CN108060227B - Amplification primer, kit and detection method for detecting PAH gene mutation - Google Patents

Abstract

The invention discloses an amplification primer, a kit and a detection method for detecting PAH gene mutation, wherein the amplification primer, the kit and the detection method are used for PCR specificity amplification detection of PAH gene mutation, 9 pairs of PCR primers are used in the group, and the PCR primers are respectively as follows: a primer for amplifying the upstream outer sequence (located at the 5' end of the gene) of the exon 1 of the PAH gene to the intron 2, wherein the forward primer is shown as SEQ ID NO. 1, and the reverse primer is shown as SEQ ID NO. 2; the primer for amplifying the region from the upstream (5 'end) of intron 2 of the PAH gene to the downstream (3' end) of intron 2 is shown as SEQ ID NO. 3 in the forward direction, and shown as SEQ ID NO. 4 in the reverse direction. The invention realizes the full coverage of PAH gene, the detected sequence length reaches 88171bp, which is beneficial to the detection of intron inner variation and structure variation, can improve the detection rate of pathogenic mutation, has simpler operation and low cost, and in addition, each amplified fragment and adjacent fragments have overlapping regions, and can be used for fragment splicing and haplotype construction.

Description

Amplification primer, kit and detection method for detecting PAH gene mutation

Technical Field

The invention relates to the technical field of PAH genes, in particular to an amplification primer, a kit and a detection method for detecting PAH gene mutation.

Background

Phenylalanine hydroxylase (PAHD) deficiency is a common amino acid metabolic disease, and the occurrence of PAHD is caused by abnormal phenylalanine metabolism due to reduction or loss of enzyme activity caused by mutation of phenylalanine hydroxylase (PAH). Untreated PAHD patients can cause severe intellectual disability and symptomatic epilepsy due to the neurotoxic effects of excess phenylalanine and bypass metabolites in the body. If early diagnosis and treatment can be obtained, nerve damage may not occur and intelligence is normal. With the development of clinical diagnosis and treatment, PAHD has become a treatable and preventable disease. Because the infant does not have symptoms in the early stage, the PAH gene mutation analysis is a method for diagnosing the PAHD. Meanwhile, PAH gene detection on the patient is also helpful for genetic counseling of the immediate relatives of the patient and provides basis for the prenatal detection of the second birth. The PAH gene is located at position 12q23.2 of human chromosome, the total length is about 90kb, the length of the coding region is 1359bp, and the coding region codes polypeptide chain of 452 amino acids and is further folded to form phenylalanine hydroxylase protein. The currently reported PAH gene has more than 800 mutations, most of which are missense mutations, and the rest of which also comprise shearing mutations, nonsense mutations and small deletion/insertion mutations, relate to the whole gene, have high genetic heterogeneity and have significant regional and ethnic differences. Meanwhile, scholars at home and abroad report that large fragment deletion or repeated mutation spanning exons and/or introns exists in the mutation part of the PAH gene. At present, about 90% of patients can be clarified by adopting a method of combining PCR amplification exon and flanking limited region sequences with DNA sequencing, and about 10% of patients can not clarify the causes, so that a detection method capable of specifically detecting the PAH full-length gene is urgently needed. Since the first pathogenic mutation of the human PAH gene was detected by DiLella et al in 1986 using the fragment cloning-Sanger nucleotide sequencing method, more and more molecular detection techniques for mutation detection of the PAH gene appeared at home and abroad. The reported gene detection methods include Polymerase chain reaction-allele specific oligonucleotide hybridization (PCR-ASO), Polymerase chain reaction-denaturing gel gradient electrophoresis (PCR-denaturing gel electrophoresis, PCR-DGGE), Polymerase chain reaction-Single strand conformation polymorphism (PCR-Single strand conformation polymorphism, PCR-SSCP), Polymerase chain reaction-restriction fragment length polymorphism (PCR-restriction fragment length polymorphism, PCR-RFLP), Polymerase chain reaction-denaturing high performance liquid chromatography (PCR-denaturing high-performance liquid chromatography, PCR-DHPLC), Polymerase chain reaction-high resolution melting curve analysis (PCR-high resolution, HRM), etc., and the methods are characterized by simple economic operation, however, they have the disadvantage that only a limited number of specific mutants can be detected or that the specific position and type of mutation cannot be defined. Currently, Sanger sequencing method is the main method clinically applied to PAH gene mutation detection. Zhang (Zhang Shi et al (J. China Youth and genetics, 2006, 14 th vol., No. 5, pages 14-16) in 2006 established a method for sequencing PCR amplification product Sanger of PAH gene exon by mutation detection and analysis of typical phenylketonuria gene full-length exon. Song 26121in 2007 (mutation constitution of phenylalanine hydroxylase gene in northern China, journal of Chinese medical genetics, 2007, No. 24, No. 3, page 241-containing 246) performed Sanger sequencing detection on all exons and flanking intron sequences of PAH genes of 230 infants suffering from phenylketonuria by using an analysis method established by Zhang et al.

For the detection of PAH gene mutation, a plurality of patents have been published and reported. CN1335408A (2002) discloses a special DNA chip for PAH gene mutation diagnosis suitable for early diagnosis and prenatal screening of phenylketonuria, but the method only detects 40 PAH gene mutations. CN1696308A (2005) also discloses a special DNA chip for PAH gene mutation diagnosis suitable for early diagnosis and prenatal screening of phenylketonuria, but the method only detects 6 PAH gene mutations. CN101481741A (2009) discloses that 13 PAH gene mutations are detected by using PCR primers that specifically amplify exon nos. 4, 5, 6, 7, 10, 11, and 12 of PAH gene and flanking intron sequences thereof, in combination with Sanger sequencing methods, enzymatic mismatch cleavage, and other methods. CN101570787A (2009) also discloses a composite chip for prenatal rapid diagnosis, but this method only detects 5 PAH gene mutations. CN101899518A (2010) discloses a kit for detecting 7 hot spot mutation sites of phenylketonuria PAH gene and a PCR amplification method thereof, and because the specificity of the product is very high, the genotype of the corresponding mutation site of the PAH gene can be judged by directly observing the existence of a product strip of gel electrophoresis. CN101693921A (2010) discloses a method for detecting mutations in exons 7 and 12 of PAH gene by specific PCR amplification of oral swab DNA and Sanger sequencing. CN201376967Y (2010) discloses a composite chip for prenatal rapid diagnosis, but this method only detects 5 PAH gene mutations. CN102533992A (2012) discloses a method for sequencing phenylalanine hydroxylase gene, that is, specific PCR amplification is performed on the target region of exon 1 to 13 of PAH gene, and the PCR product is used to construct sequencing library, and then high-throughput sequencing is performed. CN202671546U (2013) discloses a phenylketonuria pathogenic gene mutation detection kit, namely 13 exons of PAH gene are amplified by using specific PCR primers, and then Sanger sequencing is used for mutation analysis. CN103436616A (2013) discloses a kit for simultaneously detecting 12 mutation hotspots of phenylketonuria PAH genes of Chinese population, which is suitable for population screening. CN104031990A (2014) discloses a phenylketonuria PAH gene detection kit, which is mainly used for detecting 13 common mutation sites of PAH genes. CN103509865A (2014) discloses a method for detecting mutation of PAH gene by using high resolution melting curve analysis technology, which can mainly detect whether the sequences of exons 3, 6, 7, 11 and 12 of PAH gene are abnormal, but can not detect specific genotype. CN104232770A (2014) discloses a phenylketonuria gene detection kit, which utilizes a multiple connection detection reaction technology to rapidly detect genotypes of 7 loci of PAH genes. CN105177160A (2015) discloses a primer and a kit for detecting the mutation of pathogenic genes of various neonatal hereditary metabolic diseases, wherein specific primer multiplex PCR is carried out on the sequences of exons and exon intron binding regions of 40 pathogenic genes (including PAH genes) related to 22 common neonatal hereditary metabolic diseases, and then second-generation sequencing and analysis are carried out to give mutation information of detected genes. CN105755109A (2016) discloses a novel phenylketonuria gene screening and diagnosing system and kit, which utilizes a system constructed by a PCR technology, a DNA connection technology and a capillary electrophoresis technology to detect 41 common PAH gene mutation sites. The total length of the PAH gene exon is 4122bp (the reference genome is GRCh37/hg 19), while the total length of the intron is as high as 76597bp (the reference genome is GRCh37/hg 19), the above reported technology only detects mutation sites, or detects the PAH gene exon sequence and a small part of intron sequences flanking the exon, but cannot detect the whole intron sequences of the PAH gene, and the field still needs a gene detection method capable of detecting the whole length of the PAH gene, namely, the method can detect not only the whole exon sequences, but also the whole intron sequences.

Over the past 10 years, sequencing technology has progressed rapidly, with second generation sequencing technologies typified by Illumina double-ended sequencing and Ion Torrent semiconductor sequencing, and third generation sequencing technologies typified by PacBio SMRT sequencing emerging. Illumina paired-end sequencing uses cluster generation and sequencing-by-synthesis methods to achieve rapid and accurate sequencing. This process recognizes DNA bases while they are added to a nucleic acid strand. Each base emits a unique fluorescent signal while adding to the growing strand, and these signals can be used to determine the order of the DNA sequence. Ion Torrent semiconductor sequencing is a new generation of revolutionary sequencing technology based on semiconductor chips, and the technology uses a high-density semiconductor chip with holes distributed, and one hole is a sequencing reaction pool. When DNA polymerase polymerizes one of 4 dNTPs onto an extended DNA strand, a hydrogen ion is released, the pH value in the reaction cell changes, and an ion receptor located below the cell senses the signal, and the chemical signal is directly converted into a digital signal, thereby reading the DNA sequence. PacBio SMRT sequencing is also based on the principle of sequencing by synthesis, and the core of this technology is the use of Zero-order Waveguide technology (Zero-Mode Waveguide, ZMW), each ZMW holding a DNA polymerase and a DNA template, the nucleotides of fluorescently labeled phosphate groups binding to the template strand at the polymerase active site (each deoxynucleotide being labeled with a dye that is not colored), being excited to fluoresce, and the labeled phosphate groups being cleaved and released after the fluorescent pulse has ended. The polymerase moves to the next position and the next deoxynucleotide is ligated to the site to begin releasing the fluorescent pulse for the next cycle to effect sequencing. The PacBio SMRT sequencing has the characteristics of super-long reading length, small GC bias, high accuracy and the like, and can directly carry out sequencing on long-chain PCR (Long-range PCR, LR-PCR) products without fragmentation treatment and secondary PCR amplification. Because the total length of the total exons and the total introns of the PAH gene is 80719bp (the reference genome is GRCh37/hg 19), the whole length sequence of the gene is completed by the traditional Sanger sequencing, the workload is extremely high, the cost is extremely high, and the whole length of the PAH gene can be sequenced simultaneously by adopting a high-throughput sequencing technology, so that the detection period can be shortened, and the workload and the detection cost can be reduced.

Therefore, it is necessary to invent an amplification primer, a kit and a detection method for detecting PAH gene mutation to solve the above problems.

Disclosure of Invention

The invention aims to provide an amplification primer, a kit and a detection method for detecting PAH gene mutation, so as to solve the problems in the background technology.

In order to achieve the purpose, the invention provides the following technical scheme: an amplification primer, a kit and a detection method for detecting PAH gene mutation are used for PCR specificity amplification to detect PAH gene mutation primer groups, wherein the PCR primers in the group have 9 pairs, and the primer groups are respectively as follows:

a primer for amplifying the upstream outer sequence (located at the 5' end of the gene) of the exon 1 of the PAH gene to the intron 2, wherein the forward primer is shown as SEQ ID NO. 1, and the reverse primer is shown as SEQ ID NO. 2;

the primer for amplifying the upstream (5 'end) of intron 2 of the PAH gene to the downstream (3' end) of intron 2 is shown as SEQ ID NO. 3 in the forward direction and shown as SEQ ID NO. 4 in the reverse direction;

the primer for amplifying the intron 2 to the intron 3 of the PAH gene has a forward primer shown as SEQ ID NO. 5 and a reverse primer shown as SEQ ID NO. 6;

the primer for amplifying the region from the upstream of intron 3 (intron 5 'end) to the downstream of intron 3 (intron 3' end) of the PAH gene has a forward primer shown as SEQ ID NO. 7 and a reverse primer shown as SEQ ID NO. 8;

the primer for amplifying the intron 3 to the intron 4 of the PAH gene has a forward primer shown as SEQ ID NO. 9 and a reverse primer shown as SEQ ID NO. 10;

the primer for amplifying the intron 4 to the intron 5 of the PAH gene has a forward primer shown as SEQ ID NO. 11 and a reverse primer shown as SEQ ID NO. 12;

the primer for amplifying the intron 5 to the intron 7 of the PAH gene has a forward primer shown as SEQ ID NO. 13 and a reverse primer shown as SEQ ID NO. 14;

the primer for amplifying the intron 5 to the intron 11 of the PAH gene has a forward primer shown as SEQ ID NO. 15 and a reverse primer shown as SEQ ID NO. 16;

the primer for amplifying the sequence (located at the 3' end of the gene) from the intron 9 to the downstream outer side of the exon 13 of the PAH gene has the forward primer shown as SEQ ID NO. 17 and the reverse primer shown as SEQ ID NO. 18.

The kit for detecting PAH gene mutation by PCR specific amplification comprises one or more pairs of primers in a primer group.

Preferably, the kit for detecting PAH gene mutation by PCR specific amplification further comprises one or more reagents, and the reagents are used for carrying out long-chain PCR (long-range PCR, LR-PCR) reaction by using the primers; reagent DNA polymerase, buffer solution and dNTP mixture for long fragment PCR reaction by using the primer; reagents for processing amplification products to enable the amplification products to be used in high throughput sequencing technologies.

Preferably, the kit comprises reagents of one or more pairs of primers in a PCR primer set, 10 XGeneAmp High Fidelity PCR buffer, 10mmol/LdNTP, 5mol/L betaine, DMSO and 5U/. mu.L polymerase mix.

Preferably, the PCR reaction system with the total volume of 50 mul comprises the following components: 1 mul of each PCR primer, and the concentration of the primer is 10 mul mol/L; 10 XGeneAmp High Fidelity PCR buffer 5U L, 10mmol/L, dNTP 2U L, 10U mol/L forward and reverse primers each 1U L, 5mol/L betaine 5U L, DMSO 2.5U L, 5U/U L polymerase mixture 1U L, 50 ng/U L genomic DNA 2U L, water 30.5U L.

An amplification primer, a kit and a detection method for detecting PAH gene mutation comprise the following steps:

(1) amplifying the PAH gene using LR-PCR technique using human genomic DNA as a template and the primer set of claim 1 under conditions suitable for amplifying the nucleic acid of interest, wherein each pair of primers consists of a forward primer and a reverse primer.

(2) Purifying and recovering the amplified products of the 9 long fragments;

(3) mixing the purified 9 PCR products, and quantifying to obtain 1 PCR product library of the personal genome DNA sample;

(4) carrying out DNA fragmentation treatment on the obtained PCR product library; the DNA fragmentation method comprises a chemical fragmentation method and a physical fragmentation method, wherein the chemical method comprises a transposase method and a traditional enzyme digestion method, the physical method comprises an ultrasonic fragmentation method or a mechanical fragmentation method, 1 sequencing library is constructed by applying a linker tag technology to a fragmented PCR product library, different genomic DNA samples can be distinguished from each other by adding different library linkers, Barcode adapters are added, the PCR product library can also be not subjected to fragmentation treatment, and a single SMRTBell library is directly constructed;

(5) mixing the obtained sequencing libraries, selecting different sequencing platforms for sequencing according to different sequencing types of the libraries, wherein the sequencing platforms comprise Illumina double-end sequencing, Ion Torrent semiconductor sequencing, PacBio SMRT long-read sequencing and the like, and obtaining sequencing data of a PCR product library;

(6) different genome DNA samples are distinguished based on the tag Barcode sequence, DNA sequence fragments obtained by sequencing a single sample are compared to a reference PAH gene through bioinformatics analysis, SNVs and Indels are extracted, structural variation is analyzed, and polymorphic variation is eliminated to obtain PAH gene mutation information of the DNA sample.

Preferably, human genome DNA is used as a template, PAH gene is selected and 9 pairs of PCR primers are designed, LR-PCR technology is adopted to amplify the PAH gene, the amplified region comprises all exon and all intron sequences of the PAH gene, and partial exon 1 upstream outer sequences and partial exon 13 downstream outer sequences, the exon 1 upstream outer sequences are positioned at the 5 'end of the gene, the exon 13 downstream outer sequences are positioned at the 3' end of the gene, in addition, each amplified segment and adjacent amplified segments have overlapping regions, and the method can be used for segment splicing and haplotype construction and can be used for detecting large structural variation, mainly refers to gene deletion mutation and gene repetition mutation;

purifying and recycling the amplified 9 long fragment products, mixing the purified 9 PCR products, and quantifying to obtain a PCR product library of 1 detected DNA sample, wherein the PCR product library corresponds to 1 detected genome DNA sample;

and constructing a sequencing library for the obtained PCR product library, wherein the sequencing library comprises an Illumina double-end sequencing library, an Ion Torrent semiconductor sequencing library and an SMRTBell library, sequencing on a corresponding sequencing platform, and performing bioinformatics analysis after sequencing data is obtained to obtain the full-length variation information of the PAH gene.

The invention has the technical effects and advantages that: the invention designs 9 pairs of PCR primers by utilizing the sequence information specificity of the PAH gene, the length of the amplified fragments is between 11846bp and 12890bp, the average length of 12481bp of the 9 amplified fragments is a long fragment which exceeds 11kb, the common PCR reaction system and conditions are difficult to succeed, under the optimized PCR reaction system and conditions of the invention, the total exon sequences and all intron sequences of the PAH gene can be obtained by amplification by adopting 9 PCR reactions, compared with other target region capture technologies (such as a chip capture technology and an Ampliseq technology), the method can realize PAH gene full coverage, the detected sequence length reaches 88171bp, which is helpful for detecting intron inner variation and structure variation, can improve the detection rate of pathogenic mutation, and the operation is simpler, the cost is low, and in addition, each amplified fragment and the adjacent fragment have an overlapping region, so that the method can be used for fragment splicing and haplotype construction.

The invention realizes the combined use of PAH gene long-chain PCR amplification and Illumina double-end sequencing for the first time, and applies PAH gene full-length sequencing to phenylalanine hydroxylase deficiency gene detection research for the first time.

Drawings

FIG. 1 is a schematic diagram showing the amplification result of gene long-chain PCR (LR-PCR).

FIG. 2 is a schematic representation of the Agilent 2200 tape State System analysis results of sequencing libraries.

Fig. 3 is a graph of coverage depth and coverage rate for PAH gene Illumina MiSeq sequencing.

Fig. 4 is an IGV view of two PAH pathogenic mutations in Illumina MiSeq sequencing results.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The invention relates to a method for detecting PAH gene mutation, which designs 9 pairs of PCR primers by using sequence information of PAH gene, applies long-chain PCR (Long-range PCR, LR-PCR) technology to amplify the whole PAH gene in a targeted way, covers a genome sequence with the total length of 88171bp, namely a sequence between 103227356 and 103315526 of a chromosome 12 genome (the reference genome is GRCh37/hg 19), comprises the whole exon sequence (the total length is 4122 bp) and the whole intron sequence (the total length is 76597 bp) of the PAH gene, in addition, each amplified fragment and adjacent fragments have an overlapping region, can be used for fragment splicing and monomer construction, constructing a library after the amplified products are fragmented, performing high-throughput sequencing by using an Illumina gene sequencer or an Ion Torrent semiconductor sequencer, or directly constructing a single SMRTBell library after the amplified products are purified and mixed, performing sequencing by using a PacBio RSII sequencer, and (4) analyzing sequencing data through bioinformatics to obtain mutation information of the PAH gene.

The invention provides a primer group for specific PCR amplification of PAH gene, preferably the PCR primers are shown in Table 1, the PCR primer group has 9 pairs, the overlapping area of adjacent amplification fragments is about 3kb, the 9 pairs of primers are respectively as follows: a primer for amplifying the upstream outer sequence (located at the 5' end of the gene) of the exon 1 of the PAH gene to the intron 2, wherein the forward primer is shown as SEQ ID NO. 1, and the reverse primer is shown as SEQ ID NO. 2; a primer for amplifying the upstream (5 'end) of intron 2 of the PAH gene to the downstream (3' end) of intron 2, wherein the forward primer is shown as SEQ ID NO. 3, and the reverse primer is shown as SEQ ID NO. 4; a primer for amplifying the intron 2 to the intron 3 of the PAH gene, wherein the forward primer is shown as SEQ ID NO. 5, and the reverse primer is shown as SEQ ID NO. 6; a primer for amplifying the region from the upstream of intron 3 (intron 5 'end) to the downstream of intron 3 (intron 3' end) of the PAH gene, wherein the forward primer is shown as SEQ ID NO. 7, and the reverse primer is shown as SEQ ID NO. 8; a primer for amplifying the intron 3 to the intron 4 of the PAH gene, wherein the forward primer is shown as SEQ ID NO. 9, and the reverse primer is shown as SEQ ID NO. 10; a primer for amplifying the intron 4 to the intron 5 of the PAH gene, wherein the forward primer is shown as SEQ ID NO. 11, and the reverse primer is shown as SEQ ID NO. 12; a primer for amplifying the intron 5 to the intron 7 of the PAH gene, wherein the forward primer is shown as SEQ ID NO. 13, and the reverse primer is shown as SEQ ID NO. 14; a primer for amplifying the intron 5 to the intron 11 of the PAH gene, wherein the forward primer is shown as SEQ ID NO. 15, and the reverse primer is shown as SEQ ID NO. 16; a primer for amplifying a sequence (located at the 3' end) from the intron 9 of the PAH gene to the downstream outer side of the exon 13, wherein a forward primer is shown as SEQ ID NO:17, a reverse primer is shown as SEQ ID NO:18, the lengths of the amplified fragments 1 of the primer SEQ ID NO:1 and the primer SEQ ID NO:2 are 12594bp, the lengths of the amplified fragments 2 of the primer SEQ ID NO:3 and the primer SEQ ID NO:4 are 12764bp, the lengths of the amplified fragments 3 of the primer SEQ ID NO:5 and the primer SEQ ID NO:6 are 12067bp, the lengths of the amplified fragments 4 of the primer SEQ ID NO:7 and the primer SEQ ID NO:8 are 12890bp, the lengths of the amplified fragments 5 of the primer SEQ ID NO:9 and the primer SEQ ID NO:10 are 11846bp, the lengths of the amplified fragments 6 of the primer SEQ ID NO:11 and the primer SEQ ID NO:12 are 12027bp, the lengths of the amplified fragments 7 of the primers SEQ ID NO:13 and the amplified fragments 14 are 25bp, the length of the amplified fragment 8 of the primer SEQ ID NO:15 and the primer SEQ ID NO:16 is 12553bp, the length of the amplified fragment 9 of the primer SEQ ID NO:17 and the primer SEQ ID NO:18 is 12764bp, the total length of the amplified fragments 9 is 88171bp, the genome region covers all exons (the exon coverage rate is 100%) and all intron sequences (the intron coverage rate is 100%) of the PAH gene, and the length sequence of 4145bp outside the upstream of the exon 1 and the length sequence of 3307bp outside the downstream of the exon 13.

The invention also provides a kit for detecting PAH gene mutation by specific PCR amplification, which comprises one or more pairs of primers in the primer group, and the kit can also comprise one or more of the following reagents: reagents for performing a long-fragment PCR reaction using the primers; preferably wherein the reagent DNA polymerase, buffer and dNTP mixture for performing long fragment PCR reaction using the primer; reagents for treating the amplification product to enable the amplification product to be used in a High throughput sequencing technique, which may specifically be 10 × GeneAmp High Fidelity PCR buffer; 10mmol/L dNTP; 5mol/L betaine; DMSO; 5U/mul polymerase mixture, and the PCR reaction system with the total volume of 50 mul contains the following components: 10 XGeneAmp High Fidelity PCR buffer 5U L, 10mmol/LdNTP 2U L, 10U mol/L forward and reverse primers each 1U L, 5mol/L betaine 5U L, DMSO 2.5U L, 5U/U L polymerase mixture 1U L, 50 ng/U L genomic DNA 2U L, water 30.5U L.

The PCR primer group or the kit has the application of detecting PAH gene mutation and the application in detecting phenylalanine hydroxylase deficiency genes.

The invention also provides a method for detecting PAH gene mutation in vitro, which comprises the following steps: (1) using human genome DNA as a template, using a primer group consisting of 9 pairs of primers of the invention, and adopting LR-PCR technology to amplify PAH gene under the condition suitable for amplifying target nucleic acid; (2) purifying and recovering the amplified products of the 9 long fragments; (3) mixing the purified 9 PCR products, and quantifying to obtain 1 PCR product library of the personal genome DNA sample; (4) constructing a high-throughput sequencing library for the obtained PCR product library; (5) mixing a plurality of sequencing libraries, and sequencing by selecting different sequencing platforms according to different types of libraries; (6) comparing DNA sequence fragments obtained by sequencing a single sample to a reference PAH gene through bioinformatics analysis, extracting SNVs and Indels, analyzing structural variation, and eliminating polymorphic variation to obtain the PAH gene mutation information of the sample.

The method for detecting mutation of PAH gene of the present invention preferably comprises the following steps: (1) using genome DNA as a template, selecting PAH gene and designing 9 pairs of PCR primers, and amplifying the PAH gene by adopting LR-PCR technology, wherein a PCR reaction system comprises 0.5M betaine and 5% DMSO, and PCR reaction conditions adopt 2-step PCR; (2) purifying and recovering the amplified products of the 9 long fragments, and quantifying; (3) mixing the purified 9 PCR products in equal proportion, and quantifying to obtain 1 PCR product library of the personal genome DNA sample; (4) for 1 PCR product library, DNA fragmentation, end repair and adaptor ligation reactions are completed in one step by using a transposase method, and an Index1 tag sequence and an Index2 tag sequence are added to the two ends of the fragmented fragment to construct an on-machine sequencing library of each PCR product library; wherein the tag sequences of Index1 and Index2 are different; the tags used by the different PCR product libraries are different from each other to distinguish different genomic DNA samples; performing quality detection and quantification on each sequencing library; (5) loading a plurality of sequencing libraries (tags can be distinguished) on a Flow Cell chip in equal amount in a mixing manner, and performing double-end synthesis-side sequencing on an Illumina gene sequencer (Illumina misseq sequencer, Illumina NextSeq500 sequencer or Illumina HiSeq X Ten sequencer); (6) distinguishing different genome DNA samples based on the tag sequences, carrying out splitting by identifying the tag sequences in sequence results, establishing sequencing result data corresponding to each genome DNA sample, comparing DNA sequence fragments obtained by sequencing a single sample to a reference PAH gene through bioinformatics analysis, carrying out SNVs and Indels extraction, carrying out structural variation analysis, and eliminating polymorphic variation to obtain mutation information of the PAH gene of the sample.

In the detection method, the DNA fragmentation method comprises a chemical breaking method and a physical breaking method, wherein the chemical method comprises a traditional enzyme digestion method and a novel transposase method, the physical breaking method comprises an ultrasonic breaking method or a mechanical breaking method, fragments with the length of 400bp-600bp are obtained after the DNA is broken, and the purification and recovery method comprises but is not limited to magnetic bead recovery and can also be electrophoresis tapping recovery.

In the detection method of the present invention, the sequencing technology includes but is not limited to Illumina sequencing technology, and can also be other second generation sequencing technologies; third generation sequencing technologies are also possible, such as a PacBio RSII sequencer and a PacBio sequential sequencer; different library construction methods were used for different sequencers.

Example 1

Extracting genome DNA: 2ml of peripheral blood of a detected person is collected and placed in an EDTA anticoagulation tube, 0.2ml of EDTA anticoagulation peripheral blood sample is taken, DNA is extracted according to the specification of the whole blood DNA extraction kit, the DNA concentration is analyzed by using Qubit2.0, the extracted genome DNA is prepared to be used as a next PCR template, 5 gene DNA samples are detected in the research, wherein 1 gene DNA sample has known pathogenic mutation sites, and 4 gene DNA samples are normal human genome DNA.

Example 2

LR-PCR amplification: designing and synthesizing 9 pairs of PAH gene specific primers according to the PAH gene sequence, carrying out LR-PCR by adopting a GeneAmp High Fidelity PCR System, amplifying 9 large fragment sequences respectively 1, 2, 3, 4 and 5 in total, 6. 7, 8 and 9, primer sequences and amplification product sizes are shown in Table 1, using GeneAmp High Fidelity PCR System, setting reaction conditions to carry out LR-PCR on Veriti 96-well Thermal Cycler PCR instrument, the total volume of PCR reaction is 50 μ L, including 5 μ L of 10 XGeneAmp High Fidelity PCR buffer, 2 μ L of 10mmol/L dNTP, 1 μ L of each of 10 μmol/L forward and reverse primers, 5 μ L of 5mol/L betaine, 2.5 μ L of DMSO, 1 μ L of 5U/μ L polymerase mixture, 2 μ L of 50ng/μ L genomic DNA, 30.5 μ L water, PCR cycle parameters: denaturation at 98 deg.C for 1 min; 10sec at 98 ℃, 13min at 68 ℃ for 30 cycles, and finally extending for 15min at 72 ℃, wherein the PCR reaction is completed on a Veriti 96-well Thermal Cycler PCR instrument.

Table 1, LR-PCR primers for amplification of PAH gene:

note: f is a forward primer (upstream primer), R is a reverse primer (downstream primer), and the PCR primer sequences in the table are as follows: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18.

After LR-PCR is completed, 3 μ l of PCR product is detected by 0.6% agarose gel electrophoresis, and FIG. 1 shows that the sizes of 9 amplified fragments are consistent with a theoretical value, target bands are clear, non-specific amplification is avoided, and the selected LR-PCR reaction system and conditions are appropriate.

Example 3

LR-PCR product purification and quantification: an LR-PCR product is purified by a magnetic bead method, a Qubit2.0 fluorescence photometer is adopted to carry out DNA concentration quantification on the purified products No. 1-9 of each genome DNA sample, and the concentration detection results of total 45 LR-PCR amplification products of 5 genome DNA samples are as follows:

DNA concentration detection (Qubit 2.0)

As the length of the 9 amplified fragments is basically consistent, 9 PCR products corresponding to the same genome DNA sample are mixed according to equal mass, and are concentrated and enriched by adopting Agencour AMPure XP magnetic beads (Beckman Coulter company in America), and are quantified by using a Qubit2.0 fluorescence photometer.

Example 4

Library construction: the Library construction is prepared by TruePrepTM DNA Library Prep Kit V2 (Vazyme company, China), and the specific operation steps are as follows:

1. DNA fragmentation, end repair and linker ligation reactions by the transposase method:

1) thawing the 5 XTTBL solution at room temperature, and turning upside down and mixing uniformly for later use;

2) the following reagents were added to the sterilized PCR tube in order:

reagent	Volume of	Final concentration/amount
			Purified mixed PCR product	3μl(10ng/µl)	30ng
5×TTBL	10µl	1×
			TTE Mix V50	5µl	-
ddH2O	32µl	-
			Total volume	50µl

3) Lightly beating the mixed solution for 20 times, and fully mixing;

4) centrifuging by a rapid centrifuge;

5) the PCR tube was placed on a Veriti 96-well Thermal Cycler PCR instrument (ABI, USA) and the following reaction program was set up:

hot lid	105℃
		55℃	10 Min
10℃	Hold

2. The fragmentation product was purified using Agencourt AMPure XP magnetic beads:

1) transferring 50 mu l of the fragmentation product into a new 1.5mL low-adsorption centrifuge tube, vortexing, shaking and uniformly mixing Agencour AMPure XP magnetic beads, sucking 50 mu l of the Agencour AMPure XP magnetic beads, transferring the Agencure AMPure XP magnetic beads into the 1.5mL low-adsorption centrifuge tube, gently blowing and beating the Agencure AMPure XP magnetic beads for 10 times by using a pipettor, fully mixing the Agencure AMPure XP magnetic beads and the Agencure XP magnetic beads, and incubating the Agencure AMPure XP magnetic beads for 5 minutes at room temperature;

2) the low-adsorption centrifuge tube was centrifuged briefly and placed in a magnetic rack to separate the magnetic beads from the liquid. The supernatant was carefully removed until the solution was clear (about 5 minutes);

3) keeping the centrifuge tube in a magnetic frame all the time, adding 200 mul of freshly prepared 80% ethanol to rinse the magnetic beads, incubating at room temperature for 30 seconds, and carefully removing the supernatant;

4) repeating the step 3, and rinsing twice in total;

5) keeping the centrifugal tube in the magnetic frame all the time, opening the cover and drying in air for 10 minutes;

6) the tube was removed from the rack, 26. mu.l of sterile ultrapure water was added for elution, gently pipetted using a pipette and mixed well, the tube was centrifuged briefly and placed in the rack to separate the beads from the liquid, and 24. mu.l of the supernatant was carefully pipetted into the PCR tube after the solution was clarified (about 5 minutes).

3. PCR enrichment and connection of tag sequences;

1) placing the sterilized PCR tube in an ice bath, and sequentially adding the reaction components:

reagent	Volume of	Final concentration
			The product of the previous step	24µl	-
5×TAB	10µl					1×
			PPM	5µl	-
N503 star (tag primer)	5µl	-
			N705 star (tag primer)	5µl	-
TAE	1µl	-
			Total volume	50µl

TruePrepTM Index Kit V2 for Illumina (Vazyme # TD202) provides 8N 5XX and 12N 7XX, which can be selected by themselves based on sample number and Index selection strategy;

2) and (3) lightly blowing and beating by using a pipettor, fully and uniformly mixing, and placing the PCR tube in a PCR instrument for carrying out the following reaction:

4. sorting and purifying the length of the amplified product;

1) vortex, shaking and uniformly mixing Agencour AMPure XP magnetic beads, sucking 35 mul of volume into the 50 mul of PCR product, lightly blowing and beating 10 times by using a pipettor, and fully mixing, and incubating for 5 minutes at room temperature;

2) the reaction tube was centrifuged briefly and placed in a 96-well magnetic rack to separate the beads from the liquid, the supernatant was carefully transferred to a new 1.5ml low adsorption centrifuge tube after the solution was clarified (about 5 minutes), and the beads were discarded;

3) mixing Agencour AMPure XP magnetic beads uniformly by vortex oscillation, sucking a volume of 7.5 microliter into the centrifuge tube, lightly blowing and beating 10 times by using a pipettor, and fully mixing, and incubating for 5 minutes at room temperature;

4) the reaction tube was briefly centrifuged and placed in a magnetic rack to separate the beads from the liquid, and the supernatant carefully removed until the solution cleared (about 5 minutes);

5) keeping the centrifuge tube in a magnetic frame all the time, adding 200 mul of freshly prepared 80% ethanol to rinse the magnetic beads, incubating at room temperature for 30 seconds, and carefully removing the supernatant;

6) repeating the step 5, and rinsing twice in total;

7) keeping the centrifugal tube in the magnetic frame all the time, opening the cover and drying the magnetic beads in air for 10 minutes;

8) taking out the centrifugal tube from the magnetic frame, adding 22 mul of sterilized ultrapure water for elution, lightly blowing and beating the centrifugal tube by using a pipettor to be fully and uniformly mixed, centrifuging the centrifugal tube for a short time, placing the centrifugal tube in the magnetic frame to separate magnetic beads and liquid, carefully sucking 20 mul of supernatant into a new sterilized centrifugal tube after the solution is clarified (about 5 minutes), and storing the supernatant at-20 ℃.

5. Detecting the quality of the sequencing library;

1) the DNA concentration is detected by the Qubit 2.0;

2) the Agilent 2200 tape State System is used for qualitative and quantitative analysis of the sequencing library, FIG. 2 is a schematic diagram of the analysis result of the sequencing library, the distribution range of fragment sizes is shown, and the main peak position is 484 bp.

Example 5

Sequencing on an Illumina MiSeq sequencer: after setting relevant parameters such as 2x300PE and Index according to a standard operation program of an Illumina MiSeq sequencer, automatically generating DNA clusters on a sequencing Flow Cell chip, synthesizing and sequencing at the same time, loading a specific sequencing library on the Flow Cell chip, complementing joint sequences at two ends of the library with oligonucleotide sequences on a Flow Cell chip substrate, amplifying each fixed fragment by bridge PCR to form a cluster, performing reversible terminal synthesis and sequencing reaction at the same time during sequencing, namely, in the base extension process, only one correct complementary base can be extended in each cycle reaction, confirming the base type according to different fluorescent signals, and reading a nucleic acid sequence after hundreds of cycles.

Example 6

And (4) analyzing results: the sequencing result is a series of DNA reading (reads), the MiSeq Reporter (MSR) software is adopted to identify the label sequence in the sequence result, establish the data of the sequencing result of the genome DNA sample corresponding to each label, simultaneously filter and remove the linker sequence and the low-quality data, BWA (Burrows-Wheeler Aligner) is adopted to compare the sequencing reading to the reference sequence, GATK is adopted to extract SNVs and Indels, structural variation analysis is carried out, the PAH gene mutation information of the sample is obtained after polymorphic variation is eliminated, Integrative Genomics Viewer (IGV) software is applied to the obtained BAM file to carry out visual analysis of the comparison reading sequence, FIG. 3 is a schematic diagram of the coverage depth and the coverage rate of the PAH gene Illumina MiSeq sequencing, the depth of most region in the 88kb detection range of the PAH gene can reach more than 1000 x, the lowest depth is 96 x, and the coverage rate of all exons and all introns is 100 percent of sequencing, the method can realize the full-coverage sequencing of the PAH gene, FIG. 4 is an IGV view of two PAH pathogenic mutations, shows that the C.1223G > A mutation and the C.116-118 delTCT mutation of the PAH gene, judges the PAH genotype of the genome DNA sample to be C.1223G > A/C.116-118 delTCT, and adopts Sanger sequencing verification to detect results, and the results are consistent.

Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications may be made to the embodiments or portions thereof without departing from the spirit and scope of the invention.

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Claims (5)

1. An amplification primer group for detecting PAH gene mutation, which is characterized in that the amplification primer group comprises 9 pairs of primers which are respectively as follows:

the primer for amplifying the upstream outer sequence of the exon 1 of the PAH gene to the intron 2 is shown as SEQ ID NO. 1 in the forward primer and shown as SEQ ID NO. 2 in the reverse primer;

the primer for amplifying the upstream of the intron 2 of the PAH gene to the downstream of the intron 2 is shown as SEQ ID NO. 3, and the reverse primer is shown as SEQ ID NO. 4;

the primer for amplifying the intron 2 to the intron 3 of the PAH gene has a forward primer shown as SEQ ID NO. 5 and a reverse primer shown as SEQ ID NO. 6;

the primer for amplifying the upstream of the intron 3 of the PAH gene to the downstream of the intron 3 is shown as SEQ ID NO. 7 in the forward direction and shown as SEQ ID NO. 8 in the reverse direction;

the primer for amplifying the intron 3 to the intron 4 of the PAH gene has a forward primer shown as SEQ ID NO. 9 and a reverse primer shown as SEQ ID NO. 10;

the primer for amplifying the intron 4 to the intron 5 of the PAH gene has a forward primer shown as SEQ ID NO. 11 and a reverse primer shown as SEQ ID NO. 12;

the primer for amplifying the intron 5 to the intron 7 of the PAH gene has a forward primer shown as SEQ ID NO. 13 and a reverse primer shown as SEQ ID NO. 14;

the primer for amplifying the intron 5 to the intron 11 of the PAH gene has a forward primer shown as SEQ ID NO. 15 and a reverse primer shown as SEQ ID NO. 16;

the primer for amplifying the sequence from the intron 9 to the downstream outer side of the exon 13 of the PAH gene is shown as SEQ ID NO. 17 in the forward primer and as SEQ ID NO. 18 in the reverse primer.

2. A kit for detecting mutation of PAH gene, which is characterized in that: the kit comprises the amplification primer set of claim 1, and further comprises reagents for performing a long-chain PCR reaction using the amplification primer set, and reagents for processing an amplification product so that the amplification product can be used in a high-throughput sequencing technology; the reagent for the long-chain PCR reaction comprises DNA polymerase, buffer solution and dNTP mixture which are used for carrying out long-fragment PCR reaction by using the amplification primer group.

3. The kit of claim 2, wherein: the kit also contains 10 XHi-Fi PCR buffer, 10mmol/L dNTP, 5mol/L betaine, DMSO and 5U/. mu.L polymerase mixture.

4. The kit of claim 3, wherein: the PCR reaction system with the total volume of 50 muL contains the following components: 10 XHi-Fi PCR buffer 5U L, 10mmol/L dNTP 2U L, 10U mol/L forward and reverse primers 1U L, 5mol/L betaine 5U L, DMSO 2.5U L, 5U/U L polymerase mixture 1U L, 50 ng/U L genomic DNA 2U L, water 30.5L.

5. A detection method for non-disease diagnostic purposes for detecting mutations in the PAH gene comprising the steps of:

(1) amplifying the PAH gene using long-chain PCR technique using the amplification primer set of claim 1 using human genomic DNA as a template under conditions suitable for amplifying the nucleic acid of interest, wherein each pair of primers consists of a forward primer and a reverse primer;

(2) purifying and recovering the amplified products of the 9 long fragments;

(3) mixing the purified 9 PCR products, and quantifying to obtain 1 PCR product library of the personal genome DNA sample;

(4) and (3) performing DNA fragmentation treatment on the obtained PCR product library: wherein the DNA fragmentation method comprises a chemical disruption method and a physical disruption method, wherein the chemical disruption method comprises a transposase method or a restriction endonuclease method, the physical disruption method comprises a mechanical disruption method, 1 sequencing library is constructed by applying a linker tag technology to the disrupted PCR product library, and the respective sequencing libraries are distinguished by adding different library linkers Barcode Adapter to different genomic DNA samples;

or a single SMRTBell library is directly established without fragmenting the PCR product library;

(5) mixing a plurality of sequencing libraries, and sequencing by selecting different sequencing platforms according to different sequencing types of the libraries: the sequencing platform comprises Illumina double-end sequencing, Ion Torrent semiconductor sequencing and PacBio SMRT long-read sequencing to obtain sequencing data of a PCR product library;

(6) different genome DNA samples are distinguished based on the tag Barcode sequence, DNA sequence fragments obtained by sequencing a single sample are compared to a reference PAH gene through bioinformatics analysis, SNVs and Indels are extracted, structural variation is analyzed, and polymorphic variation is eliminated to obtain PAH gene mutation information of the DNA sample.

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