embodiment
Below implement to have described in more detail the present invention, these embodiment are only exemplary, but it will be understood to those of skill in the art that the following example is only for the present invention is described, and should not be considered as limiting scope of the present invention.
Utilize target area capture technique, use the exon order-checking of exon trapping chip to people DMD gene, and then carry out DMD gene extron disappearance and repeat the correlative study that suddenlys change, a current or new technology.The ultimate principle of this technology is to catch the target sequence on genome with a set of oligonucleotide probe, then use according to these sequences that capture of primer pair of DMD gene gene regions sequence and/or the design of described joint sequence and carry out pcr amplification, again these amplified productions are carried out to high-flux sequence, thereby the base sequence in identification DNA sample, by analysis of biological information method, order-checking gained sequence information is analyzed, thereby find the variation information of target sequence, comprise single nucleotide variations, insertion/deletion, repetition, the variation of exon copy number etc.
A kind of method that the invention provides the DMD of detection gene extron disappearance and/or repeat, comprises step:
1) will interrupt respectively as double chain DNA fragment from testing sample and normal control sample extraction genomic dna, and add joint sequence at the two ends of described double chain DNA fragment;
2), with the double chain DNA fragment with joint described in the first primer and the second primer amplification, obtain the first amplified production;
3), by after described the first amplified production sex change, carry out hybrid capture with nucleic acid chip;
4) nucleic acid of being caught with three-primer and the 4th primer amplification, obtains the second amplified production;
5) above-mentioned the second amplified production is checked order, obtain sequencing sequence fragment;
6) described sequencing sequence fragment is compared on the exon sequence and exon flank with reference to DMD gene;
7) by relatively testing sample and normal control sample comparison result determine whether the exon of described testing sample DMD gene has repetition and/or disappearance, if i.e. comparison to the described testing sample sequencing sequence on described gene extron reference sequences significantly more than/be less than described normal control sample sequencing sequence, represent whether the exon of described gene has repetition and/or disappearance.
At method steps 1 of the present invention) in, the described double-stranded DNA that economy-combat is had no progeny is preferably 100-1000bp, and more preferably at 150-500bp, most preferably at 200-300bp, particularly at 200-250bp, above-mentioned lengths table is shown the master tape position of double-stranded DNA electrophoresis.
At method steps 1 of the present invention) in, described double-stranded DNA economy-combat is had no progeny and is preferably had flat end, for example, cause described flat end by end reparation.In another preference, also comprise step: the 3 ' end at described flat terminal double link DNA fragmentation adds " A ", described 3 ' end adds the double chain DNA fragment of " A " and is connected with the joint of " T ", becomes two ends all with the double-stranded DNA fragmentation mixture of joint.Described joint sequence length preferably 20-150nt, particularly 50-100nt.Those skilled in the art can be according to the suitable joint sequence of sequence selection, and sequence conventional in test kit that also can be commercially available is as joint sequence.
At method steps 1 of the present invention) in, DNA fragmentation two ends of preferred described two strands are connected with joint sequence by joint catenation sequence.In another preference, described joint catenation sequence is poly (N)
n, wherein, each N is respectively independently selected from A, T, G or C, and n is the arbitrary positive integer that is selected from 1-20.In another preference, described joint catenation sequence is poly (A)
n, wherein, the positive integer that n is 1-20, preferably n=1-2.In another preference, it is poly (N ') that described joint connects complementary region sequence
m, wherein each N ' is respectively independently selected from A, T, G or C, the positive integer that m is 1-20, and poly (N)
nand poly (N ')
mfor complementary sequence.In another preference, m is the arbitrary positive integer that is selected from 1-3.In another preference, the length that described joint connects complementary district is identical with the length of joint catenation sequence, i.e. poly (N)
nand poly (N ')
mfor fully-complementary sequence.In another preference, it is poly (T) that described joint connects complementary district
m, wherein, the positive integer that m is 1-20, preferably m=1-2.Those skilled in the art can be according to the suitable joint catenation sequence of sequence selection, and sequence conventional in test kit that also can be commercially available is as joint catenation sequence.
At method steps 2 of the present invention) in, preferably described the first primer and the second primer are according to the gene regions sequence of DMD gene and/or the design of described joint sequence.In a preference, the first described primer and the second primer have the joint land corresponding to the PBR of described joint, and are positioned at the order-checking probe land in outside, joint land.In another preference, the first described primer and the second primer are the oligonucleotide of length 30-80bp.In another preference, the first primer and the second primer length are 55-65bp.In another preference, described the first primer and the second primer are different.
At method steps 3 of the present invention) in, the chip that the present invention uses can design in the following manner: pass through microarray technology, by high-density DNA fragment array sequentially or arrangement mode it is attached to as solid phase surfaces such as sheet glass, with fluorescently-labeled DNA probe, by base complementrity Hybridization principle, carry out a large amount of genetic expression and monitoring, target acquisition sequence.For example, chip can be synthetic by the design of Roche NimbleGen company of the U.S..
At method steps 3 of the present invention) in, preferably described nucleic acid chip is fixed with 5-200,000 kind of specific probe corresponding to described DMD gene.In another preference, on described chip, the kind of specific probe is 50-150,000 kind, and more preferably 500-100,000 kind, 5000-80 best, 000 kind.In another preference, the sequence of described probe corresponding to DMD gene with lower area: exon and/or the preferred 50-500nt in exon rear and front end, more preferably 100-300nt, most preferably 200nt.In another preference, the length of described specific probe is 20-120mer, preferably, and 50-100mer, more preferably, 60-80mer.In another preference, described specific probe is that full synthetic or body outer clone are synthetic.
At method steps 3 of the present invention) after, preferably include step: with sealing molecule sealing be positioned at described amplified production two ends, corresponding to the region of the first primer and the second primer, thereby obtain the mixture of the single-stranded amplification product that is closed of two ends, carry out subsequent step 4 with the mixture of the described single-stranded amplification product through sealing).In another preference, described sealing molecule seals the 70%-100% region corresponding to the first primer and the second primer in the first pcr amplification product.In another preference, described sealing molecule seals 100% region corresponding to the first primer and the second primer in the first pcr amplification product.
At method steps 4 of the present invention) in, preferably described three-primer and the 4th primer are according to the gene regions sequence of DMD gene and/or the design of described joint sequence.In a preference, described three-primer and the 4th primer respectively specificity corresponding to or be incorporated into described the first primer and the second primer.In another preference, described three-primer and the 4th primer difference specific binding are in the outside of described the first primer and the second primer, and length is less than the first primer and the second primer.In another preference, described three-primer and the 4th primer length are 15-40bp, are preferably 20-25bp.In another preference, described three-primer and the 4th primer are different.
At method steps 5 of the present invention) in, described order-checking preferably adopts s-generation sequencing technologies, for example illumina solexa, Hiseq 2000, ABI SOLiD, Roche 454 check order platform and/or Ion torrent.In another preference, order-checking probe fixing on the mixture of the second described amplified production and solid phase carrier is hybridized, and carry out solid phase bridge-type pcr amplification, form order-checking bunch; Then described order-checking bunch is checked order by " limit synthetic-Bian order-checking " method, thereby obtain the nucleotide sequence of disease associated nucleic acid molecules in sample.At present, there are some biotechnology service companies that order-checking service can be provided.
At method steps 6 of the present invention) in, described sequencing sequence is compared with reference to being undertaken by software as known in the art on DMD gene extron subsequence and exon flank, for example short oligonucleotide analysis package (Short Oligonucleotide Analysis Package, SOAP) comparison and BWA (Burrows-Wheeler Aligner) comparison; Described flank length is preferably 50-500nt, more preferably 100-300nt, most preferably 200nt.
At method steps 6 of the present invention) after, can, first to the Quality Control of sequencing result primitive sequencer sequence, remove underproof sequencing sequence, the project that wherein original read Quality Control comprises sees the following form;
At method steps 7 of the present invention) in, by sequencing result is carried out to bioinformatic analysis method, can judge between exon that person DMD gene to be measured is undergone mutation and normal people whether there is significant difference statistically.In a preference, can carry out depth calculation by suitable machine language, for example java, C++ or Perl.
In a preference, relatively the step of testing sample and normal control sample comparison result is as follows:
The first step:
For each DMD gene extron, by testing sample compare sequencing sequence number on this exon with respect to comparison the sequencing sequence number of criteria to whole exons, obtain standardized testing sample sequencing sequence number ratio,
By control sample compare sequencing sequence number on this exon with respect to comparison the sequencing sequence number of criteria to whole exons, obtain standardized control sample sequencing sequence number ratio;
Second step:
Described standardized testing sample sequencing sequence number ratio and standardized control sample sequencing sequence number ratio are compared, if their significant difference statistically,
In the time that described standardized testing sample sequencing sequence number ratio is less than standardized control sample sequencing sequence number ratio, after being multiplied by 2, described standardized testing sample sequencing sequence number ratio compares with standardized control sample sequencing sequence number ratio, if they are significant difference statistically, represent that heterozygous deletion does not occur this exon
In the time that described standardized testing sample sequencing sequence number ratio is greater than standardized control sample sequencing sequence number ratio, described standardized testing sample sequencing sequence number ratio is compared with standardized control sample sequencing sequence number ratio after 2, if they are significant difference statistically, represent that heterozygous deletion does not occur this exon.
By these two steps method of calculation, can determine the variation of the exon copy number of DMD gene, thereby judge whether to have occurred to repeat and/or disappearance.
In another preference, relatively the step of testing sample and normal control sample comparison result is as follows:
A. for an exon of DMD gene, 1. through type calculates the ratio %exonN that testing sample is compared the order-checking degree of depth exonN_depth of the sequencing sequence on this exon and compared the average order-checking degree of depth averaged_depth of the sequencing sequence on whole exons;
By %exonN substitution formula 2., calculate the Z-score of the described exon order-checking degree of depth,
...........................①,
...②;
Wherein, for described exon, utilize control sample to compare the order-checking degree of depth and the average order-checking degree of depth of comparing the sequencing sequence on whole exons of the sequencing sequence on this exon, 1. calculate %exonN (normal) according to formula, mean%exonN (normal) is the mean value of all control sample %exonN (normal), and S.D.%exonN (normal) is the standard deviation of all control sample %exonN (normal); If Z-score is greater than the first default cutoff, the order-checking degree of depth of this DMD gene extron is variant remarkable between testing sample and normal specimens, and it is further screened;
B. in two kinds of situation the significant DMD gene extron of above-mentioned order-checking depth difference is screened:
In the time that exonN is less than averaged_depth value, by %exonN be multiplied by 2 substitution formulas 2. in, the Z-score value calculating is less than the second default cutoff and represents that heterozygous deletion has occurred this exon, and the Z-score value calculating is greater than the second default cutoff and represents that heterozygous deletion does not occur this exon;
In the time that exonN is greater than averaged_depth value, by %exonN divided by 2 substitution formulas 2. in, the Z-score value calculating is less than the 3rd default cutoff and represents that repetition has occurred this exon, and the Z-score value calculating is greater than the 3rd default cutoff and represents that heterozygous deletion does not occur this exon.
By these two steps method of calculation, can determine the variation of the exon copy number of DMD gene, thereby judge whether to have occurred to repeat and/or disappearance.
Identical or different for the default cutoff of first in above-mentioned steps a and b, the second default cutoff and the 3rd default cutoff.Choosing by contriver of cutoff determined according to statistical theory carrying out after great many of experiments, specific as follows:
In the case of testing, contriver finds to utilize the normal distribution on Z value (Z scores) coincidence statistics that the inventive method obtains, and sees accompanying drawing 1.The first step that whether has repetition and/or disappearance for the exon of determining described testing sample DMD gene, in the time that Z value is 3.0, has 99.9% confidence level---and-DMD female carrier is different with normal people at the copy number of identical exon.For second step, in order to get rid of the false positive in the first step, if female carrier lacks copy, so her two times should and normal people's (normal people should be twice) there is no significant difference, so detect after will being multiplied by 2.In like manner, repeat copy situation too.
Although used in embodiments of the present invention cutoff 3.0, cutoff also can be greater than 3.0 or be less than 3.0.For example, can use cutoff 1.64 (corresponding 90% confidence level), 1.96 (corresponding 95% confidence levels) or 2.58 (corresponding 99% confidence levels), possibility percentage ratio is larger, and confidence level is higher.This area generally adopts more than 90% confidence level, and cutoff at least will be more than 1.64.
In the present invention, transgenation comprises copy number variation (CNV).
Because DMD gene is positioned on X chromosome, so method of the present invention is specially adapted to occur the women of DMD gene extron heterozygous deletion.
Therefore, it will be understood by those skilled in the art that sudden change detection method of the present invention can be for the sudden change based on detecting DMD gene.
In the present invention, carry out PCR probe used, primer can design based on current solid phase chip hybridization technique, also can be undertaken by biotechnology service company.It will be understood by those skilled in the art that on same chip and catch DMD gene region with high specific and high coverage rate.For example, 2.1M people's exon sequence of Roche NimbleGen is caught chip and can be caught approximately 180,000 exons and approximately 550 miRNA.
Utilize the inventive method to obtain DMD gene extron disappearance and duplicate message can be for for example carrying out gene type to crowd.
In embodiments of the invention, adopt the method for binding sequence capture technique of the present invention, high-flux sequence and analysis of biological information, the DMD gene of DNA sample is detected, obtain the abrupt information of DMD gene in sample.
In the present invention, sequencing sequence, sequencing sequence segment, the section of reading all refer to the data DNA sequence dna (read) that sequenator produces.The order-checking degree of depth refers to reads hop count.
embodiment
As used in the present embodiment, term " primer " refer to can with template complementary pairing, the general name of the oligonucleotide of the DNA chain of and template complementation synthetic in the effect of archaeal dna polymerase.Primer can be natural RNA, DNA, can be also any type of natural nucleotide, and primer can be even that non-natural Nucleotide is as LNA or ZNA etc.A special sequence complementation in primer " haply " (or " substantially ") and template on a chain.Primer must with template on an abundant complementation of chain could start extend, but the sequence of primer needn't with the sequence complete complementary of template.Such as, add the preceding paragraph and the not complementary sequence of template at one 3 ' end and 5 ' end of the primer of template complementation, such primer still haply with template complementation.As long as have sufficiently long primer can with the sufficient combination of template, the primer of non-complete complementary also can form primer-template composite with template, thereby increases.
In the present embodiment, the sequence of the important primer of several classes and title are in table 1.
Table 1
The first primer (SEQ ID NO.1) and the second primer (SEQ ID NO.2) increase to the DNA double chain nucleic acid fragment with joint, obtain the first pcr amplification product, the first primer and the second primer have the joint land corresponding to the PBR of described joint, and are positioned at the order-checking probe land in outside, joint land.The effect of sealing molecule 1 (SEQ ID NO.3) and sealing molecule 2 (SEQ ID NO.4) is in the time carrying out sequence capturing, with joint complementation, avoids catching non-specific sequence.The effect of three-primer (SEQ ID NO.5) and the 4th primer (SEQ ID NO.6) is the DNA fragment specific that a large amount of amplifications are caught, to carry out next step order-checking.
1 DMD female carrier and 4 female normal people are raised in this research, sign written Informed Consent Form.
Carry out chip preparation and hybridization according to the specification sheets of Roche NimbleGen, check order according to the specification sheets of Illumina, step is as follows.
1: chip design
Reference sequences is DMD gene extron subsequence and the exon front and back 200bp of NCBI build 37/hg19 (available from http://www.ncbi.nlm.nih.gov/), synthetic by the design of Roche NimbleGen company of the U.S..
2: library preparation
Get people's peripheral blood, extract genomic dna, obtain 3 μ g DNA.Human gene group DNA's sample that extracting is obtained carries out fragmentation on Covaris S2 instrument (purchased from Covaris company of the U.S.), finally interrupts and becomes the mixture of master tape in the DNA double chain fragment of 200bp.
Next, above-mentioned fragment is carried out to purifying, purge process adopts Ampure Beads method, carries out (Beckman company of the U.S.) according to Agencourt AMPure protocol.In brief: DNA fragmentation is carried out to end reparation, become the fragment mixture with flat end, and add one " A " at 3 of each strand ' end; Then in joint ligation system (PE library), connecting according to the specification sheets in test kit, is that DNA fragmentation adds the joint with " T " by this process; After connection, continue to carry out purifying according to Agencourt AMPure protocol (Beckman company of the U.S.), and except unnecessary reagent is as cushion, enzyme, ATP etc., finally obtain being connected with the DNA of joint.
Owing to being connected with, the DNA sample concentration of joint is very low, the enrichment of next increasing, PCR reaction operation on the PTC-200PCR of Bio-Rad company instrument (PCR reaction system: 98 DEG C, 30s; 98 DEG C of sex change 15s, 65 DEG C of annealing 30s, 72 DEG C are extended 30s, coamplification 4-10 circulation; Final 72 DEG C are extended 5min).
50 μ L pcr amplification reaction reaction systems contain: sample DNA after 25 μ L 2x Phusion HF Master Mix, 1 μ L the first primer (SEQ ID NO.1), 1 μ L the second primer (SEQ ID NO.2), 23 μ L jointings (buffer and taq enzyme purchased from sequenom company).
DNA through increasing, with joint, uses Ampure beads method, according to the program of Agencourt AMPure protocol (Beckman company of the U.S.) purified pcr product.
Purified product after having reacted can be preserved a couple of days at 4 DEG C, also can preserve several weeks at-20 DEG C, also can be directly used in follow-up sequence capturing.
3: sequence capturing
Ready DNA sample is placed in to 60 DEG C of evaporates to dryness of SpeedVac, then adds the ultrapure water of 11.2 μ L, fully dissolve.Centrifugal sample 30 seconds, adds respectively following two kinds of reagent: 2 × SC Hybridiation Buffer (being purchased from Roche NimbleGen company of the U.S.) of 18.5 μ L and 1 × SC Hybridiation Component A (being purchased from Roche NimbleGen company of the U.S.) of 7.3 μ L at full speed.Concussion mixes and is placed on whizzer centrifugal 30 seconds at full speed, then makes the abundant sex change of DNA in 95 DEG C, and denaturation process 10 minutes obtains the DNA library with joint of strand.
According to the test kit specification sheets of Roche NimbleGen, chip with correspondent probe is fixed on hybridization instrument (Roche NimbleGen company of the U.S.) on request, sample after sex change is added in chip and seal chip, then set hybridization program, under strict condition, hybridize 64-72 hour in 42 DEG C.In hybridization system, on gene chip, the concentration of probe molecule will be far away higher than concentration of target molecules.The hybridization system of 34 μ l contains: 300 μ gCot-1DNA, 1 μ g DNA library (preparing in step 1), 1 μ L sealing molecule 1 (SEQ ID NO.3), 1 μ L sealing molecule 2 (SEQ ID NO.4).Wherein Cot-1DNA is by Human Cot-1
-Fluorometric QC (Invitrogen) obtains according to provider's specification sheets.
After waiting to hybridize, chip is washed and sample wash-out in the following order:
By NaOH elutriant reclaim, and with 32 μ L 20% Glacial acetic acid neutralize; Above-mentioned neutralizer is purified with Qiagen MinElute PCR Purification Kit, and the sample after catching is finally dissolved in 138 μ L pure water.
Pcr amplification is carried out in above-mentioned DNA library of catching.Reaction conditions is: 98 DEG C of 30s; 15 circulation (98 DEG C of 15s; 60 DEG C of 30s; 72 DEG C of 30s); 72 DEG C of 5min; 4 DEG C leave standstill.Described amplification is divided into 6 pipe 50 μ L and carries out:
PCR product adopts Ampure Beads method, carries out (Beckman company of the U.S.) carry out purifying according to Agencourt AMPure protocol, is dissolved in 50ul EB after completing, and uses NanoDrop and Bioanalyzer 2100 detectable levels.
4: PCR (LM-PCR) amplified production and real-time fluorescence quantitative PCR (QPCR) with joint mediation detect enrichment:
The book that furnishes an explanation in NSC Assay mix test kit according to Roche NimbleGen company of the U.S. carries out following steps:
1) 4 kinds of NSC Assay mix that diluted are taken out and dissolved on ice.
2) according to before Nanodrop detectable level, (Non-Captured) that do not catch and (Captured LM-PCR) product of successfully catching are diluted to 1ng/ul, finally volume requirement > 12ul.
3) according to 4 kinds of NSC Assay of each sample, each sample comprises 2 kinds of DNA masterplates, and each sample needs 4*2=8 reaction, each dull and stereotyped 1 negative control totally 4 reaction that needs.The multipotency of 96 hole flat boards carries out 11 sample detection.
4) in the centrifuge tube of 1.5ml, preparing QPCR reaction mixture, is once each reaction reagent usage quantity, can, according to the unified configuration of concrete sample size mixed solution, need to include negative control and positive control in calculating.
5) the 12ul QPCR reaction mixture configuring is transferred in 96 hole QPCR Sptting plates, adds wherein the 1ng/ul LM-PCR product of 3ul dilution, all reagent and sample are added to rear use sealed membrane flat board is sealed.With the centrifugal 2min of 4000rpm.
6) 96 orifice plates are placed on QPCR instrument, detect according to program shown in following table 2
Table 2
7) test post analysis test-results, arranged QPCR testing data, utilized the poor Δ Ct of Ct value to calculate enrichment E=(ef)
Δ Ct, wherein ef represents the Q-PCR amplification efficiency of goal gene, desirable amplification efficiency ef value is 2.Wherein in order to calculate Δ Ct, the N-LM-PCR purified product before Non-capture Ct refers to hybridize is template, carries out Q-PCR amplification, the Ct value detecting with goal gene Auele Specific Primer; C-LM-PCR purified product after capture Ct refers to hybridize is template, carries out Q-PCR amplification, the Ct value detecting with goal gene Auele Specific Primer.See the following form:
Judge that whether library is qualified, can carry out next step test: need according to building storehouse type after average enrichment times value judgement, average enrichment times > 60 carries out next step order-checking in the present embodiment.
5: order-checking and data analysis
On a chip, carry out hybridization, in the 64-72 that sample is hybridized on chip hour, target sequence and probe complementation, thus be hunted down.Sample after wash-out is carried out to two end sequencings in Solexa order-checking platform.By the sample source of data analysis sequencing data, and the capture effect of sample is calculated.
The DMD gene extron subsequence and the exon front and back 200bp that use BWA software that the section of reading in all swimming lanes is first compared to reference sequences NCBI build 37/hg19 (available from http://www.ncbi.nlm.nih.gov/) get on.The input of comparison is the fq file after the pollutions such as transition joint, and the output of comparison is original comparison result---SAM file.Use samtools instrument need to carry out a series of processing to baseline results: after format conversion and compression, comparison result to be sorted by chromosome sequence.Secondly, the swimming lane in same library is merged together, finally more all libraries is merged together.Through this series of processes, obtain input file---the BAM formatted file of qualified energy as sudden change detection software SOAPsnp (soap.genomics.org.cn/soapsnp.html), enter the interface of sudden change detection software.
By to dropping on the hop count of reading on hop count and all exons of reading on each exon, calculate each exon degree of depth and mean depth, 1. substitution formula calculates %exonN.Taking remaining 4 women noncarriers as reference, 2. mean%exonN (normal) and S.D.%exonN (normal) substitution formula are calculated to Z-score.Calculate and find, this carrier 10,11,15,23,26,29,46-51,70,71 and 73 exon Z-score are respectively: 3.80,3.23,4.33,3.72,6.16,5.09,7.04,9.08,6.81,10.10,26.95,8.51,4.36,3.65 and 3.24, be all greater than cutoff 3.00.Then, carry out second step calculating, the result obtaining is respectively 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, and wherein 0.85,0.32,0.49,0.63,1.01,1.52 corresponding exon 46-51 are all less than 3.0.Therefore, utilize method of the present invention to show, this carrier has deficient phenomena at 46-51 exon.
As follows, in order to verify result of the present invention, by real-time fluorescence quantitative PCR, detect and analyze this carrier 46-51 exon copy number.Experimental procedure is as follows:
Design after primer, 1 DMD female carrier and 4 female normal people's sample is carried out to the upper machine amplification of qPCR.Reaction system is:
Pre-configured mother liquor
10×Buffer |
1.0ul |
MGSO4 |
0.1ul |
Tween20 100× |
0.1ul |
DMSO |
0.5ul |
H2O |
3.35ul |
Betaine |
2.0ul |
Get the above mother liquor configuring in advance and add following composition
The reaction system of preparation 2.0ml:
dNTP(2.5mM) |
0.8ul |
Eva Green |
0.5ul |
ROX |
0.2ul |
Upstream Primer |
0.2ul |
Downstream Primer |
0.2ul |
HS Taq |
0.1ul |
Experimentally machine condition is shown in Fig. 2.
After test, QPCR product is carried out to 2% agarose gel electrophoresis, by the observation to amplified band, find that DMD female carrier has disappearance at 46-51 exon, and 4 female normal people do not lack at 46-51 exon.
Therefore, the result calculating by exon of the present invention is consistent with the result of real-time fluorescence quantitative PCR, illustrates that method of the present invention is feasible.