TWI467020B - Method of detecting dmd gene exon deletion and/or repeated - Google Patents

Description

Method for detecting exon deletion and/or duplication of DMD gene

The invention relates to the field of gene detection, in particular to the analysis of DMD genes and methods thereof.

The DMD gene is the largest human gene ever discovered, and this gene sometimes mutates, such as a mutation in the gene at 1:3500 in a newborn baby boy. Large fragments of one or more exons within the DMD gene are deleted, involving both the proximal and middle hotspots of the gene (exons 3-7 and exons 44-55). Large fragments of one or more exons within the DMD gene are repetitive, accounting for approximately 6% of the DMD mutation. More DMD gene development is point mutations, deletions and insertions of small fragments.

Duchenne muscular dystrophy (DMD) is an X-linked recessive genetic disease, and the gene associated with the disease is DMD. The onset of approximately 60% of DMD cases is associated with deletion of large fragments of one or more exons in the gene, involving both proximal and intermediate hotspots (exons 3-7 and exons 44-55) . Approximately 6% of the genetic variants of DMD cases are associated with large intra-gene fragments. The remaining cases stem from point mutations in genes, deletions and insertions of small fragments.

At present, the detection methods of DMD genes mainly include the following: microarray comparative genomic hybridization technology (a-CGH), MLPA, MAPH, SCAIP, multiplex PCR, Southern blot, Sanger sequencing and second generation sequencing technology. For high-throughput detection of exon deletions and duplications of DMD genes, the disadvantages of these assays are low throughput and efficacy. Rate difference. Therefore, there is a need in the art for new high throughput methods for detecting exon deletions and duplications of DMD genes.

The present invention relates to a method for detecting exon deletion and/or duplication of a DMD gene, which utilizes a sequence message design probe of a current DMD gene to sequence a DNA fragment obtained by capture enrichment, and obtain an exon of the DMD gene by analysis. Missing message.

The present invention provides a method for detecting exon deletion and/or duplication of a DMD gene, comprising the steps of: 1) breaking genomic DNA extracted from a sample to be tested and a normal control sample into a double-stranded DNA fragment, respectively, and Adding a linker sequence to both ends of the strand DNA fragment; 2) amplifying the double-stranded DNA fragment carrying the linker with the first primer and the second primer to obtain a first amplification product; 3) the first amplification product After denaturation, the nucleic acid wafer is used for hybridization capture; 4) the captured nucleic acid is amplified by the third primer and the fourth primer to obtain a second amplification product; 5) the second amplification product is sequenced to obtain a sequencing sequence fragment. 6) aligning the sequencing sequence fragments to the exon sequence of the reference DMD gene and flanking the exon; 7) determining the DMD gene of the sample to be tested by comparing the comparison between the sample to be tested and the normal control sample Whether the exon has a repeat and/or a deletion, that is, if the sequencing sequence of the sample to be tested on the exon reference sequence of the gene is significantly more than/less than the sequencing sequence of the normal control sample, Is the exon of the gene heavy? And / or lack of Lost.

The present invention also provides a method for detecting Duchenne muscular dystrophy in a subject, comprising: detecting a mutation of a DMD gene from a sample of the subject using the method for detecting exon deletion and/or duplication of the DMD gene of the present invention If a mutation in the DMD gene is detected, the subject has Duchenne muscular dystrophy or is susceptible to Duchenne muscular dystrophy.

Because DMD is an X-linked sex disorder, for women, her DMD gene mutations can be homozygous or heterozygous, so female subjects can be diagnosed as Duchenne muscular dystrophy, or Duchenne muscular nutrition. A susceptible person or a DMD mutation carrier. It is understood that the probability of developing Duchenne muscular dystrophy in male offspring of female subjects with heterozygous DMD gene mutations is 50%.

For men, since he has only one X chromosome, if he detects a mutation in his DMD gene, he can be diagnosed as Duchenne muscular dystrophy.

The method of the invention combines sequence capture technology, high throughput sequencing and biomessage analysis to detect DMD genes. The combination of these three techniques is a very effective detection strategy for DMD gene deletion and/or duplication.

The invention is described in more detail in the following examples, which are intended to be illustrative only, but those of ordinary skill in the art will understand that the following examples are only illustrative of the invention and are not to be construed as limiting the invention. range.

Using Target Region Capture Techniques, Using Exons to Capture Wafers to Human DMD Genes The exon sequencing, and then the study of DMD gene exon deletion and repeated mutation, is still a new technology. The basic principle of this technique is to use a set of oligonucleotide probes to capture the target sequence on the genome, and then use these primers designed according to the DMD gene gene region sequence and/or the linker sequence to perform PCR amplification on these captured sequences. The high-throughput sequencing of these amplified products is performed to identify the base sequence in the DNA sample, and the sequence information obtained by the sequencing is analyzed by the biological information analysis method, thereby finding the mutation information of the target sequence, including the single nucleotide. Variation, insertion/deletion, duplication, change in exon copy number, etc.

The present invention provides a method for detecting exon deletion and/or duplication of a DMD gene, comprising the steps of: 1) breaking genomic DNA extracted from a sample to be tested and a normal control sample into a double-stranded DNA fragment, respectively, and Adding a linker sequence to both ends of the strand DNA fragment; 2) amplifying the double-stranded DNA fragment carrying the linker with the first primer and the second primer to obtain a first amplification product; 3) the first amplification product After denaturation, the nucleic acid wafer is used for hybridization capture; 4) the captured nucleic acid is amplified by the third primer and the fourth primer to obtain a second amplification product; 5) the second amplification product is sequenced to obtain a sequencing sequence fragment. 6) aligning the sequencing sequence fragments to the exon sequence of the reference DMD gene and flanking the exon; 7) determining the DMD gene of the sample to be tested by comparing the comparison between the sample to be tested and the normal control sample Whether the exon has a repeat and/or a deletion, that is, if the sequencing sequence of the sample to be tested on the exon reference sequence of the gene is significantly more than/less than A normal control sample sequencing sequence indicating whether the exon of the gene has a repeat and/or deletion.

In step 1) of the method of the invention, the double-stranded DNA after disruption is preferably from 100 to 1000 bp, more preferably from 150 to 500 bp, most preferably from 200 to 300 bp, especially from 200 to 250 bp, The above length is expressed as the main band position of double-stranded DNA electrophoresis.

In step 1) of the method of the invention, the double-stranded DNA preferably has a blunt end after being interrupted, for example by end repair. In another preferred embodiment, the method further comprises the steps of: adding "A" to the 3' end of the blunt-ended double-stranded DNA fragment, and adding a double-stranded DNA fragment of "A" to the 3' end with a "T" The linkers are joined to form a double-stranded DNA fragment mixture with a linker at both ends. The linker sequence is preferably from 20 to 150 nt in length, especially from 50 to 100 nt. Those of ordinary skill in the art can select a suitable linker sequence based on the sequence, or a sequence commonly used in commercially available kits as a linker sequence.

In step 1) of the method of the invention, preferably the two ends of the double-stranded DNA fragment are ligated to the linker sequence via a linker ligation sequence. In another preferred embodiment, the linker joining sequence is poly(N) _n , wherein each N is independently selected from A, T, G or C, and n is any positive integer selected from 1-20. In another preferred embodiment, the linker sequence is poly(A) _n , wherein n is a positive integer from 1-20, preferably n=1-2. In another preferred embodiment, the linker joining complementary region sequence is poly(N') _m , wherein each N' is independently selected from A, T, G or C, and m is a positive integer of 1-20. And poly(N) _n and poly(N') _m are complementary sequences. In another preferred embodiment, m is any positive integer selected from 1-3. In another preferred embodiment, the length of the linker joining complementary region is the same as the length of the linker ligation sequence, i.e., poly(N) _n and poly(N') _m are fully complementary sequences. In another preferred embodiment, the linker complementary region is poly(T) _m , wherein m is a positive integer from 1-20, preferably m=1-2. Those of ordinary skill in the art can select a suitable linker sequence based on the sequence, or a sequence commonly used in commercially available kits as a linker sequence.

In step 2) of the method of the invention, preferably the first primer and the second primer are designed according to the gene region sequence of the DMD gene and/or the linker sequence. In a preferred embodiment, the first primer and the second primer have a linker binding region corresponding to the primer binding region of the linker, and a sequencing probe binding region located outside the linker binding region. In another preferred embodiment, the first primer and the second primer are oligonucleotides of 30-80 bp in length. In another preferred embodiment, the first primer and the second primer are 55-65 bp in length. In another preferred embodiment, the first primer and the second primer are different.

In step 3) of the method of the present invention, the wafer used in the present invention can be designed by attaching a high-density DNA fragment array to a solid phase such as a glass sheet in a certain order or arrangement by microarray technology. On the surface, fluorescently labeled DNA probes, through the principle of base-complementary hybridization, perform a large amount of gene expression and monitoring to capture target sequences. For example, the wafer can be designed and synthesized by Roche NimbleGen, USA.

In step 3) of the method of the invention, preferably the nucleic acid wafer is immobilized with from 5 to 200,000 specific probes corresponding to the DMD gene. In another preferred embodiment, the type of the specific probe on the wafer is from 50 to 150,000, more preferably from 500 to 100,000, and most preferably from 5,000 to 80,000. In another preferred embodiment, the sequence of the probe corresponds to the following region of the DMD gene: preferably 50-500 nt, more preferably 100-300 nt, in front and rear of the exon and/or exon. Preferably 200 nt. In another preferred embodiment, the specific probe has a length of 20-120 mer, preferably 50-100 mer, more Good place, 60-80 mer. In another preferred embodiment, the specific probe is a fully synthetic or in vitro clone synthesis.

After step 3) of the method of the present invention, preferably, the method comprises the steps of: blocking a region corresponding to the first primer and the second primer at both ends of the amplification product with a blocking molecule, thereby obtaining a single-strand expansion in which both ends are closed. The mixture of product additions is subjected to the subsequent step 4) using a mixture of said blocked single-stranded amplification products. In another preferred embodiment, the blocking molecule blocks a 70%-100% region of the first PCR amplification product corresponding to the first primer and the second primer. In another preferred embodiment, the blocking molecule blocks a 100% region of the first PCR amplification product corresponding to the first primer and the second primer.

In step 4) of the method of the invention, preferably the third primer and the fourth primer are designed according to the gene region sequence of the DMD gene and/or the linker sequence. In a preferred embodiment, the third primer and the fourth primer respectively correspond to or bind to the first primer and the second primer, respectively. In another preferred embodiment, the third primer and the fourth primer are specifically bound to the outside of the first primer and the second primer, respectively, and the length is smaller than the first primer and the second primer. In another preferred embodiment, the third primer and the fourth primer are 15-40 bp in length, preferably 20-25 bp. In another preferred embodiment, the third and fourth primers are different.

In step 5) of the method of the invention, the sequencing preferably employs second generation sequencing techniques such as illumina solexa, Hiseq 2000, ABI SOLiD, Roche 454 sequencing platform and/or Ion torrent. In another preferred embodiment, the mixture of the second amplification product is hybridized with a sequencing probe immobilized on a solid phase carrier, and subjected to solid phase bridge PCR amplification to form a sequencing cluster; The sequencing cluster is sequenced by the "edge synthesis-edge sequencing" method to obtain the nucleotide sequence of the disease-related nucleic acid molecule in the sample. Currently, Some biotech service companies offer sequencing services.

In step 6) of the method of the invention, aligning the sequencing sequence to the exon of the reference DMD gene and flanking the exon can be performed by software known in the art, such as a short oligonucleotide analysis package. (Short Oligonucleotide Analysis Package, SOAP) alignment and BWA (Burrows-Wheeler Aligner) alignment; the flanking length is preferably 50-500 nt, more preferably 100-300 nt, and most preferably 200 nt.

After step 6) of the method of the present invention, the original sequencing sequence of the sequencing result can be quality-controlled to remove the unqualified sequencing sequence, wherein the items included in the original read quality control are shown in the following table;

In step 7) of the method of the present invention, by performing a bioinformatics analysis method on the sequencing result, it can be determined whether there is a statistically significant difference between the exon of the DMD gene mutation and the normal person. In a preferred embodiment, depth calculations, such as java, C++, or Perl, can be performed in a suitable computer language.

In a preferred embodiment, the steps of comparing the test sample to the normal control sample are as follows: Step 1: For each DMD gene exon, the number of sequencing sequences aligned to the exon is compared to the number of sequencing sequences aligned to all exons, and a standardized sample to be tested is obtained. Sequencing sequence number ratio, normalizing the number of sequencing sequences on the exon to the exon relative to the number of sequencing sequences aligned to all exons, obtaining a standardized control sample sequencing sequence number ratio; second step: pair Comparing the ratio of the number of normalized sample sequencing sequences to the number of normalized control sample sequencing sequences, if they are statistically significant, when: the ratio of the number of sequencing sequences of the standardized sample to be tested is smaller than the standardized control When the number of sample sequencing sequences is proportional, the ratio of the number of sequencing sequences of the standardized sample to be tested is multiplied by 2 and compared with the ratio of the number of sequencing sequences of the standardized control samples. If they are statistically significant, it indicates that the exon is not Hybridization loss occurs when the ratio of the number of sequencing sequences of the standardized sample to be tested is greater than the standard When comparing the number of sequencing sequences of the control sample, the ratio of the number of sequencing sequences of the standardized sample to be tested is divided by 2 and compared with the ratio of the number of sequencing sequences of the standardized control samples, if they are statistically significant, indicating that the ratio is No heterozygous deletions occurred in the neutrons.

Through the two-step calculation method, the change in the exon copy number of the DMD gene can be determined to determine whether a duplication and/or a deletion has occurred.

In still another preferred embodiment, the steps of comparing the test sample with the normal control sample are as follows: a. For an exon of the DMD gene, the sample to be tested is compared to the exon by Equation 1. The sequencing depth of the sequencing sequence exonN_depth and the ratio of the average sequencing depth averaged_depth of the sequencing sequence on all exons is %exonN ; the %exonN is substituted into Equation 2, and the Z-score of the exon sequencing depth is calculated. Wherein, for the exon, the sequencing depth of the sequencing sequence on the exon is compared with the sequencing depth of the sequencing sequence on the exon and the average sequencing depth of the sequencing sequence on the entire exon is compared, and %exonN is calculated according to Formula 1 ( Normal) , mean %exonN(normal) is the average of all control samples %exonN(normal) , SD%exonN(normal) is the standard deviation of all control samples %exonN(normal) ; if Z-score is greater than the first preset The cut-off value indicates that the depth of sequencing of the exon of the DMD gene is significantly different between the sample to be tested and the normal sample, and is further screened; b. The DMD gene exon with significant difference in sequencing depth is divided into two cases. Screening: When exonN is less than the averaged_depth value, multiply %exonN by 2 into Equation 2, and the calculated Z-score value is less than the second preset cutoff value, indicating that the exon has a heterozygous deletion, and the calculated Z The -score value is greater than the second preset cutoff value indicating that the exon is not heterozygous; when exonN is greater than the averaged_depth value, %exonN is divided by 2 into Equation 2, and the calculated Z-score value is less than the third pre- Let the cutoff value indicate the explicit display Repeat occurs, Z-score is greater than the value calculated by the third predetermined cutoff value indicates that exon heterozygous deletion does not occur.

Through the two-step calculation method, the change in the exon copy number of the DMD gene can be determined to determine whether a duplication and/or a deletion has occurred.

The first preset cutoff value, the second preset cutoff value, and the third preset cutoff value in the above steps a and b are the same or different. The selection of the cutoff value is determined by the inventor based on statistical theory after conducting a large number of experiments, as follows:

In the case of conducting experiments, the inventors found that the Z-values obtained by the method of the present invention conform to the statistical normal distribution map, see Figure 1. For the first step of determining whether the exon of the DMD gene of the sample to be tested has a repeat and/or deletion, when the Z value is 3.0, there is 99.9% credibility---- DMD female carrier and normal The copy number of a person in the same exon is different. For the second step, in order to exclude the false positive in the first step, if the female carrier is missing the copy, then her double should be no significant difference from the normal person (normal person should be twice), so multiply by 2 Detection. In the same way, the same is true for duplicate copies.

Although a cutoff value of 3.0 is used in the embodiment of the present invention, the cutoff value may be greater than 3.0 or less than 3.0. For example, you can use a cutoff value of 1.64 (corresponding to 90% confidence), 1.96 (corresponding to 95% confidence) or 2.58 (corresponding to 99% confidence). The higher the percentage of likelihood, the higher the credibility. . The field generally adopts a reliability of more than 90%, that is, the cutoff value must be at least 1.64 or more.

In the present invention, gene mutations include copy number variation (CNV).

Since the DMD gene is located on the X chromosome, the method of the present invention is particularly suitable for use in women who have a heterozygous deletion of the exon of the DMD gene.

Thus, it will be understood by those of ordinary skill in the art that the mutation detection methods of the present invention can be used to detect mutations in the DMD gene.

In the present invention, the probes and primers used for performing the PCR can be designed based on the current solid phase wafer hybridization technique, or can be performed by a biotechnology service company. It is understood by those of ordinary skill in the art that the DMD gene region is captured with high specificity and high coverage on the same wafer. For example, Roche NimbleGen's 2.1 M human exon sequence capture wafer captures approximately 180,000 exons and approximately 550 miRNAs.

The DMD gene exon deletion and repetitive information obtained by the method of the present invention can be used, for example, to genotype a population.

In an embodiment of the present invention, the DMD gene of the DNA sample is detected by the combined sequence capture technology, high-throughput sequencing and biological message analysis method of the present invention, and the mutation information of the DMD gene in the sample is obtained.

In the present invention, the sequencing sequence, the sequencing sequence fragment, and the read segment all refer to a data DNA sequence produced by the sequencer. The depth of sequencing refers to the number of reads.

Example

As used in the present embodiment, the term "primer" refers to a generic term for an oligonucleotide which is complementary to a template and which synthesizes a DNA strand complementary to a template in the action of a DNA polymerase. The primer may be natural RNA, DNA, or any form of natural nucleotide, and the primer may even be a non-natural nucleotide such as LNA or ZNA. The primer is "substantially" (or "substantially") complementary to a particular sequence on a chain on the template. The primer must be sufficiently complementary to a strand on the template to begin extension, but the sequence of the primer does not have to be fully complementary to the sequence of the template. For example, a sequence that is not complementary to the template is added to the 5' end of a primer complementary to the template at the 3' end, such primers are still substantially complementary to the template. As long as there is a sufficiently long primer to bind well to the template, the non-fully complementary primer can also form a primer-template complex with the template for amplification.

In this embodiment, the sequence and name of several important types of primers are shown in Table 1.

The first primer (SEQ ID NO: 1) and the second primer (SEQ ID NO: 2) amplify the DNA double-stranded nucleic acid fragment carrying the linker to obtain a first PCR amplification product, a first primer and a second primer A linker binding region having a primer binding region corresponding to the linker and a sequencing probe binding region located outside the linker binding region. The action of blocking molecule 1 (SEQ ID NO: 3) and blocking molecule 2 (SEQ ID NO: 4) is complementary to the linker when sequence capture is performed, avoiding the capture of non-specific sequences. Third primer (SEQ ID NO: 5) and The role of the fourth primer (SEQ ID NO: 6) is to amplify a large amount of the captured specific DNA fragment for subsequent sequencing.

The study recruited 1 DMD female carrier and 4 female normals and signed a written informed consent form.

Wafer preparation and hybridization were performed according to the instructions of Roche NimbleGen and sequenced according to Illumina's instructions, as follows.

1: wafer design

The reference sequence was NCBI build 37/hg19 (obtained from http://www.ncbi.nlm.nih.gov/) and the exon sequence of the DMD gene and 200 bp before and after the exon were designed and synthesized by Roche NimbleGen, USA.

2: Library preparation

Human peripheral blood was taken and genomic DNA was extracted to obtain 3 μg of DNA. The obtained human genomic DNA sample was extracted and fragmented on a Covaris S2 instrument (purchased from Covaris, USA), and finally a mixture of DNA double-stranded fragments having a main band of 200 bp was disrupted.

Next, the above fragment was purified by Ampure Beads method according to Agencourt AMPure protocol (Beckman, USA). In short: the DNA fragment is end-repaired into a mixture of fragments with blunt ends, and an "A" is added to the 3' end of each single strand; then, in the linker ligation reaction system (PE library), according to the reagent The instructions in the box are connected, and the DNA fragment is added with a "T" linker; after the connection, purification is continued according to Agencourt AMPure protocol (Beckman, USA), and excess reagents such as buffer, enzyme, ATP are removed. Etc., finally get the DNA with the linker.

Since the concentration of the DNA sample with the linker was very low, the amplification and enrichment were carried out, and the PCR reaction was run on a Bio-Rad PTC-200 PCR machine (PCR reaction system: 94 ° C, 2 min; denaturation at 94 ° C for 15 s, Annealing at 62 ° C for 30 s, extending at 72 ° C for 30 s, a total of 4 cycles; finally extending at 72 ° C for 5 min).

The 50 μL PCR amplification reaction system contains: 34 μL Nuclease-Free water, 10 μL 10×pfx Amplification Buffer, 4 μL dNTP (10 mM), 4 μL MgSO4 (50 mM), 2 μL Platimum Pfx DNA polymerase, 8 μL One primer (SEQ ID NO: 1) (10 μM), 8 μL of the second primer (SEQ ID NO: 2) (10 μM), 23 μL of the sample DNA after ligation of the linker (buffer and enzyme purchased from INTROTROGEN Platinum ® Pfx DNA Polymerase Kit).

The amplified DNA was ligated with a linker, and the PCR product was purified using the Ampure beads method according to the procedure of Agencourt AMPure protocol (Beckman, USA).

The purified product after completion of the reaction can be stored at 4 ° C for several days, or at -20 ° C for several weeks, or directly used for subsequent sequence capture.

3: Sequence capture

The prepared DNA sample was evaporated to dryness at 60 ° C in a SpeedVac, and then 11.2 μL of ultrapure water was added to fully dissolve. The samples were centrifuged at full speed for 30 seconds, and the following two reagents were added: 18.5 μL of 2×SC Hybridiation Buffer (available from Roche NimbleGen, USA) and 7.3 μL of 1×SC Hybridiation Component A (available from Roche NimbleGen, USA). After shaking and mixing, the cells were centrifuged at full speed for 30 seconds, and then the DNA was sufficiently denatured at 95 ° C for 10 minutes to obtain a single-stranded DNA library with a linker.

According to the instructions of Roche NimbleGen's kit, the wafer with the corresponding probe was fixed on the hybrid instrument (Roche NimbleGen, USA) as required, the denatured sample was added to the wafer and the wafer was closed, and then the hybridization procedure was set at 42 °C. Hybridize under stringent conditions for 64-72 hours. In hybrid systems, the concentration of probe molecules on a gene wafer is much higher than the concentration of the target molecule. The hybridization reaction system contained: 450 μg of Cot-1 DNA, 5 μg of DNA library (obtained in step 1), 10 μL of blocking molecule 1 (SEQ ID NO: 3), and 110 μL of blocking molecule 2 (SEQ ID NO: 4). Cot-1 DNA was obtained by Human Cot-1 DNA®-Fluorometric QC (Invitrogen) according to the supplier's instructions.

After the hybridization is complete, the wafer wash and sample are eluted in the following order:

The NaOH eluate was recovered and neutralized with 40 μL of 20% glacial acetic acid; The neutralized solution was purified using Qiagen MinElute PCR Purification Kit, and the captured sample was finally dissolved in 165 μL of pure water.

The above-captured DNA library was subjected to PCR amplification. The reaction conditions were: 98 ° C for 30 s; 15 cycles (98 ° C for 15 s; 60 ° C for 30 s; 72 ° C for 30 s); 72 ° C for 5 min; 4 ° C for standing.

The amplification was divided into 6 tubes of 50 μL for:

The PCR product was purified by Ampure Beads method according to Agencourt AMPure protocol (Beckman, USA), and dissolved in 32 μl of elution buffer (Elution Buffer (QIAGEN)) (Thermo Fisher Scientific Inc.; model: Nanodrop) 8000) and Bioanalyzer 2100 (Agilent; model: 2100) were tested for concentration.

4: Detection of enrichment by linker-mediated PCR (LM-PCR) amplification products and real-time fluorescent quantitative PCR (QPCR):

Follow the instructions provided in the NSC Assay mix kit from Roche NimbleGen, USA:

1) Remove the diluted 4 NSC Assay mix and dissolve on ice.

2) The uncaptured (Non-Captured) and successfully captured (Captured LM-PCR) products were diluted to 20 ng/μl according to the previous Nanodrop detection concentration, with a final volume requirement of >5 μl.

3) According to 4 NSC Assays per sample, each sample includes 2 DNA templates, each sample requires 4 × 2 = 8 reactions, and each plate requires 1 negative control for 4 reactions. A maximum of 11 samples can be detected on a 96-well plate.

4) Prepare the QPCR reaction mixture in a 1.5 ml centrifuge tube. For each reagent usage, the mixture can be uniformly configured according to the specific sample amount. Negative and positive controls need to be included in the calculation.

5) Transfer the configured 12 μl QPCR reaction mixture to a 96-well QPCR reaction plate, add 3 μl of diluted 1 ng/μl LM-PCR product, add all reagents and samples, and use a parafilm. Flat seal. Centrifuge at 4000 rpm for 2 min.

6) Place the 96-well plate on the QPCR instrument and test according to the procedure shown in Table 2 below.

7) Analyze the test results after completion of the test, sort out the QPCP test data, and calculate the enrichment E=(ef) ^△Ct using the difference ΔCt of the Ct value, where ef represents the Q-PCR amplification efficiency of the target gene, ideally expanded The efficiency ef value is 2. In order to calculate ΔCt, Non-capture Ct refers to the C-value detected by Q-PCR amplification using the N-LM-PCR purified product before hybridization as a template and the target gene-specific primer; capture Ct means The C-LM-PCR purified product after hybridization was used as a template, and the target gene-specific primer was used for Q-PCR amplification, and the detected Ct value. See the table below:

It is judged whether the library is qualified, and whether the next test can be carried out: it is necessary to judge the type of the library after the average enrichment multiplier value, and in the present embodiment, the average enrichment value > 60 is used for the next step of sequencing.

5: Sequencing and data analysis

The hybridization reaction is carried out on a wafer, and the target sequence is complementary to the probe in 64-72 hours of hybridization on the wafer, thereby being captured. The eluted samples were double-end sequenced in a Solexa sequencing platform. The sample source of the sequencing data is analyzed by data analysis, and the capture effect of the sample is calculated.

Reads from all lanes were first aligned to the DMD gene exon sequence and exon of the reference sequence NCBI build 37/hg19 (available from http://www.ncbi.nlm.nih.gov/) using BWA software. Go up and down 200 bp. The input of the comparison is the contaminated fq file such as the filter joint, and the output of the comparison is the original comparison result - SAM file. The samtools tool is used to perform a series of processing on the original results: after format conversion and compression, the alignment results are sorted by chromosome sequence. Second, merge the lanes of the same library together and finally merge all the libraries together. After this series of treatments, the qualified input file-BAM format file, which is the mutation detection software SOAPsnp (soap.genomics.org.cn/soapsnp.html), is obtained, which is the interface to enter the mutation detection software.

By calculating the number of reads on each exon and the number of reads on all exons, the depth of each exon and the average depth are calculated, and %exonN is calculated by substituting into Equation 1. Taking the remaining four female non-carriers as reference, the mean%exonN(normal) and S.D.%exonN(normal) are substituted into Equation 2 to calculate Z-score. The calculation found that the carrier was The exons Z-score of 10, 11, 15, 23, 26, 29, 46-51, 70, 71, and 73 are: 3.80, 3.23, 4.33, 3.72, 6.16, 5.09, 7.04, 9.08, 6.81, 10.10, respectively. 26.95, 8.51, 4.36, 3.65, and 3.24 are all greater than the cutoff value of 3.00. Next, the second step is performed, and the results are 19.60, 11.26, 24.77, 12.50, 37.55, 33.79, 0.85, 0.32, 0.49, 0.63, 1.01, 1.52, 60.62, 22.32, and 11.30, of which 0.85, 0.32, 0.49 The exons 46-51 corresponding to 0.63, 1.01, and 1.52 are all less than 3.0. Thus, using the method of the present invention, it was shown that the carrier had a deletion in exon 46-51.

As follows, in order to verify the results of the present invention, the carrier 46-51 exon copy number was detected and analyzed by real-time fluorescent quantitative PCR. The experimental steps are as follows:

After designing the primers, a sample of 1 DMD female carrier and 4 female normal humans was subjected to qPCR amplification. The reaction system is: pre-formed mother liquor

Take the above prepared mother liquor plus the following ingredients to prepare a 2.0 ml reaction system:

The experimental conditions were as follows: (1) amplification curve: 95 ° C, 10 s; 95 ° C, 15 s; 59 ° C, 15 s, 40 cycles above; (2) dissolution curve: 72 ° C, 30 s; 95 ° C, 15 s; 60 ° C, 60 s; 95 ° C, 15 s.

After the test, the QPCR product was subjected to 2% agarose gel electrophoresis. Through observation of the amplified bands, it was found that female DMD carriers were missing in exon 46-51, while 4 female normal persons were exposed in 46-51. The child is not missing.

Therefore, the results calculated by the exons of the present invention are consistent with the results of real-time fluorescent quantitative PCR, indicating that the method of the present invention is feasible.

The above are only the preferred embodiments of the present invention, and all changes and modifications made to the scope of the present invention should be within the scope of the present invention.

The following figures are intended to illustrate the specific embodiments of the invention and are not intended to limit the scope of the invention as defined by the appended claims.

Figure 1 shows a normal distribution map for determining the cutoff value of the present invention.

<110> Shenzhen Huada Gene Technology Co., Ltd. Shenzhen Huada Gene Research Institute

<120> A method for detecting exon deletion and/or duplication of a DMD gene

<130> FP1121166P

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Claims (20)

A method for detecting exon deletion and/or duplication of a DMD gene, comprising the steps of: 1) breaking genomic DNA extracted from a sample to be tested and a normal control sample into a double-stranded DNA fragment, respectively, and in the double-stranded DNA fragment Adding a linker sequence to both ends; 2) amplifying the double-stranded DNA fragment carrying the linker with a first primer and a second primer to obtain a first amplification product, wherein the first primer is SEQ ID NO. The second primer is SEQ ID NO. 2; 3) after denaturation of the first amplification product, hybridization capture with a nucleic acid wafer; 4) amplification of the captured nucleic acid with a third primer and a fourth primer, Obtaining a second amplification product, wherein the third primer is SEQ ID NO. 5, the fourth primer is SEQ ID NO. 6; 5) sequencing the second amplification product to obtain a sequencing sequence fragment 6) aligning the sequencing sequence fragment to the exon sequence of the reference DMD gene and flanking the exon; and 7) determining the DMD gene of the sample to be tested by comparing the comparison between the sample to be tested and the normal control sample Whether there are duplications and/or deletions in the exons, ie if the genes are aligned Reference sequence on the sample to be tested in a statistically significant sequence sequenced more / less than the normal control sample sequencing sequence, indicating whether exon gene duplication and / or deletion. The method according to claim 1, wherein the step of comparing the sample to be tested and the normal control sample in step 7) is as follows: First step: For each DMD gene exon, the number of sequencing sequences aligned to the exon is compared to the number of sequencing sequences aligned to all exons, and the ratio of the number of sequencing sequences of the sample to be tested is obtained. The number of sequencing sequences aligned to the exon of the control sample is normalized relative to the number of sequencing sequences aligned to all exons, and a standardized ratio of number of sequencing sequences of the control sample is obtained; second step: the standardized Comparing the ratio of the number of sequencing sequences of the sample to be tested to the ratio of the number of sequencing sequences of the normalized control samples, if they are statistically significant, when the ratio of the number of sequencing sequences of the standardized sample to be tested is smaller than the number of sequencing sequences of the standardized control sample In the ratio, the ratio of the number of sequencing sequences of the standardized sample to be tested is multiplied by 2 and compared with the ratio of the number of sequencing sequences of the standardized control samples. If they are statistically significant, it indicates that the exon has no heterozygous deletion. When the ratio of the number of sequencing sequences of the standardized sample to be tested is greater than the standardized control When the number of sample sequencing sequences is proportional, the ratio of the number of sequencing sequences of the standardized sample to be tested is divided by 2 and compared with the ratio of the number of sequencing sequences of the standardized control samples. If they are statistically significant, it indicates that the exon is not A heterozygous deletion has occurred. The method of claim 1, wherein the double-stranded DNA after disruption in step 1) is 100-1000 bp. The method of claim 1, wherein the double-stranded DNA has a blunt end after being interrupted, or a blunt end is caused by end repair. The method of claim 1, wherein the linker sequence is 20-150 nt in length, and the ends of the double-stranded DNA fragment are ligated to a linker sequence by a linker sequence, wherein the linker sequence is, for example, poly (N) _n , wherein each N is independently selected from A, T, G or C, n is any positive integer selected from 1 to 20, and the linker sequence is poly(A) _n , wherein n is a positive integer from 1 to 20, and the sequence of the linker joining complementary region is poly(N') _m , wherein each N' is independently selected from A, T, G or C, and m is 1-20 positive An integer is, for example, selected from any positive integer of 1-3, and poly(N) _n and poly(N') _m are complementary sequences. The method of claim 1, wherein the first primer and the second primer in step 2) are designed according to a gene region sequence of the DMD gene and/or the linker sequence. The method of claim 1, wherein the first primer and the second primer described in the step 2) have a linker binding region corresponding to the primer binding region of the linker, and a sequencing outside the linker binding region. Probe binding zone. The method of claim 1, wherein the first primer and the second primer described in the step 2) are oligonucleotides having a length of 30 to 80 bp. The method of claim 1, wherein the nucleic acid wafer in step 3) is immobilized with from 5 to 200,000 specific probes corresponding to the DMD gene. The method of claim 9, wherein the sequence of the specific probe corresponds to the following region of the DMD gene: exons and/or 50-500 nt at both ends of the exon. The method of claim 9, wherein the specific probe has a length of from 20 to 120 mer. The method of claim 1, wherein the method step 3) comprises the step of: blocking the regions at the two ends of the amplification product corresponding to the first primer and the second primer with a blocking molecule, thereby obtaining the two ends a mixture of blocked single-stranded amplification products, using the mixture of blocked single-stranded amplification products, in a subsequent step 4); wherein the blocking molecule blocks the first PCR amplification product corresponding to the first primer And a 70%-100% region of the second primer; the blocking molecule is SEQ ID NO. 3 or SEQ ID NO. The method of claim 1, wherein the third primer and the fourth primer in step 4) are designed according to a gene region sequence of the DMD gene and/or the linker sequence. The method of claim 1, wherein the third primer and the fourth primer specifically correspond to or bind to the first primer and the second primer, respectively. The method of claim 1, wherein the third primer and the fourth primer are specifically bound to the outside of the first primer and the second primer, respectively, and the length is smaller than the first primer and the second primer. Introduction. The method of claim 1, wherein the third primer and the fourth primer are 15-40 bp in length. The method of claim 1, wherein the third primer and the fourth primer are different. The method of claim 1, wherein the sequencing in step 5) employs a second generation sequencing technique. The method of claim 1, wherein the sequencing in step 5) is to hybridize the mixture of the second amplification product to a sequencing probe immobilized on a solid phase carrier, and perform a solid phase bridge. PCR amplification is performed to form a sequencing cluster; the sequencing cluster is then sequenced by a "synthesis-edge sequencing" method to obtain a nucleotide sequence of a disease-associated nucleic acid molecule in the sample. A method for detecting Duchenne muscular dystrophy in a subject, comprising: detecting a mutation of a DMD gene from a sample of the subject using the method of any one of claims 1 to 19; and if DMD gene mutation, the subject has Duchenne muscular dystrophy Symptoms or predisposition to Duchenne muscular dystrophy.