120

agagtaaagg agaagagtgt tcctaggtct ctcattttgg 160

<210>92

<211>160

<212>DNA

<213> human cells (human cells)

<400>92

agagaggact tgagggaact cccacctcat cagtggggat ccctcagagt ccctctgtat 60

gtcagcgcta ggaagccccg agcataaatg tcagaaatgc ccctaaattc ctcttcagaa 120

gtaacaggga aatgaaggtc ttagtcagat gggttagcag 160

<210>93

<211>160

<212>DNA

<213> human cells (human cells)

<400>93

ttgcctatct tctctaacct tgtccctgag tagaatattt tcaacatctg atgctctttg 60

agatacagat agtataagag ggatgtgtaa tacgttagtt atgcatctca agccatgctt 120

tatttgctgc tgccaatttc tcagttgctt gtatttaata 160

<210>94

<211>160

<212>DNA

<213> human cells (human cells)

<400>94

taccatggct ggtggaggga tcagacctga aggggaggaa gggaggagag tagtaactga 60

gaggttctag tgaagtgggg ggaaggaaag acactggaaa tagaagagaa atatgatcta 120

tctattacca ttacttatat tggttaaaat gaaaaggtcc 160

<210>95

<211>160

<212>DNA

<213> human cells (human cells)

<400>95

cagactgcta gatactaagg tgaggacccc tagtggggac gtagggacca gcgacgctag 60

aacagttacg tccagaagcg taccacccct gccgtcagcc cggaggccac gggctgccgg 120

atgtggctca tcctgacttc cgctttgaag gcgaggaccc 160

<210>96

<211>160

<212>DNA

<213> human cells (human cells)

<400>96

ctgggggtgc tgcttcaagc tctcctgctg ctggcattcc ccaggagcct cagagagccc 60

caaccactgc cgctgctgcg gctgcgggtg tttcatccac aaaatctaaa aaaggtgcca 120

agagccacca aggtgagaaa aatgcaagtt cctcccaggc 160

<210>97

<211>160

<212>DNA

<213> human cells (human cells)

<400>97

aggaatggga ggaaggcccc aaatacatat tataatgtcc tgggaaataa ttgcaattat 60

aatgaagatg taaattgttt tataaaaccc tatatgcaat gcatatttca tacaaaagga 120

gcctactaaa gaggaatggc ccagggaatg ttgtgttgaa 160

<210>98

<211>160

<212>DNA

<213> human cells (human cells)

<400>98

gcagaataag aaatgcaaaa tagttgcatt tcattatacc ctttaattgc actttacagc 60

actatgcaaa agatttcctt tataggactg aaggcacaac aatgcattta aataaaaaaa 120

tacatgctgg tgtgtcaaat tagaatgcca ctttttgcct 160

<210>99

<211>160

<212>DNA

<213> human cells (human cells)

<400>99

ccgctacgcc tgtcagtctc ctgaagatac gcttgtttgt cgccgtgacg agtttttact 60

ccccaaaata tctctcagag gtccccaagc tgaccgcaaa agcaggaaga aaaagctgct 120

caagaaagcg gccctatttt ccgagctctc gccagtacag 160

<210>100

<211>160

<212>DNA

<213> human cells (human cells)

<400>100

ctcaaggttg ctggagaaggaataatgaag caaggctcac aaagagctca ggagcccata 60

ggtggcagat agcaaaatgt gccagtagaa aacagtttct aagaatgcat gatagtgtaa 120

gaaagggatg tggagggaac tcagagattc aggctggcag 160

<210>101

<211>160

<212>DNA

<213> human cells (human cells)

<400>101

aaattatagg tgtttgttag tttcacactg atttcttcaa attaagcaat tcaattatga 60

attaccacaa tgtagcttta tttgtccata gctttttatt ttttaatgta ttttccagca 120

aattttttct tagtcaaaga catacttcag tgaatctcag 160

<210>102

<211>160

<212>DNA

<213> human cells (human cells)

<400>102

aacaaatata tgactataca actgtatcag aaccctccca tcagcctcat tcttctcttg 60

ttccagtgtt tactgggctc tggcagcctt ctcatataga ccacttgaga atgtctaact 120

tactgttcat agcttcccca aatctacctt ctcctttact 160

<210>103

<211>160

<212>DNA

<213> human cells (human cells)

<400>103

caggggccag atgcagggag gtgggcgggg cgtgggaaac gcgggaaggc cgggccgggt 60

aggtccacac agcgactcac gtcgctgcac gactctgcac agcccactgg agggaacaag 120

cagttctaca cactttgaga cccctatgga ggcgagcccc 160

<210>104

<211>160

<212>DNA

<213> human cells (human cells)

<400>104

ttttactttt accattctgt tttcccctcc tattgtagta gcattttttg tattacaaat 60

ggaaagtaga aggtaataat attcctgaaa caaaacatag tgaaaatcat ctgaagaata 120

aacaaaacga agagaaaaaa ataaagacaa tgccacaagt 160

<210>105

<211>160

<212>DNA

<213> human cells (human cells)

<400>105

ttccgagctc tcgccagtac agccagcacg gaaggcgttc gtagaggaag tggaagccca 60

gctgatgacc aagcatccct tggccatgta ccccaatctg ggaaaagata tgcctccaga 120

tctcctacta caggtgctga aacagctgga tcccgagagg 160

<210>106

<211>160

<212>DNA

<213> human cells (human cells)

<400>106

aaaataaatg aaaacaaata tatgactata caactgtatc agaaccctcc catcagcctc 60

attcttctct tgttccagtg tttactgggc tctggcagcc ttctcatata gaccacttga 120

gaatgtctaa cttactgttc atagcttccc caaatctacc 160

<210>107

<211>160

<212>DNA

<213> human cells (human cells)

<400>107

cagtaaaagc tacctgatac actgtgtgag tgacctcatg tgaatgcctt tgcaattctt 60

atccaaaaga aagtgcagct tgtttttacc tttaagaagt tggtagaaac cttaataaag 120

ataatattga aatctgggga ttgaaatctg tgattgaaat 160

<210>108

<211>160

<212>DNA

<213> human cells (human cells)

<400>108

tctataaaat ttcttccact gcaaatgcac tctgcttcag cacaggcacc tatctgtgtg 60

gatttcattc tgtccaatac actgtatccc aattttgctg cttagctctc cagcactgca 120

aaaatgtggc cacctacctc tgcatgccca gcctaccttg 160

<210>109

<211>160

<212>DNA

<213> human cells (human cells)

<400>109

tgatatatct tcaaatactt tgctgctgag acaagagaat ctaccagtgt ccttcctagg 60

gttaaaacac aggggatcaa acctcccaga tgattactcc tcccactttg acctagtatt 120

cagcacttca ttgcagcttc tctttcccca aacagcccca 160

<210>110

<211>160

<212>DNA

<213> human cells (human cells)

<400>110

tgtgcaaata aatggggtta gtgaataatg atacatagtt gagacaggcc aggaatagca 60

tgagtagtgg acgaggacac gtagctggaa ggagggagaa attgatgtcc caagcttaga 120

cggacttttc atgcttcttg aatttttgca ctacagaggg 160

<210>111

<211>160

<212>DNA

<213> human cells (human cells)

<400>111

gaggatctgg aggcgagggg cacgggatct cttcggcaga gggtgaattc cttggactgt 60

ctggagtcaa ggtcaggacc ctgaatgtgc atgaaaggga ccaccatccc ccaacctgta 120

acaaagaggg ccacactaaa tcctgccccg gaagtcttcc 160

<210>112

<211>160

<212>DNA

<213> human cells (human cells)

<400>112

ttatttaggg tattgcaaag gatagatcaa accaccttaa agggggagaa atgtggagca 60

gaagtttctc atgtaaccta tcagctttag agctagattc atggccacag aggcatcagt 120

gatctttctc acagcagacg gaatcggaga gaatcctccc 160

<210>113

<211>160

<212>DNA

<213> human cells (human cells)

<400>113

aaaatcggtt gatatagggt gatcacaaat tactaatttt tacttttacc attctgtttt 60

cccctcctat tgtagtagca ttttttgtat tacaaatgga aagtagaagg taataatatt 120

cctgaaacaa aacatagtga aaatcatctg aagaataaac 160

<210>114

<211>160

<212>DNA

<213> human cells (human cells)

<400>114

ctgaagtgag agcagctgtt aggaaatcct acttctatct atcaagaagg gtttatttaa 60

gaaaatagat gacatacacg ctataattat caatatagtt tcttccatgg gctaccaagt 120

ccaaagtatt attgcttttg cataatttat gctgataacc 160

<210>115

<211>160

<212>DNA

<213> human cells (human cells)

<400>115

ggcaagcatg ttgtgtggca aaaacagcag atatccttgc acttggaaac acagatgcca 60

cagtacttat ggtcaagtat cttctttggg tgtagaagtc cagggctttg ctttttaaac 120

attccacacg tgaacttcat agatctcctc tgggtagcat 160

<210>116

<211>160

<212>DNA

<213> human cells (human cells)

<400>116

atatatgaat agccttcaga tatctcttac tcaccactgc ctgatgaaaa ttactggata 60

atttgagtga acaggtctac ggggtgaaag aaccagagca tatcagacac cccatcctac 120

cttagactgc ttgctacata ttgtcaagca gactgactat 160

<210>117

<211>160

<212>DNA

<213> human cells (human cells)

<400>117

cgccgcaagg cccgagatga gacccggggt ctcaatgttc ctcaggtcac tgaagcagag 60

gaagaagagg ccccctgctg ttcctcttct gtttctgggg gtgctgcttc aagctctcct 120

gctgctggca ttccccagga gcctcagaga gccccaacca 160

<210>118

<211>160

<212>DNA

<213> human cells (human cells)

<400>118

tcattgtaat tacattttta cacattctga ttttctaccc tagacattcc cttcactcca 60

ctcttctaca caaacccata tccaccctcc tcttattctc ccccaacccc aacacaaaga 120

gaccaccaca gacaccatgg ataaaagccc cagactatga 160

<210>119

<211>160

<212>DNA

<213> human cells (human cells)

<400>119

taaaatattt attgaagaat taacagaggt cttctattaa tctgacatgg taaaacagaa 60

gaggaggaac aatcatagac atgagaaagg agacactgta ggtgacgtaa attaaaattt 120

tataaacaag gtattttttc tgtggattta atattgggag 160

<210>120

<211>160

<212>DNA

<213> human cells (human cells)

<400>120

gatggctgca tcaatgcttc aggaaatagg aatgttttat tgagctggga gattaaatat 60

atgtggagcc acttgtacca ctttgggagg agttttcagt ttaagcctac agcatttctg 120

gtaaaactca catgaagtgt gacaaagtaa tcagaaccgc 160

<210>121

<211>160

<212>DNA

<213> human cells (human cells)

<400>121

cattaaaagg gacatatggc tcacttatca ctaatcaact cttacccagc accctcacag 60

ccactagcaa ggcaaagaga gtgagctccc actatcccct ttagctatgc ctcttgtgtt 120

gcccatttgt gatccctttt ccctaaaatc catccttaac 160

<210>122

<211>160

<212>DNA

<213> human cells (human cells)

<400>122

aggatcctct catcagcact tttgtgctcc ctgaatcata cccctggaag gggtgttcgg 60

atccacagat actaagagtg tggagtatcc aggactgggc catgggcttc atcttccaga 120

gaagtttcct tccagctggt gatcagatgt agggacgctc 160

<210>123

<211>160

<212>DNA

<213> human cells (human cells)

<400>123

aaactggggg caagcactga ctcaattatc tcaggctggg gaatttgtta ccctttttat 60

ataaagcata acacatggtc aatttttctt ttgttgtatt ctgtgatgta gactacattt 120

caaatgataa aataaaatta gaagtggtag gggaattcca 160

<210>124

<211>160

<212>DNA

<213> human cells (human cells)

<400>124

ttgtcaaatg tacacactgc cttcttctgt gacagggaaa aattttaaat aaatacattc 60

ctactatttc tgagtatata cctaattgtt tctaggttaa taaattcgta tcttcttggc 120

ctgctataag actttttttt ttctcagagt ataataagct 160

<210>125

<211>160

<212>DNA

<213> human cells (human cells)

<400>125

ctggatgaca atggcagtgc tcatttacct caacaaaggc tttcaacagc catagaagtt 60

ggtgacaaat ttatactcta gtaagtatgt ggtttacaga taaggcaggc tgagtttaaa 120

ggtttgcagt agggattgca ccggatagtg tgaaaattgt 160

<210>126

<211>160

<212>DNA

<213> human cells (human cells)

<400>126

gaaaaaaata aaagaaattt tttggaaatt tgaacagcaa caacaaaaag aactcacatt 60

tttgactact gttgtttagt tactagagcg tgcatatagcaaggcttctt ttgaaaacag 120

aatttcacaa tttatatttt gtgtgtggca acgtactatg 160

<210>127

<211>160

<212>DNA

<213> human cells (human cells)

<400>127

atatcaaatt cagcacaagg gggagagaag aagcagggga gataagtttc ttgtccaaaa 60

gagtagatat gttagacata tgccattttt cattctaatc aaaagtcccc aagcacacag 120

aaaccattga ccagaatgta accatttgta catgccaaga 160

<210>128

<211>160

<212>DNA

<213> human cells (human cells)

<400>128

aattttcaac aatgacatgg tatgaaatta atttgtgctc aacacatcat gaagtaccac 60

aaagcctacc ctgcctttgt atcccagaat aaatttcctt ctctctccct cccagttctc 120

agaatacatc ctgaaaatgt ctctgtatct aactgatgtg 160

<210>129

<211>160

<212>DNA

<213> human cells (human cells)

<400>129

tgattcaggc tgtgggtgtg aggatggaga gtaggagtca cgtttgagtt ttggaaagtt 60

agggactatg tgaaagggcg agggacagcc tataatgact gaatcctgaa ggttggataa 120

ctaggtagaa gtttctctta gctgagattg aggataaagg 160

<210>130

<211>160

<212>DNA

<213> human cells (human cells)

<400>130

tcccctaaaa agcggggggc agggggaagc aaagactgtc aaggcagtga ttgttcaaat 60

ggttttatgg gaagaaatta ttactagaac cgcaatcaca tgttcagcct ccctatatta 120

aagcagaccc gttcctgctg aaatggtggt cagggcccca 160

<210>131

<211>160

<212>DNA

<213> human cells (human cells)

<400>131

tccagctccc ctacacagcc ctagtacttc tggttcctgg ctcaatgcca agaggggttg 60

gccttggaag tcccttgcta tagtaaatag cattcagact tagagaggta atcagagtac 120

cttctggtgt cagcctggaa taatgctgac atgccctgaa 160

<210>132

<211>160

<212>DNA

<213> human cells (human cells)

<400>132

cctgctgata tatatgtgtc tcagaaatac agaattaaaa atgtgtaatt gttcagtaaa 60

ttatagccaa tattatggtc acagtcacga tttattgttt gggtgaagag acttttgccc 120

attaacagtc tttcctgtcc tcactttcta catgttccct 160

<210>133

<211>160

<212>DNA

<213> human cells (human cells)

<400>133

gaatagatta gcagctgttt caagaggacg gagccaagtg actacttcca ctcaacttaa 60

ttcctagtac taccctgaag ttcaaaggag gtaatggctg aaatccccca aatttgtgtg 120

gtctcagggt gtttttaaaa gaaacataac agttaccagc 160

<210>134

<211>160

<212>DNA

<213> human cells (human cells)

<400>134

tgaaggctat ctttatgtct cctgaacaga ggcatttctt cccctatgaa cagtggatcc 60

tgtcccctga ggttggcact gtgcaaataa atggggttag tgaataatga tacatagttg 120

agacaggcca ggaatagcat gagtagtgga cgaggacacg 160

<210>135

<211>160

<212>DNA

<213> human cells (human cells)

<400>135

ttccttctct ctccctccca gttctcagaa tacatcctga aaatgtctct gtatctaact 60

gatgtgttta cctcctagtt accaaggagc ttttctgacc atagactttc cccacatctg 120

cctcccctcc ctacccccag caattgtcac tggtctagag 160

<210>136

<211>160

<212>DNA

<213> human cells (human cells)

<400>136

cctgtgaggc tctagagcac tgccctcaag aaaaaaactg ctggaggcag catttattgg 60

agccaccaac cgttgagttt accagatgag gccattcaca tcttccctct atccccaagt 120

tgggcttcac tgtcatctag cttcctgccc acattcctgc 160

<210>137

<211>160

<212>DNA

<213> human cells (human cells)

<400>137

ccccaaatga agaaggcccc tcacaactct gtcgttctcc ctgcagaaag gaggaatggc 60

cttaagtggt ttttcttgtc ttttgcttgg agagtgagag aatggtgagt accatagtct 120

aagaagtgtg accttcagtc tgcagaggca agaatttcag 160

<210>138

<211>160

<212>DNA

<213> human cells (human cells)

<400>138

catttttgtc atcagaaaat atttccaagt taagcaatct tacaaagaag tatctcagga 60

ctatcaacat tagtgagccg ggtaccctga agtgaggtac tctttcctca cttcatagtg 120

tggccaccat agccattctt gcctttcttc agtttgctgt 160

<210>139

<211>160

<212>DNA

<213> human cells (human cells)

<400>139

tttccctgcc acatctacat cttttctcag tcttacttcc tgagtcctga tggctccttc 60

ttggctttcc caggtaaaat taaggcgaag gggctctgca agaagggaag gggctttggg 120

gacaggaaaa ccctccctca gtgggcaaca gcatctacca 160

<210>140

<211>160

<212>DNA

<213> human cells (human cells)

<400>140

ctgaaaataa agggtatata ttacgggaag aagtacttca tctctgggga tcccaggaag 60

gtcatcacca gagatctgac gcatgaaagg tagctggact gccagcagat gccccatagt 120

gattcttccc actatgaatt cctatggggt ctgagagccc 160

<210>141

<211>160

<212>DNA

<213> human cells (human cells)

<400>141

ggattaaaga agctcagaga gttgtagaag ctcgtttgtc cttaaaatgc aggaatcatg 60

gcttctgata catataggac ggtcaaggtt tatttctccc ctgccttgcc ttatttcatt 120

gaccccagtg tctatctagc actgggtcaa tgcaactgat 160

<210>142

<211>160

<212>DNA

<213> human cells (human cells)

<400>142

gattaaagaa gctcagagag ttgtagaagc tcgtttgtcc ttaaaatgca ggaatcatgg 60

cttctgatac atataggacg gtcaaggttt atttctcccc tgccttgcct tatttcattg 120

accccagtgt ctatctagca ctgggtcaat gcaactgatt 160

<210>143

<211>160

<212>DNA

<213> human cells (human cells)

<400>143

gtattacaca ataagcattt aagttgtgtt ttctatatga gggaataatc atgcggaata 60

ataaacaaac ttgtgtcttc taaacagtgt taaaaccatg atggacgttt tcttcttggc 120

ctaaacatga ttttcccaga gtctggtatg taatgaatgg 160

<210>144

<211>160

<212>DNA

<213> human cells (human cells)

<400>144

tgaagcaggg gttctacttg ttagtaacca tgtttaaaag tctggttgtg gaagatgttg 60

acttaataag atttgaggaa tatctcaagt aaacctttat ttatttcaag taaagctttg 120

tccttctctg cctaccttct gttgcccatg attaaagcta 160

<210>145

<211>160

<212>DNA

<213> human cells (human cells)

<400>145

gctcaggccc tgccaagact tcatatgata atcctaaaag ttaactgata aaaccccaaa 60

caccagaaga caggaggccg cactgccagt acctcctgtc accatagtaa gcccaaggaa 120

gggctgacag aatgcagtct gaaactcact gtaggggttc 160

<210>146

<211>160

<212>DNA

<213> human cells (human cells)

<400>146

agagtgttcc taggtctctc attttgggtc cctcattgtg gatccagtgg gcagtgtctc 60

tggggacaat tcatgggagc gatagaggca gctcctcaag tttgcattaa aagggacata 120

tggctcactt atcactaatc aactcttacc cagcaccctc 160

<210>147

<211>160

<212>DNA

<213> human cells (human cells)

<400>147

gactgtccta ggcctgatat ttacaaaagg caattgaacc aatgaggaga taatcttgga 60

agagctgaaa ataaagggta tatattacgg gaagaagtac ttcatctctg gggatcccag 120

gaaggtcatc accagagatc tgacgcatga aaggtagctg 160

<210>148

<211>160

<212>DNA

<213> human cells (human cells)

<400>148

gaagccttcc ttacaaatct aatttataag catgaagaag tctgagcact cagtgaagca 60

gggactgcaa gcccagagtc ctttccactc atttcccctt cttccccctc aggtcagtgg 120

gccctgttac tcacagaaat cgagggagta tcctaagcac 160

<210>149

<211>160

<212>DNA

<213> human cells (human cells)

<400>149

attaaagcta tcacagagtg ctctgcacag cactgtgtca gtgggcccaa tcaatgtaca 60

caagagatta tacatgctga ttacttaatt tgacacctct taattatttg tttgtaggaa 120

ggaaaatata accctcaatt tcaatttcag atagccaaaa 160

<210>150

<211>160

<212>DNA

<213> human cells (human cells)

<400>150

agaagtgggc tgtctagtgt tctggactcc ctagcatcaa ttttccttct atttttttcc 60

taattacttt ggaagttagc aatcccctgt tccctctaga cacgttgctc ccttctgtga 120

aggtgccctt tactcccagc tatcttacca ggaaagtgct 160

<210>151

<211>160

<212>DNA

<213> human cells (human cells)

<400>151

ttcaccagtt ctaattctgg ctttgtcttg gtaagtgggt ttttactttc tcgtggaaac 60

aattattttt cttattcaac cactgtggta actgcaccct gtggccttaa taactttccc 120

agcacccaaa ccctctattt tcatccaatt tcctccagcc 160

<210>152

<211>160

<212>DNA

<213> human cells (human cells)

<400>152

gcaaaactgt gctgaggccc ttttagggcc ataggtcttt aaaatttaaa ggactccgtg 60

gtgagtctga tgtagtctct ggtaaagtag aggaactttt taaaattccc ataccccaag 120

taagcaccac acccgcactg caagcccaca ataatgaaga 160

<210>153

<211>160

<212>DNA

<213> human cells (human cells)

<400>153

catcacaggg cttcaacctg ccagccacag ctttcagcct ctggagaata tgggcattgt 60

gaccagatac agccaccctc atttcctcca ttgggtctca ggcagatgta ggccttacta 120

tgagtaaatg tcctcaggtg tagagagaac agagccctag 160

<210>154

<211>160

<212>DNA

<213> human cells (human cells)

<400>154

ctgagaaata agggtaatac cctcaaggga cgggggaggc ccagaagcca ctgtgctggt 60

actctttgcc ctgggctggg ggatccagag cctgttccat taaaagcgca ttcaattagg 120

ttacctcatt tgtgatttgg caaactctag gcaaggtcta 160

<210>155

<211>160

<212>DNA

<213> human cells (human cells)

<400>155

aatgcttcag gaaataggaa tgttttattg agctgggaga ttaaatatat gtggagccac 60

ttgtaccact ttgggaggag ttttcagttt aagcctacag catttctggt aaaactcaca 120

tgaagtgtga caaagtaatc agaaccgctt gctattgcaa 160

<210>156

<211>160

<212>DNA

<213> human cells (human cells)

<400>156

ggagacatgt gaggcccgag ggcactgccc ttaggaaaag cctagtgtaa gtggcctttg 60

tcagagctaa caatgttgag tttaccagct gaagacaccc acatcttcct tctcttcctt 120

aggttgtggt cccccattgg catacttcct accaacatgc 160

<210>157

<211>160

<212>DNA

<213> human cells (human cells)

<400>157

tgaatcctgt gatttgtcat gcattatctt tgcttttcat tttatgtgct gttctggaaa 60

attctactcc ttcaagtgcg tttactgctt ttcatatatt ctttaggcaa gatctttttc 120

ttgaccttca ggcccatatc agcacaccag gagaaatctc 160

<210>158

<211>160

<212>DNA

<213> human cells (human cells)

<400>158

cttctgccca catttcttgg tttacccagc catcatgcct cgtggtcaga agagtaagct 60

ccgtgcccgt gagaaacgcc gcaaggcccg agatgagacc cggggtctca atgttcctca 120

ggtcactgaa gcagaggaag aagaggcccc ctgctgttcc 160

<210>159

<211>160

<212>DNA

<213> human cells (human cells)

<400>159

agaaattatggatgattcta atttttcttt cagcttagct gtattttcca atttttctag 60

aatgtaaaaa ttatttgaac attaaatttt atttttctga aagagcacat gagatagctg 120

tctcatttgt ccagtagtgc tgactgggtg tgacagagtt 160

<210>160

<211>160

<212>DNA

<213> human cells (human cells)

<400>160

ctgaaatcaa tttgagggcc ctaaatgtgg actgaagaga acatctccta cccttaacaa 60

aggtggcctc actaagtctc gtccctgaag acggctctgg gaggccccag caaagctgtg 120

cctggaaagt ctcagggagg ggagggcctt gaacctagga 160

<210>161

<211>160

<212>DNA

<213> human cells (human cells)

<400>161

tggatataaa acctcaaaca aaacaaaaaa atacagtttc atgtgggttt ttaaagaatc 60

tcactgttct ggaaagagag gtcccactca gataacgggt agtttatgtt ttcttggagg 120

actatgggac tgtatgctta cctatgtctc tatttcctgt 160

<210>162

<211>160

<212>DNA

<213> human cells (human cells)

<400>162

agtttcatga tcagaatggg ggatcaaagt cagaatacca ggattagttt caagtgattt 60

taaagacacc aatattagaa tggactgaga caggagatac aggttgcaaa gagaaataaa 120

taacaagtct tcctcaagaa tgaaaagaac tctgagagac 160

<210>163

<211>160

<212>DNA

<213> human cells (human cells)

<400>163

tttccaaaca gactaaactt gaaaacataa gtgttacttc tcaggcaaat aagtgaagac 60

caagaagcac ttctggggga aaagggggtg cagagattct cgagggaagt gtaggctttc 120

tgcttacata aattctgtgt aaatcaacaa aaaacaggaa 160

<210>164

<211>160

<212>DNA

<213> human cells (human cells)

<400>164

ctttcaaatg gaagccaaca gcttacaaaa atggctacca tctgaaaaga tgattaatag 60

tagactgact tttcacaaca aataggaatc taaactggta aacacatatc cttcttaagg 120

gattatgtat gtaatcttaa tagtggttag cactctgccc 160

<210>165

<211>160

<212>DNA

<213> human cells (human cells)

<400>165

ggaagagaaa ctcaaatcgt aatcttacaa tcagagtctt cctgccacat gtgaccattt 60

gattcattgt caaagtgtat agagaatgca agctatttcc atatgctaac ccaattttct 120

cctcccccaa ccccaaatac cttccataaa tctttttttg 160

<210>166

<211>160

<212>DNA

<213> human cells (human cells)

<400>166

ttctaaaggt aatcagagtg gatcctctcc aagccagaac acagaaagcc ccactgcgag 60

ccttgttgtc acccagtcag ccccaggcag ggttggcaag ctgcagccta aggcacattg 120

taacttcctc agctggcttc tcaggggaca gaatgactaa 160

<210>167

<211>160

<212>DNA

<213> human cells (human cells)

<400>167

gatggctgtt gaggtggttg tgaaggagga gtagctgcac ttctgttctg ttcgttcata 60

aaaaatggat tgtagttggg agtagcagca acaacagctg gagctgagtg tggttcttga 120

actaaacata tcagctgttt acctgctgca tgctgaggat 160

<210>168

<211>160

<212>DNA

<213> human cells (human cells)

<400>168

gccgaggtga cactgggttt tagatggtcc caggctgaca tgacaaatgc agagcaacct 60

gacttcctct cccgggacta aagaagttga gggctacatc caagacccac atctgctgcc 120

agcaatagga aggtcagggc aaagctagca agctgagatg 160

<210>169

<211>160

<212>DNA

<213> human cells (human cells)

<400>169

acagcctgaa ctgctaaatg caaaagaaaa gagagtaaaa cttatatttt acttaagtga 60

ctatatacta aacctaggaa ttctgaaaga atcaggtgtc catcactttg taaccctcct 120

attttcccca ttctccatat aaaatttttt cccagttgca 160

<210>170

<211>160

<212>DNA

<213> human cells (human cells)

<400>170

attatttgaa cattaaattt tatttttctg aaagagcaca tgagatagct gtctcatttg 60

tccagtagtg ctgactgggt gtgacagagt tttttacctc cctcacagct tcctctgggg 120

tcaacccccc tgcccctatg cgccatcttc gtcacacctc 160

<210>171

<211>160

<212>DNA

<213> human cells (human cells)

<400>171

ggggaatcct gcccgcggcc tcccgagact ccggtgtccc gtctccgtcc tcagcttccc 60

aagactccgg tgtccagtcg ccgcccagag cctcccaaga ctcgggtgtc cagtctccgc 120

ccagagcctc ccaagactcg ggtgtccagt ctccacccgg 160

<210>172

<211>160

<212>DNA

<213> human cells (human cells)

<400>172

accaccacta atattcttta ctttcctcct cctttcattt ttctttccgt cttgctcctc 60

cttttcatat tccaattttc cccccatatt tttccttttt gtttcaactg ctcatgatac 120

tgaggtctaa agaatttact gagtaatgag cacagatgta 160

<210>173

<211>160

<212>DNA

<213> human cells (human cells)

<400>173

agtgtggaag tttgctgtct actttatagc ctaaaatttt caagtttacc attcaagggc 60

tctttttttc ctccttaggt agtttgacta tagatagaga gaacatcaat tggtggtggt 120

ggcttgattg ccatcatctt ggctttgata cttaccatcg 160

<210>174

<211>160

<212>DNA

<213> human cells (human cells)

<400>174

aagtcaagtt tgtggtttca cctgaagtaa gaaagcatta agtcttctta tcacacccaa 60

tcatccatat taggcttggg atagaaaagg cactagagac agtagggata agatttagtc 120

cagggagtaa gtacttggtt tcattaaaac taccctagaa 160

<210>175

<211>160

<212>DNA

<213> human cells (human cells)

<400>175

gtacctcaga ttcactaatt ccctacatat acttccaaat aattgacaga tgtgaatttc 60

aaatggcaca tagtactccg tgtctgagct ctttgatgag ccctatctac ctcccctgtc 120

ctgcgtcact taataaatcc acgggctttt cctttctaac 160

<210>176

<211>160

<212>DNA

<213> human cells (human cells)

<400>176

agtagctcat tggtttgacc aatgctctag ccattttggt gaaatggata gaaccacttt 60

gatgaactaa aattttaaca cagcatccac ttcccaaagt aagtaaggct tattcattct 120

aaagaatgcc tagactatcc tcctcttttt ggaggtgtta 160

<210>177

<211>160

<212>DNA

<213> human cells (human cells)

<400>177

tgattgaggg tagagaacat aaatatctgt gagtgtgata aaatacttgt tttacttggg 60

gacagatgtg aaagtagaat aataaaattg ttaggaaaat ataaatatgt gtgtagataa 120

tttctttata attcagatgt aagtatttga acaatacact 160

<210>178

<211>160

<212>DNA

<213> human cells (human cells)

<400>178

caccctgtat acagaaaaca ggccctgaat ttctttgccc tgtattattt tctcctcagg 60

gtattcaagg aagtaaggtc ctctgtctga aggacccgag ttcagcaaaa ggatacattc 120

cagactctgt caggagtaag actcataatg aggggcaagg 160

<210>179

<211>160

<212>DNA

<213> human cells (human cells)

<400>179

accctgtata cagaaaacag gccctgaatt tctttgccct gtattatttt ctcctcaggg 60

tattcaagga agtaaggtcc tctgtctgaa ggacccgagt tcagcaaaag gatacattcc 120

agactctgtc aggagtaaga ctcataatga ggggcaaggg 160

<210>180

<211>160

<212>DNA

<213> human cells (human cells)

<400>180

taactgttgt cactgctcta taatttggcg atgatatctg gttactttca gacaaaagaa 60

aatgtctcat aacaatgggg ttttgtatga ttctgctctt tcttccagaa atacaaaaaa 120

aagacacatt taagaattta tctctgttca cattgaggaa 160

<210>181

<211>160

<212>DNA

<213> human cells (human cells)

<400>181

cacgtttttc acatttctac caatcagact aaggagcaca ttctttaggc atacgattag 60

ttttcaggta attctttatg tgctttatct taatactgat atcctggtac cttgagggtt 120

tctttatgtt gacaatagcc ccacacatat tttgaaaggc 160

<210>182

<211>160

<212>DNA

<213> human cells (human cells)

<400>182

ggaaggagga catctgagaa agccttaacc ccagagagat tgcagtcttg agcaggtgag 60

gctgaggtcc ctgttgaaag gaagcaaagg tttcccacct ctgactgttt tcagcttctg 120

cccatccgtg gcactctgag gaaacttgta aagcaatttc 160

<210>183

<211>160

<212>DNA

<213> human cells (human cells)

<400>183

ttcccaggcc gaggtgacac tgggttttag atggtcccag gctgacatga caaatgcaga 60

gcaacctgac ttcctctccc gggactaaag aagttgaggg ctacatccaa gacccacatc 120

tgctgccagc aataggaagg tcagggcaaa gctagcaagc 160

<210>184

<211>160

<212>DNA

<213> human cells (human cells)

<400>184

tccagtccaa ccctttattt tcagccctag aaagctcagg aaagggttat caggtttagt 60

gtccccctct cttttttata taagatctaa gagaggtgag attcttagtc tgacggtgta 120

gccaccactc agcagaaggg ggctggtgcc aagccctaca 160

<210>185

<211>160

<212>DNA

<213> human cells (human cells)

<400>185

acacaatttg gagcaactta aagaagatat cagaaatttt ctccaaattc tgtagaaagc 60

acaaattaaa acagaggcac ttggagttag tggttgagac aaggcaaaca cttcaaaaat 120

taaaattata tcaaggcaaa gataaaaaaa taaatcattt 160

<210>186

<211>160

<212>DNA

<213> human cells (human cells)

<400>186

caaggcatgg actccaagcc ctggtactgt gacaaaccgc cttccaagta cttcgcgaag 60

cgcaagcaca ggcgcctgag gttcccgcct gtggacaccc agaactgggt atttgtgacg 120

gagggcatgg acgacttccg ctacgcctgt cagtctcctg 160

<210>187

<211>160

<212>DNA

<213> human cells (human cells)

<400>187

cacatataga gctcataaac atattcaccc attttaatag atattggcct tatatgaaag 60

atttttcttt ctaaaaaaaa gtacttattg ctgaagaatt agatataact ttatgaaact 120

gtcagatttt taagtttaag aattagcatg ataactgtac 160

<210>188

<211>160

<212>DNA

<213> human cells (human cells)

<400>188

ctggtgggag gggtgctttc tataattgaa ctatgttttc caaatctatc cctatttttt 60

ctactgccca tcaaagaatg ttctggctgc ttttgagttt ttaagcatct ggattcaggg 120

agagagaaag taaaagacac tctagccctt tattgaaccc 160

<210>189

<211>160

<212>DNA

<213> human cells (human cells)

<400>189

ccaggaactc agtgtggcca gtagattgaa aagacaagtc attattgaaa aaacccttat 60

tagctcactg tcaaagtcaa tatgataaat tattgtgcaa aaatactcca tccctctcct 120

ctacttctgt ggaaggactc tatttcacca catcttaact 160

<210>190

<211>160

<212>DNA

<213> human cells (human cells)

<400>190

acaaagccac cacactgttt aaaatcagat tcactatctt ccagggccct cttactcaga 60

aaagtgatct tcctacaacc ctttctattt tagaatatac catcaccaat gacctactta 120

tctaacctag gaacttctga gtcacccaaa ccatttatcc 160

<210>191

<211>160

<212>DNA

<213> human cells (human cells)

<400>191

agctcattag agaagctact taaagtccac agtgaagtca ccttccactt gcaggcagta 60

ggaatttata ttgggggctt gaggagcttt ctaggtttgt aacttcttct atggggagcc 120

tacctggaac agtacaagga ggaaggagca gcagcaaggg 160

<210>192

<211>160

<212>DNA

<213> human cells (human cells)

<400>192

cccctcctct ttaccccatt gtagatcaga acaaaagctt gaaaagtttg aggctaccat 60

tacagtttga aaaatttgtc ggaagcagac tcaccctagg atgcaatttg aaatctcttc 120

ctacattctg tggtcattta tttgtttttt cttcttttct 160

<210>193

<211>160

<212>DNA

<213> human cells (human cells)

<400>193

cctgaaatac ctgacctctg ctaaccatac aacatgttgg tgagaactgg ggagttccaa 60

aaaggataaa aaaggcagaa tagtgttctc ctgagaatag tattaaggac ccaattgcaa 120

atgatttttt gaaagccatt cccggccggg cacggtggct 160

<210>194

<211>160

<212>DNA

<213> human cells (human cells)

<400>194

caacagtatc ttctacatag tactaatggg cattgggatt tgaacctaag tcttctggtg 60

caatattcca tggagtttcc ccatcttcac actgtgcttt ccattgccct gagatcaata 120

tagagtcctt aatgcagtgc atattgttac catggctggt 160

<210>195

<211>160

<212>DNA

<213> human cells (human cells)

<400>195

ttcggcctcc caaagtgctg gtgggaggggtgctttctat aattgaacta tgttttccaa 60

atctatccct attttttcta ctgcccatca aagaatgttc tggctgcttt tgagttttta 120

agcatctgga ttcagggaga gagaaagtaa aagacactct 160

<210>196

<211>160

<212>DNA

<213> human cells (human cells)

<400>196

attatataaa ggttttggac aatagctggg gcttgaggga gtgagaaaag tcaagcaaaa 60

ggggatttgt ccctctgggg attaattcca ggacgacccc acccccgccg ccctcaccac 120

acacactcac cctccctacc ccgaggatac aaaaatccga 160

<210>197

<211>160

<212>DNA

<213> human cells (human cells)

<400>197

tacatctacc tagtgccaac gtccattgac aatgaagtaa aacctacctg tcacccattt 60

ttccctctgc atgccacccc gacctggtgg cttaatattc catttttttt cagtcacttt 120

cctataatct tctacctcca ccagactgtt ctgtaaaaag 160

<210>198

<211>160

<212>DNA

<213> human cells (human cells)

<400>198

tttggacata tacacagatg tgtgttgaaa gtgggtgata gaatctgaga cccagatctg 60

agagtcgatc cacagagggc gtaactgaag tcaatttcct caatattctc ttagtgaagg 120

tatgtaaaga ggtgtgaaaa tgaacaatta ctaagcatgc 160

<210>199

<211>160

<212>DNA

<213> human cells (human cells)

<400>199

aagtccctaa aaggaagaag gagtaagatt ccagtactaa aattaagatc aatggaccta 60

agaacaagag tagattcaag gatcaatccg cagaactaga gggatgtggg aaacatggct 120

ggtatcttgt cttagtccat ttttgctact ataacagaat 160

Claims

1. A kit for pre-implantation embryonic genetic diagnosis and prenatal diagnosis of DMD, the kit comprising a DMD gene-linked SNP site capture probe set, wherein the DMD gene-linked SNP site capture probe set is an oligonucleotide probe with a base sequence shown as SEQ ID No.1-199, and does not comprise other oligonucleotide probes.

2. The kit of claim 1, wherein the oligonucleotide probes are all single-stranded DNA.

3. The kit of claim 1 or 2, wherein the oligonucleotide probes are each labeled with biotin.

4. The kit according to claim 1 or 2, further comprising linked SNP site capture probes located in a small number of cell genome amplification preferential regions in the upstream and downstream 2M inner regions of the DMD gene, enrichment buffer BL, enrichment buffer HY, library enrichment binding buffer, washing buffer WB1, washing buffer WB3, library enrichment eluate, buffer NE, PCR reaction solution, and product purification eluate.

5. Use of a kit according to any one of claims 1 to 4 for the preparation of a kit for the prenatal and/or prenatal genetic diagnosis of DMD.

6. Use of the kit according to any one of claims 1 to 4 for the preparation of a kit for pre-implantation embryonic genetic diagnosis and prenatal diagnosis of DMD, wherein the pre-implantation embryonic genetic diagnosis and prenatal diagnosis of DMD refer to family verification, pre-implantation embryonic genetic diagnosis and prenatal diagnosis performed simultaneously.