CN109971845A - A kind of PCR primer group and amplification system expanding mankind PKD1 gene 1-33 exon - Google Patents
A kind of PCR primer group and amplification system expanding mankind PKD1 gene 1-33 exon Download PDFInfo
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Abstract
The invention discloses a kind of PCR primer groups and amplification system for expanding mankind PKD1 (Gene ID:5310) gene 1-33 exon, primer sets including PKD1 gene He its pseudogene can be distinguished, and utilize PCR reaction buffer required for primer sets amplification PKD1 gene 1-33 exon.The present invention is compared with traditional nest-type PRC and Long fragment PCR amplification PKD1 gene, have many advantages, such as easy to operate, rapidly and efficiently, it can be used for the direct detection of autosomal dominant polycystic kidney disease Disease-causing gene PKD1 gene mutation, it can also be used to the verifying to PKD1 gene mutation in two generation sequencing results.
Description
Technical field
The present invention relates to hereditary disease genetic test fields, can distinguish autosomal dominant polycystic kidney disease more particularly, to one kind and control
The primer sets and amplification system of ospc gene PKD1 gene and its pseudogene.
Background technique
Autosomal dominant polycystic kidney disease (ADPKD) is usually a kind of Delayed onset multisystem disease, and disease incidence is in 1/400-
1/1000, often there is vesica not of uniform size in 30-50 years old bilateral renal in patient, it is characterised in that: bilateral renal cyst can also relate to
And other organ tumours including liver, seminal vesicle, pancreas and arachnoid；Aberrant angiogenesis includes intracranial aneurysm, aorta
The exception of root expansion and aorta pectoralis；Mitral valve prolapse and stomach wall hernia.Performance in terms of kidney includes hypertension, nephralgia and
Renal insufficiency.About 50% ADPKD patient is developed after 60 years old as end-stage renal disease (ESRD).Hepatic cyst is ADPKD
The most common Extrarenal manifestations, increase with advancing age, and may be ignored by supersonic sounding.Under aneurysm or arachnoid
The intracranial aneurysm illness rate (22%) of chamber bleeding patients is higher than patient's (6%) of no this family history.Up to 25% by
Know from experience for tired and mitral valve prolapse occurs, is the most common valve abnormalities.The kidney trouble of different severity and other kidney appearances
Now even occur in the same family.
The diagnosis of ADPKD is mainly determined by the iconography of kidney.With ADPKD individual in, in PKD1 85%
Gene mutation is pathogenic, and only about 15% gene mutation is pathogenic in PKD2.ADPKD is with autosomal dominant
Mode heredity.The parent of about 95% ADPKD patient will receive influence；At least 10% family's discovery has new hair mutation, by
The each child for influencing individual has 50% chance to inherit variation of causing a disease.ADPKD has disease incidence height, is in a bad way, prognosis
Difference, the features such as renal failure is fast, it is antenatal, transplant before in the fields such as embryo's detection, infertile, genetic screening using gene
Detection is very important.
PKD1 gene is located on No. 16 the short arm of a chromosome, shares 46 exons, wherein 1-33 exon is in genome
It is upper there are the pseudogene of 6 very high homologies, up to 97.7%, regular-PCR and high throughput sequencing technologies are difficult to distinguish similarity
PKD1 gene and pseudogene bring puzzlement to clinical diagnosis.The method of existing detection PKD1 gene mutation often has long segment
PCR and nested PCR amplification.The disclosure of the invention that number of patent application is CN201510017500.0 it is a kind of by Long fragment PCR and
The method of high throughput sequencing technologies detection PKD1 gene mutation.This method needs to obtain Long fragment PCR product first, then will
Enrichment builds library and carries out high-flux sequence after amplified production digestion interrupts.This method needs individually to be built for PKD1 gene
Then library carries out high-flux sequence, be difficult to build library at panel with other assortments of genes, undoubtedly increase the cost of sequencing.And nest
Formula PCR amplification needs to carry out specific amplification to PKD1 gene with two pairs or more of primer combination.This method needs a site
PCR amplification more than twice is carried out, operating process is relatively complicated, not easy enough.
Currently, the application trend of genetic screening is increasingly intended to the medium-and-large-sized panel using multiple genes, previous this
Kind in such a way that the true gene magnification of PKD1 is sequenced again out for Long fragment PCR and nest-type PRC, can not very well with it is existing
Two generations sequencing gene cooperate, do not meet the technology trends of current genetic screening, more efficiently screening mode yet
Should widely be screened first with the sequencing of two generations, go to test if there is PKD1 related mutation, then with sanger sequencing
Whether the mutation occurs on true gene card.Therefore, this field, which still needs, carries out more simple and effective to PKD1 gene mutation, low
Honest and clean detection.
Summary of the invention
In order to overcome the problems, such as the prior art, there are pseudogenes to interfere, with high costs, operating process is complicated, poor in timeliness,
The method of PKD1 gene mutation is detected the invention discloses a kind of simple and effective.This method only needs pair of primers, by one
Secondary PCR amplification can specific amplification go out PKD1 gene extron short-movie section, which can directly carry out sanger sequencing
Verify PKD1 gene mutation.This method can find have first in conjunction with existing gene detecting kit from high-throughput data
Then the PKD1 gene mutation site of meaning selects suitable primer pair that will expand by a PCR amplification using this method
Product carries out sanger verifying.
The invention also discloses the primer sets that one group can distinguish PKD1 gene 1-33 exon and its pseudogene, this draws
Object group includes 18 pairs of primers altogether, as follows respectively: for expanding the primer of PKD1 gene extron 1, forward primer such as SEQ ID
Shown in NO:1, reverse primer is as shown in SEQ ID NO:2；For expanding the primer of PKD1 gene extron 2-5a, forward direction is drawn
Object is as shown in SEQ ID NO:3, and reverse primer is as shown in SEQ ID NO:4；For expanding drawing for PKD1 gene extron 5b-6
Object, forward primer is as shown in SEQ ID NO:5, and reverse primer is as shown in SEQ ID NO:6；For expanding outside PKD1 gene
The primer of sub- 7-8 is shown, forward primer is as shown in SEQ ID NO:7, and reverse primer is as shown in SEQ ID NO:8；For expanding
The primer of PKD1 gene extron 9-10, forward primer is as shown in SEQ ID NO:9, reverse primer such as SEQ ID NO:10
It is shown；For expanding the primer of PKD1 gene extron 11, forward primer is as shown in SEQ ID NO:11, and reverse primer is such as
Shown in SEQ ID NO:12；For expanding the primer of PKD1 gene extron 12, forward primer as shown in SEQ ID NO:13,
Reverse primer is as shown in SEQ ID NO:14；For expanding the primer of PKD1 gene extron 13-15a, forward primer is such as
Shown in SEQ ID NO:15, reverse primer is as shown in SEQ ID NO:16；For expanding the primer of PKD1 gene extron 15b,
Its forward primer is as shown in SEQ ID NO:17, and reverse primer is as shown in SEQ ID NO:18；For expanding PKD1 gene extron
The primer of sub- 15c, forward primer is as shown in SEQ ID NO:19, and reverse primer is as shown in SEQ ID NO:20；For expanding
The primer of PKD1 gene extron 15d-16, forward primer is as shown in SEQ ID NO:21, reverse primer such as SEQ ID NO:
Shown in 22；For expanding the primer of PKD1 gene extron 17-20, forward primer reversely draws as shown in SEQ ID NO:23
Object is as shown in SEQ ID NO:24；For expanding the primer of PKD1 gene extron 21, forward primer such as SEQ ID NO:25
Shown, reverse primer is as shown in SEQ ID NO:26；For expanding the primer of PKD1 gene extron 22, forward primer is such as
Shown in SEQ ID NO:27, reverse primer is as shown in SEQ ID NO:28；For expanding drawing for PKD1 gene extron 23-24
Object, forward primer is as shown in SEQ ID NO:29, and reverse primer is as shown in SEQ ID NO:30；For expanding PKD1 gene
The primer of exon 2 5-26, forward primer is as shown in SEQ ID NO:31, and reverse primer is as shown in SEQ ID NO:32；With
In the primer of amplification PKD1 gene extron 27-30, forward primer is as shown in SEQ ID NO:33, reverse primer such as SEQ
Shown in ID NO:34；For expanding the primer of PKD1 gene extron 31-33, forward primer as shown in SEQ ID NO:35,
Reverse primer is as shown in SEQ ID NO:36.
A kind of PCR reaction system for expanding PKD1 gene 1-33 exon is also disclosed in the present invention, draws including above-mentioned
Any pair of primer in object group further includes archaeal dna polymerase, PCR buffer, Rnase enzyme, dNTP mixture.Preferred real
It applies in scheme, the archaeal dna polymerase is TaKaRa LA Taq archaeal dna polymerase.In the PCR reaction system that total volume is 20 μ L
Contain following compositions: the 10 μ L of forward primer and 2 × Buffer of each 0.4 μ L, TaKaRa of reverse primer of 10 μm of ol/L,
3.2 0.2 μ L, 10ng/ μ L DNA profiling of μ L, 5U/ μ L TaKaRa LA Taq of 2.5mmol/L dNTP mixture, 1 μ L.
Detailed description of the invention
Fig. 1 is PKD1 gene 1-33 exon pcr amplification product electrophoretogram.Wherein 1 and 20 swimming lane M are 1500 bp
Marker, fragment length are from top to bottom followed successively by 1500bp, 1000bp, 900bp, 800bp, 700 bp, 600bp, 500bp,
400bp, 300bp, 200bp, 100bp；2 swimming lanes are the amplified production of exons 1, size 1312bp；3 swimming lanes are exon 2-
The amplified production of 5a, size 1224bp；4 swimming lanes are the amplified production of exon 5b-6, size 904bp；5 swimming lanes are outer
Show the amplified production of sub- 7-8, size 1214bp；6 swimming lanes are the amplified production of exon 9-10, size 1084bp；7 swimming
Road is the amplified production of exons 11, size 1014bp；8 swimming lanes are the amplified production of exons 12, size 863bp；9 swimming
Road is the amplified production of exons 1 3-15a, size 1390bp；10 swimming lanes are the amplified production of exons 1 5b, and size is
1543bp；11 swimming lanes are the amplified production of exons 1 5c, size 1495bp；The amplification that 12 swimming lanes are exons 1 5d-16 produces
Object, size 1468bp；13 swimming lanes are the amplified production of exons 1 7-20, size 1243bp；14 swimming lanes are exon 21
Amplified production, size 475bp；15 swimming lanes are the amplified production of exon 22, size 470bp；16 swimming lanes are exon 2 3-
24 amplified production, size 1280bp；17 swimming lanes are the amplified production of exon 2 5-26, size 1329bp；18 swimming lanes are
The amplified production of exon 2 7-30, size 1249bp；19 swimming lanes are the amplified production of exon 3 1-33, and size is
686bp。
Fig. 2 is that exons 1 amplified production Sanger method sequencing result and PKD1 gene and its pseudogene sequences comparison are illustrated
Figure.Wherein upper figure is Sanger method sequencing result, and region of the following figure the first row with shade is PKD1 gene reference sequence, the following figure the
2 to 4 behavior pseudogene sequences.
Fig. 3 is that exon 2-5a amplified production Sanger method sequencing result shows with PKD1 gene and its pseudogene sequences comparison
It is intended to.Wherein upper figure is Sanger method sequencing result, and region of the following figure the first row with shade is PKD1 gene reference sequence, under
The 2nd to 5 behavior pseudogene sequences of figure.
Fig. 4 is that exon 5b-6 amplified production Sanger method sequencing result shows with PKD1 gene and its pseudogene sequences comparison
It is intended to.Wherein upper figure is Sanger method sequencing result, and region of the following figure the first row with shade is PKD1 gene reference sequence, under
Figure the 2nd, 3 behavior pseudogene sequences.
Fig. 5 is that -8 amplified production Sanger method sequencing result of exon 7 shows with PKD1 gene and its pseudogene sequences comparison
It is intended to.Wherein upper figure is Sanger method sequencing result, and region of the following figure the first row with shade is PKD1 gene reference sequence, the following figure
2nd to 7 behavior pseudogene sequences.
Fig. 6 is that exon 9-10 amplified production Sanger method sequencing result shows with PKD1 gene and its pseudogene sequences comparison
It is intended to.Wherein upper figure is Sanger method sequencing result, and region of the following figure the first row with shade is PKD1 gene reference sequence, under
The 2nd to 5 behavior pseudogene sequences of figure.
Fig. 7 is that 1 amplified production Sanger method sequencing result of exons 1 and PKD1 gene and its pseudogene sequences comparison are illustrated
Figure.Wherein upper figure is Sanger method sequencing result, and region of the following figure the first row with shade is PKD1 gene reference sequence, the following figure the
2 to 5 behavior pseudogene sequences.
Fig. 8 is that 2 amplified production Sanger method sequencing result of exons 1 and PKD1 gene and its pseudogene sequences comparison are illustrated
Figure.Wherein upper figure is Sanger method sequencing result, and region of the following figure the first row with shade is PKD1 gene reference sequence, the following figure the
2 to 5 behavior pseudogene sequences.
Fig. 9 is that exons 1 3-15a amplified production Sanger method sequencing result and PKD1 gene and its pseudogene sequences compare
Schematic diagram.Wherein upper figure is Sanger method sequencing result, and region of the following figure the first row with shade is PKD1 gene reference sequence,
The 2nd to 5 behavior pseudogene sequences of the following figure.
Figure 10 is that exons 1 5b amplified production Sanger method sequencing result shows with PKD1 gene and its pseudogene sequences comparison
It is intended to.Wherein upper figure is Sanger method sequencing result, and region of the following figure the first row with shade is PKD1 gene reference sequence, under
The 2nd to 5 behavior pseudogene sequences of figure.
Figure 11 is that exons 1 5c amplified production Sanger method sequencing result shows with PKD1 gene and its pseudogene sequences comparison
It is intended to.Wherein upper figure is Sanger method sequencing result, and region of the following figure the first row with shade is PKD1 gene reference sequence, under
The 2nd to 5 behavior pseudogene sequences of figure.
Figure 12 is exons 1 5d-16 amplified production Sanger method sequencing result and PKD1 gene and its pseudogene sequences pair
Compare schematic diagram.Wherein upper figure is Sanger method sequencing result, and region of the following figure the first row with shade is PKD1 gene reference sequence
Column, the 2nd to 5 behavior pseudogene sequences of the following figure.
Figure 13 is that exons 1 7-20 amplified production Sanger method sequencing result and PKD1 gene and its pseudogene sequences compare
Schematic diagram.Wherein upper figure is Sanger method sequencing result, and region of the following figure the first row with shade is PKD1 gene reference sequence,
The 2nd to 6 behavior pseudogene sequences of the following figure.
Figure 14 is that 1 amplified production Sanger method sequencing result of exon 2 shows with PKD1 gene and its pseudogene sequences comparison
It is intended to.Wherein upper figure is Sanger method sequencing result, and region of the following figure the first row with shade is PKD1 gene reference sequence, the following figure
2nd to 5 behavior pseudogene sequences.
Figure 15 is that 2 amplified production Sanger method sequencing result of exon 2 shows with PKD1 gene and its pseudogene sequences comparison
It is intended to.Wherein upper figure is Sanger method sequencing result, and region of the following figure the first row with shade is PKD1 gene reference sequence, the following figure
2nd, 3 behavior pseudogene sequences.
Figure 16 is that exon 2 3-24 amplified production Sanger method sequencing result and PKD1 gene and its pseudogene sequences compare
Schematic diagram.Wherein upper figure is Sanger method sequencing result, and region of the following figure the first row with shade is PKD1 gene reference sequence,
The 2nd to 5 behavior pseudogene sequences of the following figure.
Figure 17 is that exon 2 5-26 amplified production Sanger method sequencing result and PKD1 gene and its pseudogene sequences compare
Schematic diagram.Wherein upper figure is Sanger method sequencing result, and region of the following figure the first row with shade is PKD1 gene reference sequence,
Following figure 2-5 behavior pseudogene sequences.
Figure 18 is that exon 2 7-30 amplified production Sanger method sequencing result and PKD1 gene and its pseudogene sequences compare
Schematic diagram.Wherein upper figure is Sanger method sequencing result, and region of the following figure the first row with shade is PKD1 gene reference sequence,
The following figure the 2nd, 3 behavior pseudogene sequences.
Figure 19 is that exon 3 1-33 amplified production Sanger method sequencing result and PKD1 gene and its pseudogene sequences compare
Schematic diagram.Wherein upper figure is Sanger method sequencing result, and region of the following figure the first row with shade is PKD1 gene reference sequence,
The following figure the 2nd, 3 behavior pseudogene sequences.
Figure 20 is the schematic diagram of PKD1 gene 1-33 exon design of primers.According to the sequence of PKD1 gene and its pseudogene
Column feature, and grope by many experiments of inventor, PKD1 gene is had to the 1-33 extra of the pseudogene of very high homology
Aobvious son is divided into 18 amplification regions, and the design of primers of specificity is carried out for each amplification region.
Specific embodiment
The preferred embodiment of the present invention will be described in detail belows.
Embodiment one (the present embodiment be suitable for amplification PKD1 gene extron 2-5a, exon 5b-6, exon 7-8,
Exons 11, exons 1 3-15a, exons 1 5b, exons 1 5c, exons 1 5d-16, exons 1 7-20, exon 21,
Exon 22, exon 2 3-24, exon 2 5-26, exon 2 7-30, exon 3 1-33)
1, the sample and reagent for needing to prepare have:
The genomic DNA (gDNA) that sample to be tested extracts, A260-280 is between 1.8-2.0, with nuclease free
Water is diluted to 10ng/ μ L；The primer of the base sequence as shown in SEQ ID NO:1-36, with nuclease free water
It is diluted to 10 μm of mol/L；Rnase enzyme is diluted to 5mU/ μ L；5U/ μ L TaKaRa LA Taq archaeal dna polymerase；2.5mmol/L
DNTP mixture；TaKaRa 2×GC Buffer Ⅰ.
2, using sample to be tested gDNA as template, expand PKD1 gene 1-33 exon.According to following system in sterilizing PCR
PCR reaction system is prepared in pipe.
20 μ L PCR reaction systems are as follows:
| PCR ingredient | It is added volume (μ L) |
| gDNA(10ng/μL) | 1 |
| 2×GC BufferⅠ | 10 |
| 2.5mmol/L dNTP mixture | 3.2 |
| Forward primer (10 μm of mol/L) | 0.4 |
| Reverse primer (10 μm of mol/L) | 0.4 |
| Rnase enzyme (5mU/ μ L) | 2 |
| TaKaRa LA Taq | 0.2 |
| nuclease free water | 2.8 |
3, will PCR pipe mix centrifugation after be placed in PCR thermal cycler, following amplification program is set and to start PCR anti-
It answers.
Amplification program:
94 DEG C of thermal startings and initial denaturation 1min；94 DEG C of denaturation 30sec, 65 DEG C of annealing and extension 1min, totally 40 recycle；4
DEG C save.
4, PCR product is subjected to agarose gel electrophoresis, as seen from Figure 1, amplified production size and expected purpose segment are big
It is small consistent, target fragment is recycled with plastic recovery kit and carries out sanger sequencing.
The primer and product sheet segment length of the amplification PKD1 gene 1-33 exon of table 1
Embodiment two (the present embodiment is suitable for amplification PKD1 gene extron 1, exon 9-10, exons 1 2) 1, needs
The sample and reagent to be prepared have:
The genomic DNA (gDNA) that sample to be tested extracts, A260-280 is between 1.8-2.0, with nuclease free
Water is diluted to 10ng/ μ L；The primer of the base sequence as shown in SEQ ID NO:1-36, with nuclease free water
It is diluted to 10 μm of mol/L；Rnase enzyme is diluted to 5mU/ μ L；5U/ μ L TaKaRa LA Taq archaeal dna polymerase；2.5mmol/L
DNTP mixture；TaKaRa 2×GC Buffer Ⅱ.
2, using sample to be tested gDNA as template, expand PKD1 gene 1-33 exon.According to following system in sterilizing PCR
PCR reaction system is prepared in pipe.
20 μ L PCR reaction systems are as follows:
| PCR ingredient | It is added volume (μ L) |
| gDNA(10ng/μL) | 1 |
| 2×GC BufferⅡ | 10 |
| 2.5mmol/L dNTP mixture | 3.2 |
| Forward primer (10 μm of mol/L) | 0.4 |
| Reverse primer (10 μm of mol/L) | 0.4 |
| Rnase(5mU/μL) | 2 |
| TaKaRa LA Taq | 0.2 |
| nuclease free water | 2.8 |
3, will PCR pipe mix centrifugation after be placed in PCR thermal cycler, following amplification program is set and to start PCR anti-
It answers.
Amplification program:
94 DEG C of thermal startings and initial denaturation 1min；94 DEG C of denaturation 30sec, 65 DEG C of annealing and extension 2min, totally 40 recycle；4
DEG C save.
4, PCR product is subjected to agarose gel electrophoresis, as seen from Figure 1, amplified production size and expected purpose segment are big
It is small consistent, target fragment is recycled with plastic recovery kit and carries out sanger sequencing.
Embodiment three (the present embodiment respectively verifies NGS data with nest-type PRC and amplification system of the present invention)
1, polycystic kindey illness family involved in the present embodiment, including propositus, younger brother propositus and first demonstrate,proves father propositus
Mother person totally four members.Wherein the clinical symptoms of propositus are polycystic kindey with ejaculation tubular cyst, azoospermia；Younger brother propositus
Clinical symptoms be polycystic kindey, sperm motility and density are normal, rate of teratosperm 99%；Father's polycystic kindey, mother's phenotype are normal.
Biological information credit is carried out by the full exon two generations high throughput data to four samples of the family and a check sample
It analyses and genetic counselling analysis is carried out according to " ACMG hereditary variation classification standard and guide ", find a variation of causing a disease, such as Figure 20
It is shown.The variation of causing a disease are as follows: NM_000296.3:c.4997G > A of PKD1 gene makes a variation (p.W1666X), genomic locations
Chr16:2160171, is located at the 15th exon of PKD1 gene, which is stopgain nonsense mutation.It is found in analysis
C.4997G > A variation is evaluated as pathogenicity variation (pathogenic) according to " ACMG hereditary variation classification standard and guide ",
Meet PVS1 (LOF variation), PM2 is (ESP database, thousand personal data libraries, not found in normal control population in EXAC database
Variation), (statistical methods predict the variation to cause harmful influence, including conservative to gene or gene product to PP3
Prediction, Evolution Forecasting, splice site influence etc.).The variation (is suffered from propositus, father propositus (patient) and younger brother propositus
Person) in have heterozygous variance, show that the variation will lead to the hair of autosomal dominant POLYCYSTIC KIDNEY DISEASE through above-mentioned assessment
It is raw.The variation is not found in mother's propositus sample simultaneously, illustrates that the variation meets family and isolates.
2, two wheel PCR amplifications are carried out to the variant sites of causing a disease using nest-type PRC, the second wheel PCR product is carried out
Sanger sequencing, Sanger sequencing result show propositus, and the PKD1 gene of father propositus, younger brother propositus have occurred
C.4997G > A heterozygous mutant, mother of propositus is normal, consistent with NGS data.
A) archaeal dna polymerase used in is 2 × KAPA HiFi Ready Mix, the primer such as SEQ ID NO:37-SEQ ID
Shown in NO:40,10 μm of mol/L are diluted to nuclease free water, are extracted sample gene to be tested group DNA (gDNA),
A260-280 is diluted to 20ng/ μ L between 1.8-2.0, and with nuclease free water.It is going out according to following system
First round PCR reaction system is prepared in bacterium PCR pipe.
25 μ L PCR reaction systems are as follows:
B) it is placed in PCR thermal cycler after PCR pipe being mixed centrifugation, following amplification program is set and starts the first round
PCR reaction.Amplification program:
C) 95 DEG C of thermal startings and initial denaturation 5min；95 DEG C of denaturation 15sec, 64 DEG C of annealing 20sec, 72 DEG C of extension 15sec, altogether
35 circulations；72 DEG C of final extension 10min；4 DEG C of preservations.
D) DNA concentration is purified and is measured to a wheel PCR product, be diluted to 1ng/ μ with nuclease free water
L。
E) the second wheel PCR reaction system is prepared in sterilizing PCR pipe according to following system.50 μ L PCR reaction systems are such as
Under:
F) it is placed in PCR thermal cycler after PCR pipe being mixed centrifugation, following amplification program is set and starts the second wheel
PCR reaction.Amplification program: 95 DEG C of thermal startings and initial denaturation 5min；95 DEG C of denaturation 15sec, 65 DEG C of annealing 20sec, 72 DEG C extend
15sec, totally 25 recycle；72 DEG C of final extension 10min；4 DEG C of preservations.
Table 2 verify PKD1 gene c.4997G > A variation nest-type PRC primer sequence
3, in order to compare with the result of nest-type PRC, we use the amplification system and amplification program in embodiment one
The region PKD1 gene extron 15c of four members of the family is expanded, Sanger sequencing is carried out to PCR product,
Sanger sequencing result shows propositus, and father propositus, it is miscellaneous that c.4997G > A has occurred in the PKD1 gene of younger brother propositus
Mutation is closed, mother of propositus is normal, consistent with NGS data.
A) sample gene to be tested group DNA (gDNA) is extracted, A260-280 is between 1.8-2.0, with nuclease free
Water is diluted to 10ng/ μ L；Prepare the primer of the base sequence as shown in SEQ ID NO:19 and SEQ ID NO:20, uses
Nuclease free water is diluted to 10 μm of mol/L；Rnase enzyme is diluted to 5mU/ μ L；5U/μL TaKaRa LA Taq
Archaeal dna polymerase；2.5mmol/L dNTP mixture；TaKaRa 2×GC BufferⅠ.According to following system in sterilizing PCR pipe
Middle preparation PCR reaction system.20 μ L PCR reaction systems are as follows:
B) will PCR pipe mix centrifugation after be placed in PCR thermal cycler, following amplification program is set and to start PCR anti-
It answers.
C) amplification program: 94 DEG C of thermal startings and initial denaturation 1min；94 DEG C of denaturation 30sec, 65 DEG C of annealing and extension 1min, altogether
40 circulations；4 DEG C of preservations.
Example IV
1, the present embodiment uses amplification system and amplification program in embodiment one for the mutational site of 9 different samples
Amplification is carried out to corresponding exon region (as shown in table 2) and Sanger sequencing, these mutational sites are carried out to PCR product
Information be all it is detected from high-throughput two generation sequencing datas, Sanger sequencing result is consistent with NGS data.
2, it extracts sample gene to be tested group DNA (gDNA), A260-280 is between 1.8-2.0, with nuclease free
Water is diluted to 10ng/ μ L；Prepare the primer of the base sequence as shown in SEQ ID NO:19 and SEQ ID NO:20, uses
Nuclease free water is diluted to 10 μm of mol/L；Rnase enzyme is diluted to 5mU/ μ L；5U/μL TaKaRa LA Taq
Archaeal dna polymerase；2.5mmol/L dNTP mixture；TaKaRa 2×GC BufferⅠ.
3, PCR reaction system is prepared in sterilizing PCR pipe according to following system.
20 μ L PCR reaction systems are as follows:
4, will PCR pipe mix centrifugation after be placed in PCR thermal cycler, following amplification program is set and to start PCR anti-
It answers.
Amplification program:
94 DEG C of thermal startings and initial denaturation 1min；94 DEG C of denaturation 30sec, 65 DEG C of annealing and extension 1min,
Totally 40 circulations；4 DEG C of preservations.
The site of PKD1 gene mutation and position on chromosome in 29 samples of table
Sequence table
<110>your gene technology (Suzhou) Co., Ltd is read
<120>a kind of PCR primer group and amplification system for expanding mankind PKD1 gene 1-33 exon
<160> 36
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Claims (9)
1. a kind of PCR primer group for expanding mankind PKD1 gene 1-33 exon, which is characterized in that the primer sets include 18 altogether
It is as follows respectively to primer:
For expanding the primer of PKD1 gene extron 1, forward primer is as shown in SEQ ID NO:1, reverse primer such as SEQ
Shown in ID NO:2；
For expanding the primer of PKD1 gene extron 2-5a, forward primer is as shown in SEQ ID NO:3, and reverse primer is such as
Shown in SEQ ID NO:4；
For expanding the primer of PKD1 gene extron 5b-6, forward primer is as shown in SEQ ID NO:5, and reverse primer is such as
Shown in SEQ ID NO:6；
For expanding the primer of PKD1 gene extron 7-8, forward primer is as shown in SEQ ID NO:7, reverse primer such as SEQ
Shown in ID NO:8；
For expanding the primer of PKD1 gene extron 9-10, forward primer is as shown in SEQ ID NO:9, and reverse primer is such as
Shown in SEQ ID NO:10；
For expanding the primer of PKD1 gene extron 11, forward primer is as shown in SEQ ID NO:11, reverse primer such as SEQ
Shown in ID NO:12；
For expanding the primer of PKD1 gene extron 12, forward primer is as shown in SEQ ID NO:13, reverse primer such as SEQ
Shown in ID NO:14；
For expanding the primer of PKD1 gene extron 13-15a, forward primer is as shown in SEQ ID NO:15, reverse primer
As shown in SEQ ID NO:16；
For expanding the primer of PKD1 gene extron 15b, forward primer is as shown in SEQ ID NO:17, and reverse primer is such as
Shown in SEQ ID NO:18；
For expanding the primer of PKD1 gene extron 15c, forward primer is as shown in SEQ ID NO:19, and reverse primer is such as
Shown in SEQ ID NO:20；
For expanding the primer of PKD1 gene extron 15d-16, forward primer is as shown in SEQ ID NO:21, reverse primer
As shown in SEQ ID NO:22；
For expanding the primer of PKD1 gene extron 17-20, forward primer is as shown in SEQ ID NO:23, and reverse primer is such as
Shown in SEQ ID NO:24；
For expanding the primer of PKD1 gene extron 21, forward primer is as shown in SEQ ID NO:25, reverse primer such as SEQ
Shown in ID NO:26；
For expanding the primer of PKD1 gene extron 22, forward primer is as shown in SEQ ID NO:27, reverse primer such as SEQ
Shown in ID NO:28；
For expanding the primer of PKD1 gene extron 23-24, forward primer is as shown in SEQ ID NO:29, and reverse primer is such as
Shown in SEQ ID NO:30；
For expanding the primer of PKD1 gene extron 25-26, forward primer is as shown in SEQ ID NO:31, and reverse primer is such as
Shown in SEQ ID NO:32；
For expanding the primer of PKD1 gene extron 27-30, forward primer is as shown in SEQ ID NO:33, and reverse primer is such as
Shown in SEQ ID NO:34；
For expanding the primer of PKD1 gene extron 31-33, forward primer is as shown in SEQ ID NO:35, and reverse primer is such as
Shown in SEQ ID NO:36.
2. a kind of PCR primer group for expanding mankind PKD1 gene 1-33 exon according to claim 1, feature exist
In the modification on primer base is: fluorophor modification, RNA base, 2F-RNA base, XNA base, C3spacer, C6
Spacer, Spacer 9, Spacer 18, PO3, Biotin, SH C6 and 5- nitroindoline.
3. a kind of PCR primer group for expanding mankind PKD1 gene 1-33 exon according to claim 1, feature exist
In, the fluorophor be 6-FAM, TET, JOE, HEX, Cy3, TAMRA, ROX, Texas Red, Quasar 670, Cy5,
CY5.5, Methylene Blue, BHQ1, BHQ2 or Dabcyl modification.
4. a kind of PCR primer group amplification system for expanding mankind PKD1 gene 1-33 exon, which is characterized in that for expanding
The PCR reaction system of PKD1 gene 1-33 exon, include any 1 primer in the primer sets of claim 1 or its
Any combination.
5. a kind of PCR primer group amplification system for expanding mankind PKD1 gene 1-33 exon according to claim 4,
It is characterised in that it includes archaeal dna polymerase, buffer, Rnase enzyme, dNTP mixture.
6. a kind of PCR primer group amplification system for expanding mankind PKD1 gene 1-33 exon according to claim 5,
It is characterized in that, the archaeal dna polymerase is TaKaRa LA Taq archaeal dna polymerase.
7. a kind of PCR primer group amplification system for expanding mankind PKD1 gene 1-33 exon according to claim 5,
It is characterized in that, containing following compositions in the PCR reaction system that total volume is 20 μ L: 2 × Buffer of TaKaRa, 10 μ L,
3.2 μ L of 2.5mmol/L dNTP mixture, the forward primer and each 0.4 μ L, 5U/ μ L TaKaRa LA of reverse primer of 10 μm of ol/L
Taq 0.2μL。
8. a kind of PCR primer group for expanding mankind PKD1 gene 1-33 exon and amplification system are for detecting and verifying PKD1
The application of gene mutation situation.
9. a kind of PCR primer group for expanding mankind PKD1 gene 1-33 exon according to claim 8 and amplification body
The application of system is applied to generation Sanger sequencing, high-flux sequence, PCR amplification, molecule hybridization, enzyme linked immunological.
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| CN115725716A (en) * | 2022-09-20 | 2023-03-03 | 湖南家辉生物技术有限公司 | PKD1 pathogenic mutant gene and application thereof in preparation of polycystic kidney disease diagnostic kit |
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