CN108060227A - A kind of amplimer, kit and its detection method for detecting PAH gene mutations - Google Patents

Abstract

The invention discloses a kind of amplimer, kit and its detection methods for detecting PAH gene mutations, and for the primer sets of PCR specific amplifications detection PAH gene mutations, totally 9 pairs of this group of PCR primer is as follows respectively：For expanding 1 upstream outer sequence of PAH gene extrons（Positioned at gene 5 ' end）To the primer of introne 2, forward primer such as SEQ ID NO:Shown in 1, reverse primer such as SEQ ID NO:Shown in 2；For expanding 2 upstream of PAH gene introns（5 ' ends）To 2 downstream of introne（3 ' ends）Primer, forward primer such as SEQ ID NO:Shown in 3, reverse primer such as SEQ ID NO:Shown in 4 etc..The present invention realizes PAH gene all standings, the sequence length detected reaches 88171bp, contribute to variation and the detection of structure variation in introne, the recall rate of pathogenic mutation can be improved, and operation is more simple, at low cost, in addition each amplified fragments have overlay region with adjacent segment, available for fragment assembly and haplotype reconstruction.

Description

A kind of amplimer, kit and its detection method for detecting PAH gene mutations

Technical field

The present invention relates to PAH gene technology field, more particularly to a kind of amplimer, reagent for detecting PAH gene mutations Box and its detection method.

Background technology

Phenylalanine hydroxylase deficiency（phenylalanine hydroxylase deficiency,PAHD）It is common Aminoacidopathy, the morbidity of PAHD is due to phenylalanine hydroxylase（phenylalanine hydroxylase,PAH） Mutation causes enzymatic activity to reduce or lose, and makes phenylalanine metabolic disorder.The PAHD infants of untreated are due to internal excess The neurotoxic effect of phenylalanine and bypass metabolite can cause serious dysnoesia and symptomatic epilepsy.If it can obtain To early diagnosis and therapy, then neurotrosis can not then occur, normal intelligence.With the development of clinical diagnosis and treatment, PAHD has become It can treat, preventible disease.Since infant not occurring symptom, PAH gene mutation analysis is in early days the diagnosis method of PAHD. Meanwhile PAH genetic tests are carried out to patient and also contribute to its lineal relative's genetic counselling, and provided to give birth to antenatal detection again Foundation.PAH genes are located at human chromosome position 12q23.2, overall length about 90kb, and containing 13 extrons, coding section length is 1359bp encodes the polypeptide chain of 452 amino acid and further folds composition phenylalanine hydroxylase albumen.It reports at present PAH gene mutation species is more, most of for missense mutation there are about more than 800 kinds, remaining further include shearing mutation, nonsense mutation and Small missing/insertion mutation, is related to whole gene, has high genetic heterogeneous, there is significant area and racial difference.Together When, domestic and foreign scholars have been reported that PAH gene mutations part has the large fragment deletion or again across extron and/or introne Multiple mutation.It at present can clear and definite 90% or so using the method for PCR amplification extron and flank finite region sequence combination DNA sequencing Patient, the patient for still having 10% or so cannot specify the cause of disease, therefore there is an urgent need for be capable of the detection of specific detection PAH full-length genes Method.- Sanger nucleotide sequencing methods are cloned using segment detect mankind's PAH genes from DiLella in 1986 et al. After first pathogenic mutation, occur abrupt climatic change of more and more molecular detection technologies for PAH genes both at home and abroad.It has reported Gene tester have the hybridization of polymerase chain reaction-allele specific oligonucleotide chain（Polymerase chain reaction--allele specific oligonucleotides, PCR-ASO）, polymerase chain reaction-denaturant gel ladder Spend electrophoresis（PCR-denaturing gradient gel electrophoresis,PCR-DGGE）, polymerase chain reaction-mono- Chain conformation polymorphism（PCR-Single strand conformation polymorphism,PCR-SSCP）, polymerase chain it is anti- Should-restriction fragment length polymorphism（PCR-restriction fragment length polymorphism, PCR- RFLP）, polymerase chain reaction-denaturing high-performance chromatography（PCR-denaturing high-performance liquid chromatography, PCR-DHPLC）, polymerase chain reaction-high-resolution melting curve analysis（PCR-high resolution melting,PCR-HRM）Deng having the features such as easy to operate economic and practical, but the drawback is that being only capable of detection has The specific mutation type of limit or the specific location and type that can not be clearly mutated.At present, Sanger sequencing approaches be clinical practice in The main method of PAH detection in Gene Mutation.Zhang Zhi in 2006 etc.（The mutation inspection of Classic PKU full length gene extron It surveys and analyzes,《Chinese eugenic and Journal of Heredity》, 2006, the 5th phase of volume 14,14-16 pages）It establishes to PAH gene extrons The method of sub- pcr amplification product Sanger sequencings.2007 Nian Song Fang etc.（Northern Part of China Phenylalanine Hydroxylase Gene is dashed forward Become and form,《Chinese Journal of Medical Genetics》, 2007, the 3rd phase of volume 24,241-246 pages）Point established using Zhang Zhi et al. Analysis method has carried out Sanger surveys to 230 PKU patients gene whole extrons and its flanking intron sequence Sequence detects.

For PAH detection in Gene Mutation, have multinomial patent and disclose report.CN1335408A（2002）It discloses suitable In early diagnosis and the PAH gene mutation DNA chip dedicated for diagnosing of Prenatal Screening phenylketonuria, but this method is only to 40 kinds PAH gene mutations are detected.CN1696308A（2005）Also disclose and be suitable for early diagnosis and Prenatal Screening propiophenone The PAH gene mutation DNA chip dedicated for diagnosing of disease is urinated, but this method is only detected 6 kinds of PAH gene mutations. CN101481741A（2009）Disclose the exon of the 4th, 5,6,7,10,11 and 12 using specific amplification PAH genes And its PCR primer of flanking intron sequence, detect 13 kinds of PAH in conjunction with the methods of Sanger PCR sequencing PCRs, enzymatic mispairing cutting Gene mutation.CN101570787A（2009）Also a kind of compound chip for antenatal quick diagnosis, but this method are disclosed Only 5 kinds of PAH gene mutations are detected.CN101899518A（2010）Disclose a kind of detection phenylketonuria PAH bases Because of the kit and its PCR amplification method of 7 hot mutant sites, because the specificity of its product is very high, can directly observe solidifying The presence or absence of product band of gel electrophoresis judges the genotype in the corresponding mutational site of PAH genes.CN101693921A（2010） One kind is disclosed for buccal swab DNA, using specific PCR amplification PAH genes the 7th and the 12nd exon, then is passed through Sanger sequencing technologies detect the catastrophe of the two extrons.CN201376967Y（2010）It discloses for antenatal fast The compound chip of speed diagnosis, but this method is only detected 5 kinds of PAH gene mutations.CN102533992A（2012）It announces A kind of method that Phenylalanine Hydroxylase Gene is sequenced, i.e., simultaneously to the mesh of the 1st to 13 exon of PAH genes It marks region and carries out specific PCR amplification, PCR product is used to build sequencing library, then carries out high-flux sequence. CN202671546U（2013）A kind of phenylketonuria Disease-causing gene mutation detection kit is disclosed, that is, utilizes specificity PCR primer expands 13 extrons of PAH genes, reuses Sanger sequencings and carries out mutation analysis.CN103436616A（2013 Year）Kit that is a kind of while detecting Chinese population phenylketonuria PAH 12 mutantional hotspots of gene is disclosed, is suitable for group Property examination.CN104031990A（2014）A kind of phenylketonuria PAH gene detecting kits are disclosed, predominantly detect PAH 13 common mutations sites of gene.CN103509865A（2014）It discloses a kind of utilization high-resolution fusion curve and analyzes skill The sequence of the method that art detects PAH gene mutations, main detectable PAH genes the 3rd, 6,7,11 and 12 exons whether there is It is abnormal, but specific genotype can not be detected.CN104232770A（2014）Disclose a kind of phenylketonuria genetic test Kit, the kit quickly detect the genotype in 7 sites of PAH genes using multiple connecting detection reaction technology. CN105177160A（2015）Disclose a kind of primer for detecting a variety of newborn's Inherited Metabolic Disorders Disease-causing gene mutation and examination Agent box, 40 Disease-causing genes related to 22 kinds of common newborn's Inherited Metabolic Disorders（Include PAH genes）Extron and extron The sequence of introne calmodulin binding domain CaM carries out specific primer multiplex PCR, then carries out the sequencing of two generations and analysis, provides detection gene Abrupt information.CN105755109A（2016）Disclose a kind of new phenylketonuria gene screening and diagnosis system and reagent Box, using the system constructed by round pcr, DNA interconnection techniques and capillary electrophoresis technique, the PAH gene more typical to 41 Mutational site is detected.PAH gene extrons total length is 4122bp（Reference gene group is GRCh37/hg19）, and include Sub- total length is but up to 76597bp（Reference gene group is GRCh37/hg19）, above-mentioned report technology is just for mutational site progress Detection or detection PAH gene extrons subsequence and small part are located at the intron sequences of extron flank, and cannot be directed to PAH Gene whole intron sequences are detected, and this field stills need a kind of gene tester that can detect PAH full length genes, I.e. this method can not only detect whole exon sequences, moreover it is possible to the whole intron sequences of detection.

In past 10 years, sequencing technologies have developed rapidly, and the sequencing of Illumina both-ends and Ion Torrent half occurs Conductor sequencing is the second generation sequencing technologies represented and the third generation sequencing technologies as representative is sequenced with PacBio SMRT. The sequencing of Illumina both-ends is fast and accurately sequenced using fasciation into the method being sequenced in synthesis to realize.This process It is identified while DNA base is added in nucleic acid chains.Each base is sent while the chain constantly extended is added in Unique fluorescence signal, these signals can be used to determine the order of DNA sequence dna.The sequencing of Ion Torrent semiconductors is base In the revolutionary sequencing technologies of a new generation of semiconductor chip, which has used a kind of high-density semiconductor core for being covered with aperture Piece, an aperture are exactly a sequencing reaction pond.When archaeal dna polymerase is a kind of DNA chain being aggregated in extension in 4 kinds of dNTP When upper, a hydrogen ion can be discharged, the pH value in reaction tank can change, and the ion receptor under pond experiences this letter Number, chemical signal is converted into digital signal, so as to read DNA sequence dna.PacBio SMRT sequencings are also based on side synthesis The principle of side sequencing, the core of this technology are to have used zero level guide technology（Zero-Mode Waveguide, ZMW), often A ZMW fixes an archaeal dna polymerase and a DNA profiling, is fluorescently labeled the nucleotide of phosphoric acid group in polymerase activity position It is combined on point with template strand（Each deoxynucleotide is by the dye marker without color）, fluorescence is inspired, in fluorescent pulse After, labeled phosphoric acid group is cut and discharges.Polymerase is transferred to next position, and next deoxynucleotide connects It is connected on site and starts to discharge fluorescent pulse, carry out next cycling, realize sequencing.The characteristics of PacBio SMRT are sequenced has reading Long overlength, the features such as GC skewed popularities are small, accuracy is high, it can directly dock long-chain PCR（long-range PCR,LR-PCR）Production Object is sequenced, without fragmentation processing and PCR amplification again.Due to PAH gene whole extrons and whole intrones It is added together total length 80719bp（Reference gene group is GRCh37/hg19）, traditional Sanger, which is sequenced, will complete the full length gene Sequence, workload is very big, and expense is high, and uses high throughput sequencing technologies that can be carried out simultaneously to PAH full length genes Sequencing can shorten detection cycle, reduce workload and testing cost.

Therefore, a kind of amplimer, kit and its detection method for detecting PAH gene mutations is invented to solve above-mentioned ask It inscribes necessary.

The content of the invention

It is an object of the invention to provide it is a kind of detect PAH gene mutations amplimer, kit and its detection method, To solve the problems mentioned in the above background technology.

To achieve the above object, the present invention provides following technical solution：A kind of amplimer for detecting PAH gene mutations, Kit and its detection method, for PCR specific amplifications detection PAH gene mutations primer sets, totally 9 pairs of this group of PCR primer, It is as follows respectively：

For expanding 1 upstream outer sequence of PAH gene extrons（Positioned at gene 5 ' end）To the primer of introne 2, forward direction is drawn Object such as SEQ ID NO:Shown in 1, reverse primer such as SEQ ID NO:Shown in 2；

For expanding 2 upstream of PAH gene introns（5 ' ends）To 2 downstream of introne（3 ' ends）Primer, forward primer such as SEQ ID NO:Shown in 3, reverse primer such as SEQ ID NO:Shown in 4；

For expanding the primer that PAH gene introns 2 arrive introne 3, forward primer such as SEQ ID NO:Shown in 5, reversely draw Object such as SEQ ID NO:Shown in 6；

For expanding 3 upstream of PAH gene introns（Introne 5 ' is held）To introne 3 downstream（Introne 3 ' end）Primer, Forward primer such as SEQ ID NO:Shown in 7, reverse primer such as SEQ ID NO:Shown in 8；

For expanding the primer that PAH gene introns 3 arrive introne 4, forward primer such as SEQ ID NO:Shown in 9, reversely draw Object such as SEQ ID NO:Shown in 10；

For expanding the primer that PAH gene introns 4 arrive introne 5, forward primer such as SEQ ID NO:Shown in 11, reversely draw Object such as SEQ ID NO:Shown in 12；

For expanding the primer that PAH gene introns 5 arrive introne 7, forward primer such as SEQ ID NO:Shown in 13, reversely draw Object such as SEQ ID NO:Shown in 14；

For expanding the primer that PAH gene introns 5 arrive introne 11, forward primer such as SEQ ID NO:Shown in 15, reversely Primer such as SEQ ID NO:Shown in 16；

For expanding PAH gene introns 9 to sequence on the outside of 3 downstream of exons 1（It is held positioned at gene 3 '）Primer, forward direction draws Object such as SEQ ID NO:Shown in 17, reverse primer such as SEQ ID NO:Shown in 18.

For the kit of PCR specific amplifications detection PAH gene mutations, draw comprising one or more pairs of in primer sets Object.

Preferably, the kit for the detection PAH gene mutations of PCR specific amplifications is also comprising one or more examinations Agent carries out the long-chain PCR " reagents of long-range PCR, LR-PCR " reactions using the primer；It is carried out using the primer Reagent D NA polymerases, buffer solution and the dNTP mixtures of Long fragment PCR reaction；For handling amplified production so that amplification production Object can be used for the reagent in high throughput sequencing technologies.

Preferably, the kit includes following reagents, one or more pairs of primers, 10 × GeneAmp in PCR primer group High Fidelity PCR buffer solutions, 10mmol/LdNTP, 5mol/L glycine betaine, DMSO and 5U/ μ L polymerase mixtures.

Preferably, following components are contained in the PCR reaction systems for being 50 μ l in total volume：Each 1 μ l of each PCR primer, draw The concentration of object is 10 μm of ol/L；10 × GeneAmp High Fidelity PCR buffer solutions 5 μ l, 10mmol/L, dNTP2 μ l, 10 μm each 5 μ l, DMSO2.5 μ l, 5U/ μ l polymerase mixtures of 1 μ l, 5mol/L glycine betaine, 1 μ l, 50ng/ μ l of the forward and reverse primers of ol/L 2 μ l of genomic DNA, 30.5 μ l of water.

A kind of amplimer, kit and its detection method for detecting PAH gene mutations, comprises the following steps：

（1）Using human gene group DNA as template, the primer sets of usage right requirement 1, in the condition suitable for amplification purpose nucleic acid Under, PAH genes are expanded using LR-PCR technologies, wherein being made of per pair of primers forward primer and reverse primer.

（2）To 9 long segment products of amplification, purifying recycling is carried out；

（3）9 PCR products of purifying are mixed, and are quantified, obtain the PCR product of 1 people's genome DNA sample Library；

（4）DNA fragmentation processing is carried out to obtaining PCR product library；Wherein described DNA fragmentation method includes the chemistry side of interrupting Method and physics interrupt method, wherein the chemical method includes swivel base enzyme process and traditional enzymatic cleavage methods, the physical method includes Ultrasound interrupts method or machinery interrupts method, and the PCR product library interrupted is built 1 sequencing text using splice tag technology Storehouse, can be respective to distinguish by adding different library connector Barcode Adapter to different genome DNA samples Sequencing library also can handle PCR product library without fragmentation, directly build single SMRTBell libraries；

（5）Obtained multiple sequencing libraries are mixed, it is different to be sequenced type according to library, select different microarray datasets into Row sequencing is read long sequencing etc. including the sequencing of Illumina both-ends, the sequencing of Ion Torrent semiconductors and PacBio SMRT long, is obtained Obtain the sequencing data in PCR product library；

（6）Different genome DNA sample is distinguished based on label B arcode sequences, by bioinformatic analysis by single sample The DNA sequencing fragment that product sequencing obtains is compared onto reference PAH genes, is carried out SNVs and Indels extractions, and is carried out structural The analysis of variation excludes the PAH gene mutation information that polymorphic variation obtains the DNA sample.

Preferably, PAH genes are chosen and design 9 pairs of PCR primers, using LR-PCR as template using human gene group DNA Technology expands PAH genes, and the region of amplification includes PAH gene whole extrons and whole intron sequences and part Sequence on the outside of 3 downstream of exons 1 upstream outer sequence and exons 1, exons 1 upstream outer sequence are located at gene 5 ' end, outside Sequence is located at the end of gene 3 ' on the outside of 13 downstreams of aobvious son, and in addition each amplified fragments have overlay region with adjacent amplified fragments, can use In fragment assembly and haplotype reconstruction, and available for big structure variation is detected, refer mainly to deletion mutant and gene weight Multiple mutation；

To 9 long segment products of amplification, purifying recycling is carried out, 9 PCR products of purifying are mixed, and is quantified, 1 PCR product library by inspection DNA sample is obtained, corresponding 1 by inspection genome DNA sample；

To obtained PCR product library construction sequencing library, partly led including Illumina both-ends sequencing library, Ion Torrent Body sequencing library and SMRTBell libraries, are sequenced in corresponding microarray dataset, after obtaining sequencing data, carry out biological letter Credit analysis is ceased, obtains PAH full length genes variation information.

The technique effect and advantage of the present invention：The present invention goes out 9 couples of PCR using the sequence information specific designs of PAH genes Primer, for expanding fragment length in 11846bp between 12890bp, the average length 12481bp of 9 amplified fragments, is to be more than The long segment of 11kb, common PCR reaction systems and condition are difficult success, and in the SSR-PCR optimization and item of the present invention Under part, it can be expanded using 9 PCR reactions and obtain PAH gene whole exon sequences and all intron sequences, with other mesh Mark areas captured technology is compared（Such as chip capture technique and Ampliseq technologies）, this method can realize PAH gene all standings, institute The sequence length of detection reaches 88171bp, contributes to variation and the detection of structure variation in introne, can improve the inspection of pathogenic mutation Extracting rate, and operation is more simple, and at low cost, in addition each amplified fragments have overlay region with adjacent segment, available for segment Splicing and haplotype reconstruction.

The present invention realizes PAH gene long-chain PCR amplifications for the first time and the sequencing of Illumina both-ends is used in combination, and for the first time The sequencing of PAH full length genes is applied in phenylalanine hydroxylase deficiency genetic test research, the present invention is by devising one The simplified primer of group, the targeting amplification of PAH gene complete sequences is realized based on LR-PCR technologies, and the PCR product of amplification directly turns One step of seat enzyme process completes DNA fragmentation, end is repaired and connector coupled reaction step, is significantly reduced compared with chip capture technique The demand of starting DNA, enormously simplifies experimental implementation, shortens the sequencing library structure time, is phenylalanine hydroxylase Deficiency disease genetic test provides new technical method.

Description of the drawings

Fig. 1 is gene long-chain PCR（LR-PCR）Amplification schematic diagram.

Fig. 2 is the schematic diagram of the 2200 TapeStation System analysis results of Agilent of sequencing library.

Fig. 3 is the overburden depth and coverage rate figure of PAH gene Illumina MiSeq sequencings.

Fig. 4 is the IGV views of two PAH pathogenic mutations in Illumina MiSeq sequencing results.

Specific embodiment

Below in conjunction with the attached drawing in the embodiment of the present invention, the technical solution in the embodiment of the present invention is carried out clear, complete Site preparation describes, it is clear that described embodiment is only part of the embodiment of the present invention, instead of all the embodiments.It is based on Embodiment in the present invention, those of ordinary skill in the art are obtained every other without making creative work Embodiment belongs to the scope of protection of the invention.

The present invention relates to a kind of detection PAH gene mutation methods, the method designs 9 using the sequence information of PAH genes To PCR primer, using long-chain PCR（long-range PCR,LR-PCR）Technology targeting expands entire PAH genes, covers overall length Spend the genome sequence for 88171bp, i.e. sequence between 12 genomic locations 103227356 to 103315526 of chromosome（Ginseng Genome is examined as GRCh37/hg19）, which includes PAH gene whole exon sequences（Total length is 4122bp）And whole Intron sequences（Total length 76597bp）, in addition each amplified fragments and adjacent segment have overlay region, available for fragment assembly And haplotype reconstruction, storehouse is built after amplified production fragmentation, using Illumina gene sequencers or Ion Torrent semiconductors Sequenator directly builds single SMRTBell libraries after carrying out high-flux sequence or amplified production purifying, mixing, using PacBio RSII sequenators are sequenced, and sequencing data obtains PAH gene mutation information by bioinformatic analysis.

The present invention provides a kind of primer sets for specific PCR amplification PAH genes, preferred PCR primer such as 1 institutes of table Show, totally 9 pairs of the PCR primer group, adjacent amplified fragments overlapping areas about 3kb or so, 9 pairs of primer difference of the group are as follows：With In amplification 1 upstream outer sequence of PAH gene extrons（Positioned at gene 5 ' end）To the primer of introne 2, forward primer such as SEQ ID NO:Shown in 1, reverse primer such as SEQ ID NO:Shown in 2；For expanding 2 upstream of PAH gene introns（5 ' ends）To including Sub 2 downstreams（3 ' ends）Primer, forward primer such as SEQ ID NO:Shown in 3, reverse primer such as SEQ ID NO:Shown in 4；For Expand the primer that PAH gene introns 2 arrive introne 3, forward primer such as SEQ ID NO:Shown in 5, reverse primer such as SEQ ID NO:Shown in 6；For expanding 3 upstream of PAH gene introns（Introne 5 ' is held）To introne 3 downstream（Introne 3 ' end）Draw Object, forward primer such as SEQ ID NO:Shown in 7, reverse primer such as SEQ ID NO:Shown in 8；For expanding PAH gene introns 3 To the primer of introne 4, forward primer such as SEQ ID NO:Shown in 9, reverse primer such as SEQ ID NO:Shown in 10；For expanding PAH gene introns 4 arrive the primer of introne 5, forward primer such as SEQ ID NO:Shown in 11, reverse primer such as SEQ ID NO: Shown in 12；For expanding the primer that PAH gene introns 5 arrive introne 7, forward primer such as SEQ ID NO:Shown in 13, reversely Primer such as SEQ ID NO:Shown in 14；For expanding the primer that PAH gene introns 5 arrive introne 11, forward primer such as SEQ ID NO:Shown in 15, reverse primer such as SEQ ID NO:Shown in 16；For expanding PAH gene introns 9 to 3 downstream of exons 1 Outside sequence（It is held positioned at gene 3 '）Primer, forward primer such as SEQ ID NO:Shown in 17, reverse primer such as SEQ ID NO: Shown in 18, primer SEQ ID NO:1 and primer SEQ ID NO:2 amplified fragments, 1 length is 12594bp, primer SEQ ID NO:3 With primer SEQ ID NO:4 amplified fragments, 2 length is 12764bp, primer SEQ ID NO:5 and primer SEQ ID NO:6 amplifications 3 length of segment is 12067bp, primer SEQ ID NO:7 and primer SEQ ID NO:8 amplified fragments, 4 length is 12890bp, is drawn Object SEQ ID NO:9 and primer SEQ ID NO:10 amplified fragments, 5 length is 11846bp, primer SEQ ID NO:11 and primer SEQ ID NO:12 amplified fragments, 6 length is 12027bp, primer SEQ ID NO:13 and primer SEQ ID NO:14 amplified fragments 7 length are 12825bp, primer SEQ ID NO:15 and primer SEQ ID NO:16 amplified fragments, 8 length be 12553bp, primer SEQ ID NO:17 and primer SEQ ID NO:18 amplified fragments, 9 length is 12764bp, the total coamplification 88171bp's of 9 segments Genome area covers PAH gene whole extrons（Extron coverage rate 100%）With whole intron sequences（Introne covers Rate 100%）And 3307bp length sequences on the outside of 3 downstream of exons 1 upstream outer 4145bp length sequences and exons 1.

The present invention also provides a kind of kit for specific PCR augmentation detection PAH gene mutations, comprising above-mentioned One or more pairs of primers in primer sets can also include one or more of reagent in the kit：Utilize the primer Carry out the reagent of Long fragment PCR reaction；The reagent D NA that Long fragment PCR reaction is carried out using the primer is gathered preferably wherein Synthase, buffer solution and dNTP mixtures；For handling amplified production so that amplified production can be used in high throughput sequencing technologies Reagent, mentioned reagent can be specifically 10 × GeneAmp High Fidelity PCR buffer solutions；10mmol/L dNTP； 5mol/L glycine betaines；DMSO；5U/ μ l polymerase mixtures contain following groups in total volume is the PCR reaction system of 50 μ l Point：10 × GeneAmp High Fidelity PCR buffer solutions, 5 μ l, 10mmol/LdNTP2 μ l, 10 μm of forward and reverse primers of ol/L Each 51 μ l, 50ng/ μ l genomic DNAs of μ l, DMSO2.5 μ l, 5U/ μ l polymerase mixtures of 1 μ l, 5mol/L glycine betaine, 2 μ l, water 30.5μl。

The PCR primer group or kit of the present invention has the purposes of detection PAH gene mutations and in phenylalanine hydroxylase Purposes in enzyme deficiency disease genetic test.

The present invention also provides a kind of methods for vitro detection PAH gene mutations, comprise the following steps：（1）Using Human gene group DNA is as template, the primer sets formed using 9 pairs of primers of the present invention, under conditions of suitable for amplification purpose nucleic acid, PAH genes are expanded using LR-PCR technologies；（2）To 9 long segment products of amplification, purifying recycling is carried out；（3）It will be pure 9 PCR products changed are mixed, and are quantified, and obtain the PCR product library of 1 people's genome DNA sample；（4）To The PCR product library arrived carries out high-throughput sequencing library structure；（5）Obtained multiple sequencing libraries are mixed, according to text Storehouse type is different, and different microarray datasets is selected to be sequenced；（6）Single sample is sequenced by bioinformatic analysis and is obtained DNA sequencing fragment compare to reference on PAH genes, carrying out SNVs and Indels extractions, and carry out the analysis of structure variation, arrange Except polymorphic variation obtains sample P AH gene mutation information.

The method of the detection PAH gene mutations of the present invention, preferably includes following step：（1）Using genomic DNA as mould Plate is chosen PAH genes and designs 9 pairs of PCR primers, PAH genes expanded using LR-PCR technologies, PCR reaction system bags Glycine betaine containing 0.5M and 5%DMSO, PCR reaction condition use 2 footwork PCR；（2）To 9 long segment products of amplification, carry out pure Change recycling, and quantified；（3）9 PCR products of purifying are subjected to equal proportion mixing, and are quantified, obtain 1 people's base Because of the PCR product library of group DNA sample；（4）For 1 PCR product library, using one step of swivel base enzyme process complete DNA fragmentation, End is repaired and connector coupled reaction, and Index1 sequence labels and Index2 sequence labels are added at the both ends for interrupting post-fragment, To build the upper machine sequencing library in each PCR product library；Wherein Index1 sequence labels and Index2 sequence labels is included Label it is different；The label that different PCR product libraries use is different from each other, to distinguish different genes group DNA sample；To each survey Preface storehouse carries out quality inspection and quantifies；（5）By multiple sequencing libraries（Label can be distinguished）Mixed in equal amounts is loaded in Flow Cell chips On, in Illumina gene sequencers（Illumina Miseq sequenators, Illumina NextSeq500 sequenators or Illumina HiSeq X Ten sequenators）Upper progress both-end is sequenced in synthesis；（6）Different bases is distinguished based on sequence label It because of a group DNA sample, is split by the sequence label identified in sequence results, it is corresponding to establish each genome DNA sample Sequencing result data are compared the DNA sequencing fragment that single sample sequencing obtains to reference to PAH bases by bioinformatic analysis Because upper, progress SNVs and Indels extractions, and the analysis of structure variation is carried out, it excludes polymorphic variation and obtains sample P AH bases Because of abrupt information.

In the detection method of the present invention, the DNA fragmentation method interrupts method including chemistry and physics interrupts method, Described in chemical method include traditional enzymatic cleavage methods and new swivel base enzyme process, the physics interrupt method and interrupts method including ultrasound Or machinery interrupts method, after the DNA is interrupted, obtains the segment of length 400bp-600bp, the method for purifying and recycling include but It is not limited to magnetic bead recycling or electrophoresis is tapped and recovered.

The present invention detection method in, the sequencing technologies include but not limited to Illumina sequencing technologies or Other two generations sequencing technologies；Can also be three generations's sequencing technologies, such as PacBio RSII sequenators and PacBio Sequel are surveyed Sequence instrument；Different banking process is used for different sequenators.

Embodiment 1

Extracting genome DNA：Person under inspection peripheral blood 2ml is gathered, is placed in EDTA anticoagulant tubes, takes EDTA anticoagulation cirumferential blood samples 0.2ml extracts DNA by whole blood DNA extracts kit specification, and DNA concentration is analyzed with Qubit2.0, the genome of extraction DNA goes to next step pcr template use, this research is altogether detected 5 gene DNA samples, wherein 1 gene DNA sample For product there are known pathogenic mutation site, 4 are normal human gene group DNA.

Embodiment 2

LR-PCR is expanded：According to PAH gene orders, design 9 pairs of PAH gene-specific primers of synthesis, using GeneAmp High Fidelity PCR System carry out LR-PCR, and 9 larger sequence fragments of coamplification are respectively 1,2,3,4,5,6,7,8 and 9, draw Object sequence and amplified production size are as shown in table 1, using GeneAmp High Fidelity PCR System, setting reaction item Part carries out LR-PCR in Veriti 96-well Thermal Cycler PCR instruments, and PCR reaction total volumes are 50 μ l, including 10 × GeneAmp High Fidelity PCR buffer solutions, 5 μ l, 10mmol/L dNTP, 2 μ l, 10 μm of ol/L are forward and reverse to be drawn Each 5 2.5 μ l, 5U/ μ L polymerase mixtures of μ l, DMSO, 1 μ l, 50ng/ μ l genomic DNAs of 1 μ l, 5mol/L glycine betaine, 2 μ l of object, 30.5 μ l of water, PCR cycle parameter：98 DEG C of denaturation 1min；98 DEG C of 10sec, 68 DEG C of 13min, totally 30 cycle, last 72 DEG C of extensions 15min, PCR reaction are completed in Veriti 96-well Thermal Cycler PCR instruments.

Table 1 expands the LR-PCR primers of PAH genes:

Note：F is forward primer（Sense primer）, R is reverse primer（Anti-sense primer）, the PCR primer sequence in table is followed successively by： SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18。

After the completion of LR-PCR, 3 μ lPCR products is taken to be detected through 0.6% agarose gel electrophoresis, Fig. 1 shows 9 amplifications Clip size is consistent with theoretical value, and target stripe is apparent from, and without non-specific amplification, illustrates the LR-PCR reactions of selection System and condition are suitable.

Embodiment 3

LR-PCR product purifications and quantitative：LR-PCR products are purified by paramagnetic particle method, using 2.0 fluophotometers of Qubit to every 1st ~ No. 9 product after purification of a genome DNA sample carries out DNA concentration and quantifies, 5 genome DNA samples, 45 LR- in total The Concentration Testing result of pcr amplification product is as follows：

DNA concentration detects（Qubit 2.0）

Since the length scale of 9 amplified fragments is basically identical, 9 PCR products corresponding to same gene group DNA sample are pressed Etc. quality mixed, and use Agencourt AMPure XP magnetic beads（Beckman Coulter companies of the U.S.）It is concentrated Enrichment, is quantified with 2.0 fluophotometers of Qubit.

Embodiment 4

Library construction：Library construction uses TruePrepTM DNA Library Prep Kit V2（Chinese Vazyme companies）Into Prepared by row, concrete operation step is as follows：

1st, the DNA fragmentation of swivel base enzyme process, end reparation and connector coupled reaction：

1）It will be spare after thaw at RT 5 × TTBL solution, the mixing that turns upside down；

2）Following reagent is sequentially added in the PCR pipe that sterilizes：

Reagent	Volume	Final concentration/amount
			Mixing PCR product after purification	3μl(10ng/µl)	30ng
5×TTBL	10µl	1×
			TTE Mix V50	5µl	-
ddH2O	32µl	-
			Total volume	50µl

3）Mixed liquor is gently blown and beaten to 20 abundant mixings；

4）Quick centrifuge；

5）PCR pipe is placed in Veriti 96-well Thermal Cycler PCR instruments（American AB I companies）On, it sets as follows Response procedures：

Heat lid	105℃
		55℃	10 Min
10℃	Hold

2nd, fragmentation products are purified using Agencourt AMPure XP magnetic beads：

1）50 μ l fragmentation products are transferred in new 1.5mL low adsorption centrifuge tubes, the concussion mixing that is vortexed Agencourt AMPure XP magnetic beads are simultaneously drawn 50 μ l and are moved in 1.5mL low adsorption centrifuge tubes, gently blown and beaten using pipettor 10 times it is fully mixed It is even, it is incubated at room temperature 5 minutes；

2）The of short duration centrifugation of low adsorption centrifuge tube is placed in separating magnetic bead and liquid in magnetic frame.Treat that solution is clarified（About 5 minutes） Carefully remove supernatant；

3）It keeps centrifuge tube always in magnetic frame, adds in the 80% ethyl alcohol rinsing magnetic bead of 200 μ l Fresh, be placed in room temperature Lower incubation carefully removes supernatant after 30 seconds；

4）Step 3 is repeated, amounts to rinsing twice；

5）Centrifuge tube is kept in magnetic frame, to uncap and be air-dried 10 minutes always；

6）Centrifuge tube from magnetic frame is taken out, adds in the ultrapure water elution of 26 μ l sterilizings, is gently blown and beaten using pipettor fully mixed It is even, the of short duration centrifugation of centrifuge tube is placed in separating magnetic bead and liquid in magnetic frame, treats that solution is clarified（About 5 minutes）It is careful to draw 24 In the PCR pipe of μ l supernatants extremely.

3rd, PCR enrichments and connection sequence label;

1）Above-mentioned sterilizing PCR pipe is placed in ice bath, adds each reactive component successively：

Reagent	Volume	Final concentration
			The product of previous step	24µl	-
5×TAB	10µl	1×
			PPM	5µl	-
N503*（Tag primer）	5µl	-
			N705*（Tag primer）	5µl	-
TAE	1µl	-
			Total volume	50µl

*, 8 kinds of N5XX and 12 kinds are provided in TruePrepTM Index Kit V2 for Illumina (Vazyme #TD202) N7XX can voluntarily be selected according to sample size and Index selection strategies；

2）Abundant mixing is gently blown and beaten using pipettor, PCR pipe is placed in PCR instrument and is reacted as follows：

4th, the sorting of amplified production length and purifying;

1）It is vortexed and shakes mixing Agencourt AMPure XP magnetic beads and draw 35 μ l volumes into above-mentioned 50 μ lPCR products, make 10 abundant mixings are gently blown and beaten with pipettor, are incubated at room temperature 5 minutes；

2）The of short duration centrifugation of reaction tube is placed in separating magnetic bead and liquid in 96 hole magnetic frames, treats that solution is clarified（About 5 minutes）It is small The heart shifts supernatant into new 1.5ml low adsorption centrifuge tubes, abandons magnetic bead；

3）It is vortexed and shakes mixing Agencourt AMPure XP magnetic beads and draw 7.5 μ l volumes into above-mentioned centrifuge tube, use shifting Liquid device gently blows and beats 10 abundant mixings, is incubated at room temperature 5 minutes；

4）The of short duration centrifugation of reaction tube is placed in separating magnetic bead and liquid in magnetic frame, treats that solution is clarified（About 5 minutes）It is careful to move Except supernatant；

5）It keeps centrifuge tube always in magnetic frame, adds in the 80% ethyl alcohol rinsing magnetic bead of 200 μ l Fresh, incubation at room temperature Supernatant is carefully removed after 30 seconds；

6）Step 5 is repeated, amounts to rinsing twice；

7）Centrifuge tube is kept in magnetic frame, to uncap always and be air-dried magnetic bead 10 minutes；

8）Centrifuge tube from magnetic frame is taken out, adds in the ultrapure water elution of 22 μ l sterilizings, is gently blown and beaten using pipettor fully mixed It is even, the of short duration centrifugation of centrifuge tube is placed in separating magnetic bead and liquid in magnetic frame, treats that solution is clarified（About 5 minutes）It is careful to draw 20 μ l supernatants are into new sterile centrifugation tube, in -20 DEG C of preservations.

5th, sequencing library quality testing;

1）Qubit 2.0 detects DNA concentration；

2）Qualitative and quantitative analysis is carried out to sequencing library with 2200 TapeStation System of Agilent, Fig. 2 is survey The schematic diagram of preface storehouse analysis result shows fragment size distribution scope, peak position 484bp.

Embodiment 5

Machine is sequenced on Illumina MiSeq sequenators：It is carried out according to the S.O.P. of Illumina MiSeq sequenators After the setting of the relevant parameters such as 2x300PE, Index, instrument carries out DNA fasciations on sequencing Flow Cell chips into, side automatically Side sequencing is synthesized, specific sequencing library is loaded on Flow Cell chips, joint sequence and the Flow Cell cores at library both ends Oligonucleotide sequence in piece substrate is complementary, fixed all to form a cluster through bridge type PCR amplification per bar segment, and when sequencing adopts Side sequencing reaction is synthesized with reversible distal edge, i.e., during base extension, each circular response can only extend one just Really complementary base confirms base species according to different fluorescence signals, after hundreds of Xun Huans, reads nucleotide sequence.

Embodiment 6

Interpretation of result：Sequencing result is that a series of DNA read sequence（reads）, passed through using MiSeq Reporter (MSR) software It identifies the sequence label in sequence results, establishes the data that each label corresponds to genome DNA sample sequencing result, filter off simultaneously Except joint sequence and low quality data, it will be sequenced by BWA (Burrows-Wheeler Aligner) and read sequence comparison to reference to sequence On row, SNVs and Indels is carried out using GATK and is extracted, and carry out structure variation analysis, the sample is obtained after excluding polymorphic variation Product PAH gene mutation information, to the BAM file application Integrative Genomics Viewer of acquisition（IGV) software carries out The visual analyzing for reading sequence is compared, Fig. 3 is the overburden depth and coverage rate schematic diagram of PAH gene Illumina MiSeq sequencings, In the detection range of PAH genes 88kb most of region sequencing depth up to 1000 × more than, minimum sequencing depth 96 ×, own The coverage rate of extron and all intrones is 100%, illustrates that this method can realize all standing sequencing of PAH genes, Fig. 4 is The IGV views of two PAH pathogenic mutations, display PAH genes are c.1223G>A is mutated and is c.116_118delTCT mutated, and judging should C.1223G the PAH genotype of genome DNA sample is>A/c.116_118delTCT, the testing result are sequenced using Sanger Verification, as a result unanimously.

Finally it should be noted that：The foregoing is only a preferred embodiment of the present invention, is not intended to limit the invention, Although the present invention is described in detail with reference to the foregoing embodiments, for those skilled in the art, still may be used To modify to the technical solution recorded in foregoing embodiments or carry out equivalent substitution to which part technical characteristic, Within the spirit and principles of the invention, any modifications, equivalent replacements and improvements are made should be included in the present invention's Within protection domain.

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Claims (6)

1. a kind of amplimer, kit and its detection method for detecting PAH gene mutations, which is characterized in that special for PCR Property augmentation detection PAH gene mutations primer sets, totally 9 pairs of this group of PCR primer, respectively it is as follows：

For expanding 1 upstream outer sequence of PAH gene extrons（Positioned at gene 5 ' end）To the primer of introne 2, forward direction is drawn Object such as SEQ ID NO:Shown in 1, reverse primer such as SEQ ID NO:Shown in 2；

For expanding 2 upstream of PAH gene introns（5 ' ends）To 2 downstream of introne（3 ' ends）Primer, forward primer such as SEQ ID NO:Shown in 3, reverse primer such as SEQ ID NO:Shown in 4；

For expanding the primer that PAH gene introns 2 arrive introne 3, forward primer such as SEQ ID NO:Shown in 5, reversely draw Object such as SEQ ID NO:Shown in 6；

For expanding 3 upstream of PAH gene introns（Introne 5 ' is held）To introne 3 downstream（Introne 3 ' end）Primer, Forward primer such as SEQ ID NO:Shown in 7, reverse primer such as SEQ ID NO:Shown in 8；

For expanding the primer that PAH gene introns 3 arrive introne 4, forward primer such as SEQ ID NO:Shown in 9, reversely draw Object such as SEQ ID NO:Shown in 10；

For expanding the primer that PAH gene introns 4 arrive introne 5, forward primer such as SEQ ID NO:Shown in 11, reversely draw Object such as SEQ ID NO:Shown in 12；

For expanding the primer that PAH gene introns 5 arrive introne 7, forward primer such as SEQ ID NO:Shown in 13, reversely draw Object such as SEQ ID NO:Shown in 14；

For expanding the primer that PAH gene introns 5 arrive introne 11, forward primer such as SEQ ID NO:Shown in 15, reversely Primer such as SEQ ID NO:Shown in 16；

For expanding PAH gene introns 9 to sequence on the outside of 3 downstream of exons 1（It is held positioned at gene 3 '）Primer, forward direction draws Object such as SEQ ID NO:Shown in 17, reverse primer such as SEQ ID NO:Shown in 18；

For the kit of PCR specific amplifications detection PAH gene mutations, one or more pairs of primers in primer sets are included.

2. a kind of amplimer, kit and its detection method for detecting PAH gene mutations according to claim 1, It is characterized in that：The kit for the detection PAH gene mutations of PCR specific amplifications is also comprising one or more reagents, profit Long-chain PCR " the reagents of long-range PCR, LR-PCR " reactions are carried out with the primer；Long segment is carried out using the primer Reagent D NA polymerases, buffer solution and the dNTP mixtures of PCR reactions；For handling amplified production so that amplified production can be used Reagent in high throughput sequencing technologies.

3. a kind of amplimer, kit and its detection method for detecting PAH gene mutations according to claim 2, It is characterized in that：The kit includes following reagents, one or more pairs of primers, 10 × GeneAmp High in PCR primer group Fidelity PCR buffer solutions, 10mmol/LdNTP, 5mol/L glycine betaine, DMSO and 5U/ μ L polymerase mixtures.

4. a kind of amplimer, kit and its detection method for detecting PAH gene mutations according to claim 3, It is characterized in that：Contain following components in the PCR reaction systems for being 50 μ l in total volume：Each 1 μ l of each PCR primer, primer it is dense It spends for 10 μm of ol/L；10 × GeneAmp High Fidelity PCR buffer solutions 5 μ l, 10mmol/L, dNTP2 μ l, 10 μm of ol/L Forward and reverse each 5 μ l, DMSO2.5 μ l, 5U/ μ l polymerase mixtures of 1 μ l, 5mol/L glycine betaine, 1 μ l, 50ng/ μ l genomes of primer DNA2 μ l, 30.5 μ l of water.

5. amplimer, kit and its detection method of a kind of detection PAH gene mutations according to claim 1-4, Comprise the following steps：

（1）Using human gene group DNA as template, the primer sets of usage right requirement 1, in the condition suitable for amplification purpose nucleic acid Under, PAH genes are expanded using LR-PCR technologies, wherein being made of per pair of primers forward primer and reverse primer；

（2）To 9 long segment products of amplification, purifying recycling is carried out；

（3）9 PCR products of purifying are mixed, and are quantified, obtain the PCR product of 1 people's genome DNA sample Library；

（4）DNA fragmentation processing is carried out to obtaining PCR product library；Wherein described DNA fragmentation method includes the chemistry side of interrupting Method and physics interrupt method, wherein the chemical method includes swivel base enzyme process and traditional enzymatic cleavage methods, the physical method includes Ultrasound interrupts method or machinery interrupts method, and the PCR product library interrupted is built 1 sequencing text using splice tag technology Storehouse, can be respective to distinguish by adding different library connector Barcode Adapter to different genome DNA samples Sequencing library also can handle PCR product library without fragmentation, directly build single SMRTBell libraries；

（5）Obtained multiple sequencing libraries are mixed, it is different to be sequenced type according to library, select different microarray datasets into Row sequencing is read long sequencing etc. including the sequencing of Illumina both-ends, the sequencing of Ion Torrent semiconductors and PacBio SMRT long, is obtained Obtain the sequencing data in PCR product library；

（6）Different genome DNA sample is distinguished based on label B arcode sequences, by bioinformatic analysis by single sample The DNA sequencing fragment that product sequencing obtains is compared onto reference PAH genes, is carried out SNVs and Indels extractions, and is carried out structural The analysis of variation excludes the PAH gene mutation information that polymorphic variation obtains the DNA sample.

6. a kind of amplimer, kit and its detection method for detecting PAH gene mutations according to claim 5, It is characterized in that：Using human gene group DNA as template, choose PAH genes and design 9 pairs of PCR primers, using LR-PCR technologies pair PAH genes are expanded, and the region of amplification includes PAH gene whole extrons and whole intron sequences and part of exon 1 Sequence on the outside of 3 downstream of upstream outer sequence and exons 1, exons 1 upstream outer sequence are located at gene 5 ' end, under exons 13 Trip outside sequence is located at the end of gene 3 ', and in addition each amplified fragments have overlay region with adjacent amplified fragments, are spelled available for segment It connects and haplotype reconstruction, and available for big structure variation is detected, refers mainly to deletion mutant and Duplication mutation；

To 9 long segment products of amplification, purifying recycling is carried out, 9 PCR products of purifying are mixed, and is quantified, 1 PCR product library by inspection DNA sample is obtained, corresponding 1 by inspection genome DNA sample；

To obtained PCR product library construction sequencing library, partly led including Illumina both-ends sequencing library, Ion Torrent Body sequencing library and SMRTBell libraries, are sequenced in corresponding microarray dataset, after obtaining sequencing data, carry out biological letter Credit analysis is ceased, obtains PAH full length genes variation information.