CN108707648A - The detection method and detection kit of PKD1 genes and PKD2 gene mutations - Google Patents
The detection method and detection kit of PKD1 genes and PKD2 gene mutations Download PDFInfo
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Abstract
The invention discloses the detection methods and detection kit of a kind of PKD1 genes and PKD2 gene mutations.The detection method and detection kit of the PKD1 genes and PKD2 gene mutations, by creatively designing multipair specific Long fragment PCR primer to be expanded respectively, when being expanded as template using human gene group DNA, it can be to avoid the high pseudogene of amplification homology, the false positive and false negative result that pseudogene is brought thus can be avoided the occurrence of, greatly improve the detection accuracy of PKD1 genes and PKD2 gene mutations, and in conjunction with high throughput sequencing technologies, it can simplify techniqueflow compared with aobvious capture technique outside traditional medicine, reduce testing cost, shorten detection cycle.
Description
Technical field
The present invention relates to biochemistry detection fields, more particularly, to a kind of detection method of PKD1 genes and PKD2 gene mutations
And detection kit.
Background technology
PKD1 genes are located at No. 16 the short arm of a chromosome (16p13.3-p13.12) of the mankind, and mrna length 52kb contains 46
Exon, for structure as shown in Fig. 2, its mRNA length is 14kb, the albumen of coding is known as 1 (polycystin- of polycystins
1).There are 6 pseudogenes homologous with it, the 1-33 extras of the pseudogene and PKD1 genes on No. 16 chromosomes for PKD1 genes
Aobvious subsequence homology is up to 97.7%, and about 80% pathogenic mutation occurs in this region, and partial dna sequence G/C content
Up to 85%, gene magnification is very difficult, and gene mutation type is more, is related to whole gene, and without mutantional hotspot, therefore, special
Opposite sex detection PKD1 gene mutations are relatively difficult.It is long-armed (4q22-23) that PKD2 is located at mankind's rice chromosome, mrna length
68kb has 15 exons, structure as shown in figure 3, its mRNA length about 2.9kb, the albumen of coding is known as polycystins 2
(polycysti-2)。
PKD1 the and PKD2 gene mutation forms of hitherto reported include missense mutation, nonsense mutation, shearing mistake, missing,
Be inserted into and repeat etc..The prior art is studies have shown that PKD1 genes and PKD2 gene pathogenic mutations easily lead to and suffer from autosomal dominant
Polycystic Kidney (autosomal dominant polycystic kidney disease, ADPKD), wherein PKD1 genes
ADPKD accounts for about 5%~15% caused by ADPKD caused by mutation accounts for about 85%~90%, PKD2 gene mutations.As shown in Figure 1,
ADPKD is a kind of genetic syndrome, and (PC-1 and PC-2 are co-expressed in kidney cilium and formed more capsules by PC-1 and PC-2
Mechanical stimulus is changed into chemical signal by protein complexes, is adjusted stream in cell chloride, is adjusted cell cycle and division) structure
And dysfunction or kidney ciliary structures can lead to the generation of renal cyst disease extremely, therefore, when ADPKD makes a definite diagnosis need to tie
The hereditary information of race and clinical manifestation carry out comprehensive analysis of the whole family, such as whether clinical manifestation has polycystic kindey, hepatic cyst disease to patient
Shape, and whether parental generation also has similar symptom etc., simple PKD1 genes and PKD2 gene mutations to be not sufficient to show to suffer from
ADPKD, but the abrupt climatic change result can be as intermediate diagnostic result to carry out final medical diagnosis on disease in conjunction with other information.So
And the technique of gene detection such as aobvious capture technique be easy to cause missing inspection outside traditional medicine, in amplification PKD1 genes and PKD2 genes
Shi Jiyi is expanded and the high pseudogene of its homology, thus will appear the result of false positive and false negative.
Invention content
Based on this, it is necessary to provide a kind of PKD1 genes that detection accuracy is high and PKD2 gene mutations detection method and
Detection kit.
A kind of detection method of PKD1 genes and PKD2 gene mutations, includes the following steps：
Step 1：Extract the genomic DNA of sample to be tested；
Step 2：Using the genomic DNA of extraction as template, the respective segments of PKD1 genes and PKD2 genes are carried out
Long fragment PCR expands, wherein Long fragment PCR amplification primer pair used is selected from following ten kinds of primer pairs：Sequence such as SEQ ID
NO:Sense primer shown in 1 and sequence such as SEQ ID NO:Such as SEQ ID NO of downstream primer, sequence shown in 2:On shown in 3
Swim primer and sequence such as SEQ ID NO:Such as SEQ ID NO of downstream primer, sequence shown in 4:Sense primer and sequence shown in 5
Such as SEQ ID NO:Such as SEQ ID NO of downstream primer, sequence shown in 6:Sense primer shown in 7 and sequence such as SEQ ID NO:
Such as SEQ ID NO of downstream primer, sequence shown in 8:Sense primer shown in 9 and sequence such as SEQ ID NO:Downstream shown in 10
Primer, sequence such as SEQ ID NO:Sense primer shown in 11 and sequence such as SEQ ID NO:Downstream primer, sequence shown in 12
Such as SEQ ID NO:Sense primer shown in 13 and sequence such as SEQ ID NO:Such as SEQ ID of downstream primer, sequence shown in 14
NO:Sense primer shown in 15 and sequence such as SEQ ID NO:Such as SEQ ID NO of downstream primer, sequence shown in 16:Shown in 17
Sense primer and sequence such as SEQ ID NO:Such as SEQ ID NO of downstream primer and sequence shown in 18:Draw upstream shown in 19
Object and sequence such as SEQ ID NO:Downstream primer shown in 20；
Step 3：The product that Long fragment PCR is expanded carries out high-flux sequence detection after mixing.
Above-mentioned ten pairs of primer pairs are respectively used for amplifying ten different zones of whole gene, this ten pairs of primers can form full base
The covering of cause can prevent the phenomenon that being mutated missing inspection, be conducive to the recall rate for improving mutation.
In one of the embodiments, in the step 1, the genomic DNA of the extraction sample to be tested is to use salt
The extraction of analysis method whole blood DNA, the extraction of paramagnetic particle method whole blood DNA or the extraction of embrane method whole blood DNA.
In one of the embodiments, in the step 2, sequence such as SEQ ID NO are used:Primer shown in 1~12
When the corresponding primer pair constituted carries out Long fragment PCR amplification, amplification system is to be added to have 5 10 × LA of μ l in every 50 μ l systems
PCR BufferⅡ(Mg2+Plus), 8 each concentration of μ l are the dNTP mixtures of 2.5mM, 2.5 μ l DMSO, 5 a concentration of 5M of μ l
The genomic DNA mould of glycine betaine, the sense primer of final concentration of 200nM, the downstream primer of final concentration of 200nM, 50~200ng
The TakaRa LA Taq enzymes of plate and 0.5 μ l, remaining is water；
Use sequence such as SEQ ID NO:The corresponding primer pair that primer shown in 13~20 is constituted carries out Long fragment PCR expansion
When increasing, amplification system is to be added to have 5 μ l 10 × LA PCR Buffer, II (Mg in every 50 μ l systems2+Plus), 8 each concentration of μ l
Be the dNTP mixtures of 2.5mM, the sense primer of final concentration of 200nM, the downstream primer of final concentration of 200nM, 50~
The TakaRa LA Taq enzymes of the genomic DNA template of 200ng and 0.5 μ l, remaining is water.
In one of the embodiments, in the step 2, for sequence such as SEQ ID NO:Sense primer shown in 1
With sequence such as SEQ ID NO:The primer pair and sequence such as SEQ ID NO that downstream primer shown in 2 is constituted:Draw upstream shown in 11
Object and sequence such as SEQ ID NO:Shown in 12 downstream primer constitute primer pair carry out Long fragment PCR amplification when condition be：
98 DEG C, 98 DEG C of 3min -, 30sec, 60 DEG C, 15sec totally 35 recycle -68 DEG C, 72 DEG C of 1.5min -, 7min；
For sequence such as SEQ ID NO:Sense primer shown in 3 and sequence such as SEQ ID NO:Downstream primer shown in 4
The primer pair of composition, sequence such as SEQ ID NO:Sense primer shown in 5 and sequence such as SEQ ID NO:Downstream primer shown in 6
The primer pair and sequence of composition such as SEQ ID NO:Sense primer shown in 7 and sequence such as SEQ ID NO:Draw in downstream shown in 8
Object constitute primer pair carry out Long fragment PCR amplification when condition be：94 DEG C, 94 DEG C of 2min-, 15sec, 69 DEG C, 30sec it is total
14 cycles, and the corresponding link temperature of the more previous cycle of each link of next cycle reduces by 0.5 DEG C -68 DEG C, 6min -
94 DEG C, 15sec, 62 DEG C, 30sec totally 25 recycle -68 DEG C, 72 DEG C of 6min -, 10min；
For sequence such as SEQ ID NO:Sense primer shown in 9 and sequence such as SEQ ID NO:Downstream primer shown in 10
The condition when primer pair of composition carries out Long fragment PCR amplification is：95 DEG C, 98 DEG C of 2min-, 20sec, 69 DEG C, 15sec totally 14
A cycle, and the corresponding link temperature of the more previous cycle of each link of next cycle reduces by 0.5 DEG C -72 DEG C, 3min -98
DEG C, 20sec, 62 DEG C, 15sec totally 25 recycle -72 DEG C, 72 DEG C of 3min -, 5min；
For sequence such as SEQ ID NO:Sense primer shown in 13 and sequence such as SEQ ID NO:Draw in downstream shown in 14
Object constitute primer pair carry out Long fragment PCR amplification when condition be：98 DEG C, 10sec, 60 DEG C, 15sec totally 35 cycle -68
℃,3min—72℃,5min；
For sequence such as SEQ ID NO:Sense primer shown in 15 and sequence such as SEQ ID NO:Draw in downstream shown in 16
The primer pair of object composition, sequence such as SEQ ID NO:Sense primer shown in 17 and sequence such as SEQ ID NO:Downstream shown in 18
The primer pair and sequence such as SEQ ID NO that primer is constituted:Sense primer shown in 19 and sequence such as SEQ ID NO:Shown in 20
Downstream primer constitute primer pair carry out Long fragment PCR amplification when condition be：98 DEG C, 10sec totally 35 recycle -68 DEG C,
10min—72℃、5min。
In one of the embodiments, in the step 3, the product mixing that Long fragment PCR is expanded is
After Long fragment PCR amplification terminates, the product equimolar that ten groups of long segment PCR amplifications are obtained mixes.
In one of the embodiments, in the step 3, after the product mixing that Long fragment PCR is expanded
High-flux sequence detection is carried out to include the following steps：
DNA is interrupted：The product that the Long fragment PCR expands is broken into DNA fragmentation；
It repairs end：End reparation is carried out to the DNA fragmentation；
Add A：A bases are added at 3 ' ends of the DNA fragmentation that end is repaired；
Connect sequence measuring joints：Sequence measuring joints are connected on the DNA fragmentation of the upper A bases of connection；
Pre-PCR is expanded：DNA fragmentation to being connected with sequence measuring joints carries out Pre-PCR amplifications, in addition specific molecular mark
Label build DNA library；
The DNA library of structure is sequenced, by different specific molecular labels to sequencing result after the completion of sequencing
It distinguishes, and sequencing result is compared on reference to target-gene sequence, obtain abrupt information.
It is to be broken into that the product that the Long fragment PCR is expanded, which is broken into DNA fragmentation, in one of the embodiments,
Of length no more than 500bp, peak value 350bp DNA fragmentation.
In one of the embodiments, after DNA is interrupted, end repair after, plus A after, connection sequence measuring joints it
Further include the steps that afterwards and after Pre-PCR amplifications that purification process is carried out to the product that processing obtains.
The PKD1 genes and the detection method of PKD2 gene mutations further include in sequencing in one of the embodiments,
Product after the preceding amplification to Pre-PCR is into the step of row agarose gel electrophoresis detection and fluorogenic quantitative detection.
A kind of detection kit of PKD1 genes and PKD2 gene mutations, including Long fragment PCR amplimer reagent, it is described
Primer pair in Long fragment PCR amplimer reagent is selected from following ten kinds of primer pairs：Sequence such as SEQ ID NO:Upstream shown in 1
Primer and sequence such as SEQ ID NO:Such as SEQ ID NO of downstream primer, sequence shown in 2:Sense primer shown in 3 and sequence are such as
SEQ ID NO:Such as SEQ ID NO of downstream primer, sequence shown in 4:Sense primer shown in 5 and sequence such as SEQ ID NO:6
Shown in downstream primer, sequence such as SEQ ID NO:Sense primer shown in 7 and sequence such as SEQ ID NO:Draw in downstream shown in 8
Object, sequence such as SEQ ID NO:Sense primer shown in 9 and sequence such as SEQ ID NO:Downstream primer, sequence shown in 10 are such as
SEQ ID NO:Sense primer shown in 11 and sequence such as SEQ ID NO:Such as SEQ ID NO of downstream primer, sequence shown in 12:
Sense primer shown in 13 and sequence such as SEQ ID NO:Such as SEQ ID NO of downstream primer, sequence shown in 14:On shown in 15
Swim primer and sequence such as SEQ ID NO:Such as SEQ ID NO of downstream primer, sequence shown in 16:Sense primer and sequence shown in 17
Row such as SEQ ID NO:Such as SEQ ID NO of downstream primer and sequence shown in 18:Sense primer shown in 19 and sequence such as SEQ
ID NO:Downstream primer shown in 20.
The detection method and detection kit of above-mentioned PKD1 genes and PKD2 gene mutations, it is multipair by creatively designing
Specific Long fragment PCR primer, can be to avoid expansion when being expanded as template using human gene group DNA to be expanded respectively
Increase the high pseudogene of homology, thus false positive and false negative result that pseudogene is brought can be avoided the occurrence of, greatly improves
The detection accuracy of PKD1 genes and PKD2 gene mutations, and combine high throughput sequencing technologies, compared with traditional medicine outside
Aobvious capture technique can simplify techniqueflow, reduce testing cost, shorten detection cycle.
Description of the drawings
Fig. 1 is ADPKD hereditary pattern schematic diagrames；
Fig. 2 is PKD1 gene schematic diagrames；
Fig. 3 is PKD2 gene schematic diagrames；
Fig. 4 is the product electrophoresis detection schematic diagram of Long fragment PCR amplification；
Fig. 5 is No. 1 subject's high-flux sequence analysis result；
Fig. 6 is No. 2 subject's high-flux sequence analysis results；
Fig. 7 is No. 1 subject's Sanger sequence verification result.
Specific implementation mode
To facilitate the understanding of the present invention, below with reference to relevant drawings to invention is more fully described.In attached drawing
Give presently preferred embodiments of the present invention.But the present invention can realize in many different forms, however it is not limited to this paper institutes
The embodiment of description.Keep the understanding to the disclosure more thorough on the contrary, purpose of providing these embodiments is
Comprehensively.
Unless otherwise defined, all of technologies and scientific terms used here by the article and belong to the technical field of the present invention
The normally understood meaning of technical staff is identical.Used term is intended merely to description tool in the description of the invention herein
The purpose of the embodiment of body, it is not intended that in the limitation present invention.Term as used herein "and/or" includes one or more phases
Any and all combinations of the Listed Items of pass.
Embodiment 1
1) extracting genome DNA
Acquisition subject's peripheral blood 2ml is placed in EDTA anticoagulant tubes, and 300 μ l EDTA anticoagulations is taken to use in superclean bench
In extracting genome DNA.According to salting out method whole blood DNA purification kit (producer：The Guangzhou bio tech ltd Mei Ji, goods
Number：D3311-02 extracting genome DNA) is carried out, is used in combination Nanodrop to measure DNA concentration, has recorded respective concentration and 260/280
And 260/230 ratio.This test is altogether detected 2 samples.
2) Long fragment PCR expands
Incorporated by reference to Fig. 2 and Fig. 3, journey is expanded according to the primer pair in the following table 1, the reaction system in the following table 2 and accordingly
The whole exons and most of introne of ordered pair PKD1 genes and PKD2 genes carry out Long fragment PCR amplification, and (LR-PCR expands
Increase).
1 LR-PCR the primer lists of table
Note：F indicates that sense primer (forward primer), R indicate downstream primer (reverse primer) in Primer in table 1.
The kit that Long fragment PCR amplification uses is TaKaRa LAHot Start Version comprising LA
II (Mg of Taq HS (5U/ μ l), 10 × LA PCR Buffer2+Plus) (II this pipe reagent of Plus i.e. 10 × LA PCR Buffer
In, contain certain density Mg2+, Mg need not be additionally added in experimentation2+, corresponding Mg2+Final concentration of 2mM), dNTP
Mixture (2.5mM each), producer：TaKaRa, article No. RR042A.
The component of 2 LR-PCR reaction systems 1 of table
Note：Reaction system 1 is suitable for amplified fragments 1 (PKD1_EX01), segment 2 (PKD1_Ex02-12), segment 3
(PKD1_Ex13-21), segment 4 (PKD1_Ex22-34), segment 5 (PKD1_Ex35-46), segment 6 (PKD2_EX01).
Because PKD1 genes and PKD2 Gene Partial region template G/C contents are very high, reach 80% under extreme case, passed through
DMSO and Betaine is added and matches the amplification that can effectively enhance high GC content template.
The component of 3 LR-PCR reaction systems 2 of table
Note：Reaction system 2 is suitable for amplified fragments 7 (PKD2_Ex02), segment 8 (PKD2_EX03-06), segment 9
(PKD2_EX07-10), segment 10 (PKD2_EX11-15).
LR-PCR amplification programs are selected from following 5 kinds of programs：
98 DEG C, 98 DEG C of 3min -, 30sec, 60 DEG C, 15sec totally 35 recycle -68 DEG C, 72 DEG C of 1.5min -, 7min, it is right
Applied to amplification PKD1_Ex01 and PKD2_Ex01；
94 DEG C, 94 DEG C of 2min-, 15sec, 69 DEG C, 30sec totally 14 cycles, and each link of next cycle is more previous
The corresponding link temperature of a cycle reduce by 0.5 DEG C -68 DEG C, 94 DEG C of 6min -, 15sec, 62 DEG C, 30sec totally 25 cycles -68
DEG C, 72 DEG C of 6min-, 10min, to be applied to expand PKD1_Ex02-12, PKD1_Ex13-21 and PKD1_Ex22-34；
95 DEG C, 98 DEG C of 2min-, 20sec, 69 DEG C, 15sec totally 14 cycles, and each link of next cycle is more previous
The corresponding link temperature of a cycle reduce by 0.5 DEG C -72 DEG C, 98 DEG C of 3min -, 20sec, 62 DEG C, 15sec totally 25 cycles -72
DEG C, 72 DEG C of 3min-, 5min, to be applied to expand PKD1_Ex35-46；
98 DEG C, 10sec, 60 DEG C, 15sec totally 35 recycle -68 DEG C, 72 DEG C of 3min -, 5min, to being applied to expand
PKD2_Ex02；
98 DEG C, 10sec totally 35 recycle -68 DEG C, 72 DEG C of 10min -, 5min, to be applied to expand PKD2_Ex03-06,
PKD2_Ex07-10 and PKD2_Ex11-15.
After the completion of LR-PCR amplifications, take 2 μ l LR-PCR amplified productions, be added 1 μ l 6 × Loading Buffer mixings into
1% agarose gel electrophoresis of row detects, and observes gene magnification situation, the results are shown in Figure 4.
3) structure of high-throughput sequencing library
High-flux sequence (NGS) kit used in the present embodiment is KAPA HTP Library Preparation
Kit, the NGS technologies described in the present embodiment include but not limited to that in synthesis both-end sequencing technologies are sequenced in illumina, also may be used
So that Ion Torrent semiconductor sequencing technologies, different banking process need to be used to the different flat lifts of sequencing.Below with
It is illustrated for illumina both-end sequencing approaches.
(1) DNA is purified：Using Beckman companies of the U.S. AMPure XP beads to Long fragment PCR amplified production into
Row purifying.
(2) DNA is quantitative：DNA after purification is subjected to concentration mensuration using Qubit dye methods to it.
(3) different DNA fragmentations carry out equimolar mixing：According to concentration of the DNA after quantitative, 10 DNA fragmentations are carried out etc.
Mole mixing, total amount reaches 2 μ g.
(4) DNA fragmentation：It takes equimolar to mix 2 μ g of PCR product, DNA fragmentation is carried out by setting program is interrupted.With
Q800R Ultrasonic Cell Disruptors interrupt to make a call within 20 minutes 20 seconds and stop 30 seconds into Break Row, amplitude：80%.
(5) DNA fragmentation electrophoresis quality inspection：2 μ l of the DNA after fragmentation are taken, 1.5% agarose gel electrophoresis detection is carried out,
The fragment length of final every sample should be in 500bp hereinafter, peak value in 350bp is qualification.
(6) DNA end-fillings reparation：It is formulated as follows reaction system shown in table 4, by the cohesive end after DNA fragmentation
Filling-in reparation is flat end, and reaction condition is 20 DEG C, 30min.
End-filling reparation uses the article No. DGLS70002 kits of KAPA companies, contains 10 × KAPA End
Repair Buffer and KAPA End Repair Enzyme Mix.
4 DNA end-fillings of table repair system
(7) paramagnetic particle method DNA is purified：It is added into above-mentioned reaction tubeXP reagent
(Beckman Coulter, A63881) 120 μ l are inhaled with liquid-transfering gun and are beaten repeatedly up and down, 10 points of incubation at room temperature standing after mixing well
Clock makes DNA fully be combined with magnetic bead, then pipe is placed on magnetic frame until solution turned clear, carefully discards supernatant.By Guan Ji
It is continuous to stay on magnetic frame, the incubation at room temperature of 200 μ l, 80% alcohol is added and washes twice within 30 seconds, dries 30min at room temperature.It will reaction
Pipe takes out from magnetic frame, carries out in next step.
(8) DNA fragmentation adds A to react：Each reaction tube is formulated as follows reaction system shown in table 5, and reaction condition is 30 DEG C,
It is incubated 30min.
Add A to use the article No. DGLS70002 kits of KAPA companies, contains 10 × KAPA End Repair Buffer
With KAPA End Repair Enzyme Mix.
5 DNA fragmentation of table adds the reaction system of A
(9) DNA is purified：PEG/NaCl SPRIR Solution (producers are added into above-mentioned reaction tube：KAPA, article No.
DGLS70002) 90 μ l are inhaled and are beaten, are incubated at room temperature 10 minutes, set and clarified on magnetic frame, then abandon supernatant repeatedly.200 μ l, 80% wine
Essence incubation at room temperature washes twice for 30 seconds, dries at room temperature.
(10) adjunction head：Ligation Master Mix (producers are added：KAPA, article No. DGLS70002) 50 μ l, repeatedly
Suction is beaten, 20 DEG C, is incubated 15min.
The reaction system of 6 DNA adjunction heads of table
(11) DNA is purified：Add 50 μ l of PEG/NaCl SPRIR Solution, inhales and beat repeatedly, stand 10 minutes, set magnetic force
It is clarified on frame, then abandons supernatant.200 μ l, 80% alcohol is incubated at room temperature 30 seconds and washes twice, drying at room temperature.By pipe from magnetic frame
It takes out, with 50 μ l 10mM Tris-HCl PH, 8.0 eluted dnas, is incubated at room temperature 2min, DNA is made to come with Beads enrichment.It will be from
Heart pipe is put back on magnetic frame up to solution turned clear, supernatant is transferred in new 0.5ml pipes, and latter acts are continued.
(12) Pre-PCR amplifications are carried out, specific molecular label, different samples is added to add different specific molecular labels
(Index, the specific molecular label of Sangon Biotech's synthesis is distinguishing different samples), instead
It is 50 μ l to answer system.
7 Pre-PCR amplification reaction systems of table
Pre-PCR response procedures：Lid:105 DEG C, Wait, Auto —s >98℃,45sec—>98 DEG C, 15sec, 60 DEG C,
30sec, totally 6 cycle — >72℃,30sec—>72℃,1min—>4 DEG C, hold is not less than 1h.
(13) it purifies：It is added90 μ l of XP reagent stand 10 minutes, set magnetic frame
Upper clarification, then abandons supernatant.200 μ l, 80% alcohol is incubated at room temperature 30 seconds and washes twice, and dries at room temperature.By pipe from magnetic frame
It takes out, with 52 μ l ddH2O eluted dnas are incubated at room temperature 2min, 50 μ l supernatants are transferred in a new pipe.
(14) quality inspection：DNA concentration and segment after amplification are measured with Qubit dyestuffs and 1.5% agarose gel electrophoresis
Size.
(15) library is quantitative：The two generation sequencing libraries built are quantified using quantitative fluorescent PCR.
(16) library is diluted：The library built is diluted to 10nM.And the data volume generated on demand is mixed.
(17) machine is sequenced on：Sample is sequenced using illumina Hiseq X10 bis- generations microarray datasets, has been sequenced
At rear by being split to it to the special sequence label that different samples add.
(18) sequencing data is converted to FASTQ files using bioinformatic analysis software NextGENe, line number of going forward side by side
According to analysis, the DNA sequencing fragment that sequencing is obtained is compared to reference on target-gene sequence, carries out the extraction of SNVs and Indels,
The abrupt information that gene polymorphism sites obtain target gene PKD1, PKD2 is excluded, as a result as shown in Figure 5 and Figure 6.
(19) genetic test result is understood：
8 No. 1 subject's genetic test results of table
Table 8 is the genetic test of No. 1 subject as a result, incorporated by reference to Fig. 5, and the result of this genetic test needs to combine inspection person
Clinical symptoms and family history understood.Recognize that inspection person's clinical manifestation has polycystic kindey, hepatic cyst, and inspection person father
There is corresponding clinical manifestation.In this time genetic test, the detection and analysis of polycystic kidney disease related gene is carried out to inspection person, is detected
One pathogenic heterozygous mutant of PKD1 genes is c.8095C>T(p.Q2699X).The variation is nonsense mutation, and prediction can lead to amino acid
Synthesis terminates in advance, and (PMID is reported in clinical case:18837007).And up to the present, which is joining
It examines in crowd's gene database and is not all reported.
As shown in fig. 7, being verified in conjunction with the catastrophe point that No. 1 subject is investigated in Sanger sequencings, can see
It is consistent with Sanger sequencings as a result, illustrating that the method for the present embodiment is feasible to having obtained.
9 No. 2 subject's genetic test results of table
In No. 2 subjects, the detection and analysis of polycystic kidney disease related gene is carried out to subject, detects PKD1 genes
One pathogenic heterozygous variance c.4797delC (p.Y1599*), incorporated by reference to Fig. 6.Confirmatory experiment shows that inspection person parent does not have later
The evidence of this variation is carried, this variation is new.This variation is frameshift mutation, and prediction can cause protein synthesis to carry
Preceding termination.And up to the present, which is not all reported in our reference crowd's gene database.But it is same
Site is c.4797C>G (p.Y1599*) has had been reported that (PMID in relevant clinical case：22508176).
Each technical characteristic of embodiment described above can be combined arbitrarily, to keep description succinct, not to above-mentioned reality
It applies all possible combination of each technical characteristic in example to be all described, as long as however, the combination of these technical characteristics is not deposited
In contradiction, it is all considered to be the range of this specification record.
Several embodiments of the invention above described embodiment only expresses, the description thereof is more specific and detailed, but simultaneously
It cannot therefore be construed as limiting the scope of the patent.It should be pointed out that coming for those of ordinary skill in the art
It says, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to the protection of the present invention
Range.Therefore, the protection domain of patent of the present invention should be determined by the appended claims.
SEQUENCE LISTING
<110>Zhang Wei
<120>The detection method and detection kit of PKD1 genes and PKD2 gene mutations
<160> 20
<170> PatentIn version 3.3
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<210> 7
<211> 27
<212> DNA
<213>Artificial sequence
<400> 7
atgcttagtg aggaggctgt gggggtc 27
<210> 8
<211> 30
<212> DNA
<213>Artificial sequence
<400> 8
atgaggctct ttccacagac aacagaggtt 30
<210> 9
<211> 25
<212> DNA
<213>Artificial sequence
<400> 9
ctgtgggcga tgggtttatc agcag 25
<210> 10
<211> 26
<212> DNA
<213>Artificial sequence
<400> 10
gagacggtgc agggagtacg gtagga 26
<210> 11
<211> 25
<212> DNA
<213>Artificial sequence
<400> 11
gtggagacag aagccaacca aagag 25
<210> 12
<211> 19
<212> DNA
<213>Artificial sequence
<400> 12
ggatgcgaga tggagcccg 19
<210> 13
<211> 27
<212> DNA
<213>Artificial sequence
<400> 13
tttctttcca tttgcaatgt ttcattc 27
<210> 14
<211> 28
<212> DNA
<213>Artificial sequence
<400> 14
ggaagatagt caataaacaa atgcccaa 28
<210> 15
<211> 26
<212> DNA
<213>Artificial sequence
<400> 15
gagaagacct tgtgtgaatt tgtcca 26
<210> 16
<211> 28
<212> DNA
<213>Artificial sequence
<400> 16
tcatactcag caaagttact catgcaaa 28
<210> 17
<211> 25
<212> DNA
<213>Artificial sequence
<400> 17
tcgggtaagt ataatggtga gccct 25
<210> 18
<211> 28
<212> DNA
<213>Artificial sequence
<400> 18
catcaagact ccaagatagg gaacattt 28
<210> 19
<211> 25
<212> DNA
<213>Artificial sequence
<400> 19
cacgtacttg ttgaatggcc aatgt 25
<210> 20
<211> 27
<212> DNA
<213>Artificial sequence
<400> 20
atgaaactca gaagcccttt gacagtt 27
Claims (10)
1. a kind of detection method of PKD1 genes and PKD2 gene mutations, which is characterized in that include the following steps：
Step 1：Extract the genomic DNA of sample to be tested；
Step 2：Using the genomic DNA of extraction as template, lengthy motion picture is carried out to the respective segments of PKD1 genes and PKD2 genes
Section PCR amplification, wherein Long fragment PCR amplification primer pair used is selected from following ten kinds of primer pairs：Sequence such as SEQ ID NO:1
Shown in sense primer and sequence such as SEQ ID NO:Such as SEQ ID NO of downstream primer, sequence shown in 2:Draw upstream shown in 3
Object and sequence such as SEQ ID NO:Such as SEQ ID NO of downstream primer, sequence shown in 4:Sense primer shown in 5 and sequence are such as
SEQ ID NO:Such as SEQ ID NO of downstream primer, sequence shown in 6:Sense primer shown in 7 and sequence such as SEQ ID NO:8
Shown in downstream primer, sequence such as SEQ ID NO:Sense primer shown in 9 and sequence such as SEQ ID NO:Downstream shown in 10
Primer, sequence such as SEQ ID NO:Sense primer shown in 11 and sequence such as SEQ ID NO:Downstream primer, sequence shown in 12
Such as SEQ ID NO:Sense primer shown in 13 and sequence such as SEQ ID NO:Such as SEQ ID of downstream primer, sequence shown in 14
NO:Sense primer shown in 15 and sequence such as SEQ ID NO:Such as SEQ ID NO of downstream primer, sequence shown in 16:Shown in 17
Sense primer and sequence such as SEQ ID NO:Such as SEQ ID NO of downstream primer and sequence shown in 18:Draw upstream shown in 19
Object and sequence such as SEQ ID NO:Downstream primer shown in 20；
Step 3：The product that Long fragment PCR is expanded carries out high-flux sequence detection after mixing.
2. the detection method of PKD1 genes and PKD2 gene mutations as described in claim 1, which is characterized in that in the step
In 1, the genomic DNA of the extraction sample to be tested is using the extraction of salting out method whole blood DNA, the extraction of paramagnetic particle method whole blood DNA or film
Method whole blood DNA extracts.
3. the detection method of PKD1 genes and PKD2 gene mutations as described in claim 1, which is characterized in that in the step
In 2, sequence such as SEQ ID NO are used:When the corresponding primer pair that primer shown in 1~12 is constituted carries out Long fragment PCR amplification,
Amplification system is to be added to have 5 μ l 10 × LA PCR Buffer, II (Mg in every 50 μ l systems2+Plus), 8 each concentration of μ l are
The dNTP mixtures of 2.5mM, 2.5 μ l DMSO, the glycine betaine of 5 a concentration of 5M of μ l, the sense primer of final concentration of 200nM, end are dense
Degree is downstream primer, the genomic DNA template of 50~200ng and the TakaRa LA Taq enzyme of 0.5 μ l of 200nM, remaining is
Water；
Use sequence such as SEQ ID NO:The corresponding primer pair that primer shown in 13~20 is constituted carries out Long fragment PCR amplification
When, amplification system is to be added to have 5 μ l 10 × LA PCR Buffer, II (Mg in every 50 μ l systems2+Plus), 8 each concentration of μ l are
The dNTP mixtures of 2.5mM, the sense primer of final concentration of 200nM, the downstream primer of final concentration of 200nM, 50~200ng
The TakaRa LA Taq enzymes of genomic DNA template and 0.5 μ l, remaining is water.
4. the detection method of PKD1 genes and PKD2 gene mutations as claimed in claim 3, which is characterized in that in the step
In 2, for sequence such as SEQ ID NO:Sense primer shown in 1 and sequence such as SEQ ID NO:Downstream primer shown in 2 is constituted
Primer pair and sequence such as SEQ ID NO:Sense primer shown in 11 and sequence such as SEQ ID NO:Downstream primer shown in 12
The condition when primer pair of composition carries out Long fragment PCR amplification is：98 DEG C, 98 DEG C of 3min-, 30sec, 60 DEG C, 15sec totally 35
A -68 DEG C of cycle, 72 DEG C of 1.5min -, 7min；
For sequence such as SEQ ID NO:Sense primer shown in 3 and sequence such as SEQ ID NO:Downstream primer shown in 4 is constituted
Primer pair, sequence such as SEQ ID NO:Sense primer shown in 5 and sequence such as SEQ ID NO:Downstream primer shown in 6 is constituted
Primer pair and sequence such as SEQ ID NO:Sense primer shown in 7 and sequence such as SEQ ID NO:Downstream primer structure shown in 8
At primer pair carry out Long fragment PCR amplification when condition be：94 DEG C, 94 DEG C of 2min-, 15sec, 69 DEG C, 30sec totally 14
Cycle, and the corresponding link temperature of the more previous cycle of each link of next cycle reduces by 0.5 DEG C -68 DEG C, 6min -94
DEG C, 15sec, 62 DEG C, 30sec totally 25 recycle -68 DEG C, 72 DEG C of 6min -, 10min；
For sequence such as SEQ ID NO:Sense primer shown in 9 and sequence such as SEQ ID NO:Downstream primer shown in 10 is constituted
Primer pair carry out Long fragment PCR amplification when condition be：95 DEG C, 98 DEG C of 2min-, 20sec, 69 DEG C, 15sec follows for 14 totally
Ring, and the more previous cycle of each link of next cycle corresponding link temperature reduce by 0.5 DEG C -72 DEG C, 98 DEG C of 3min -,
20sec, 62 DEG C, 15sec totally 25 recycle -72 DEG C, 72 DEG C of 3min -, 5min；
For sequence such as SEQ ID NO:Sense primer shown in 13 and sequence such as SEQ ID NO:Downstream primer structure shown in 14
At primer pair carry out Long fragment PCR amplification when condition be：98 DEG C, 10sec, 60 DEG C, 15sec totally 35 recycle -68 DEG C,
3min—72℃,5min；
For sequence such as SEQ ID NO:Sense primer shown in 15 and sequence such as SEQ ID NO:Downstream primer structure shown in 16
At primer pair, sequence such as SEQ ID NO:Sense primer shown in 17 and sequence such as SEQ ID NO:Downstream primer shown in 18
The primer pair and sequence of composition such as SEQ ID NO:Sense primer shown in 19 and sequence such as SEQ ID NO:Downstream shown in 20
Primer constitute primer pair carry out Long fragment PCR amplification when condition be：98 DEG C, 10sec totally 35 recycle -68 DEG C,
10min—72℃、5min。
5. the detection method of PKD1 genes and PKD2 gene mutations as described in claim 1, which is characterized in that in the step
In 3, the product mixing that Long fragment PCR is expanded is after Long fragment PCR amplification terminates, by ten groups of long segments
The product equimolar mixing that PCR amplification obtains.
6. the detection method of PKD1 genes and PKD2 gene mutations as described in claim 1, which is characterized in that in the step
High-flux sequence detection is carried out in 3, after the product mixing that Long fragment PCR is expanded to include the following steps：
DNA is interrupted：The product that the Long fragment PCR expands is broken into DNA fragmentation；
It repairs end：End reparation is carried out to the DNA fragmentation；
Add A：A bases are added at 3 ' ends of the DNA fragmentation that end is repaired；
Connect sequence measuring joints：Sequence measuring joints are connected on the DNA fragmentation of the upper A bases of connection；
Pre-PCR is expanded：DNA fragmentation to being connected with sequence measuring joints carries out Pre-PCR amplifications, in addition specific molecular label,
Build DNA library；
The DNA library of structure is sequenced, sequencing result is carried out by different specific molecular labels after the completion of sequencing
It distinguishes, and sequencing result is compared on reference to target-gene sequence, obtain abrupt information.
7. the detection method of PKD1 genes and PKD2 gene mutations as claimed in claim 6, which is characterized in that it is described will be described
Long fragment PCR amplification product be broken into DNA fragmentation be broken into of length no more than 500bp, peak value 350bp DNA fragmentation.
8. the detection method of PKD1 genes and PKD2 gene mutations as claimed in claim 6, which is characterized in that interrupted in DNA
Later, end repair after, plus A after, connection sequence measuring joints after and Pre-PCR amplification after further include to processing obtain
Product carry out purification process the step of.
9. the detection method of PKD1 genes and PKD2 gene mutations as claimed in claim 6, which is characterized in that further include surveying
Product after being expanded to Pre-PCR before sequence is into the step of row agarose gel electrophoresis detection and fluorogenic quantitative detection.
10. a kind of detection kit of PKD1 genes and PKD2 gene mutations, which is characterized in that draw including Long fragment PCR amplification
Object reagent, the primer pair in the Long fragment PCR amplimer reagent are selected from following ten kinds of primer pairs：Sequence such as SEQ ID NO:
Sense primer shown in 1 and sequence such as SEQ ID NO:Such as SEQ ID NO of downstream primer, sequence shown in 2:Upstream shown in 3
Primer and sequence such as SEQ ID NO:Such as SEQ ID NO of downstream primer, sequence shown in 4:Sense primer shown in 5 and sequence are such as
SEQ ID NO:Such as SEQ ID NO of downstream primer, sequence shown in 6:Sense primer shown in 7 and sequence such as SEQ ID NO:8
Shown in downstream primer, sequence such as SEQ ID NO:Sense primer shown in 9 and sequence such as SEQ ID NO:Downstream shown in 10
Primer, sequence such as SEQ ID NO:Sense primer shown in 11 and sequence such as SEQ ID NO:Downstream primer, sequence shown in 12
Such as SEQ ID NO:Sense primer shown in 13 and sequence such as SEQ ID NO:Such as SEQ ID of downstream primer, sequence shown in 14
NO:Sense primer shown in 15 and sequence such as SEQ ID NO:Such as SEQ ID NO of downstream primer, sequence shown in 16:Shown in 17
Sense primer and sequence such as SEQ ID NO:Such as SEQ ID NO of downstream primer and sequence shown in 18:Draw upstream shown in 19
Object and sequence such as SEQ ID NO:Downstream primer shown in 20.
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