CN111057758A - Primer and method for detecting PSEN1 gene mutation and application thereof - Google Patents
Primer and method for detecting PSEN1 gene mutation and application thereof Download PDFInfo
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Abstract
The invention discloses a primer and a method for detecting PSEN1 gene mutation of Alzheimer disease patients and application thereof, wherein the primer comprises a primer for amplifying PSEN1 gene full exon sequence; sanger sequencing technology and sequencing primers were used. The invention can quickly detect the mutation of the PSEN1 gene full exon in the body of the patient with Alzheimer disease. The detection result completed by the method is accurate, the Alzheimer disease can be diagnosed in an auxiliary way, and the method has important reference significance for risk prediction of diseases and targeted customization of treatment schemes.
Description
Technical Field
The invention belongs to the fields of bioscience and biotechnology, and particularly relates to a primer and a method for total exon mutation of an Alzheimer disease related gene PSEN1 and application thereof.
Background
Alzheimer's Disease (AD) is a common neurodegenerative disease with multifactorial combined action, high heritability and heterogeneity in neurology department. The clinical manifestations are the reduction and the loss of the cognitive function level in multiple fields and the abnormal mental behaviors. Statistically, the prevalence of age groups greater than or equal to 85 years is between 24% and 33% in western countries. Although developing countries lack authoritative and representative numerical statistics, it is estimated that 60% of dementia patients in the world are present in developing countries. With the advent of aging society in china, alzheimer's disease will become a social problem, placing a great burden on families and society, and becoming one of the most serious medical problems faced by current aged medicine.
Alzheimer's disease has a clear genetic predisposition, particularly Familial AD (FAD) appears as an autosomal dominant inheritance. PSEN1 is one of four established AD pathogenetic genes, and the gene mutation rate is 15.2%. To date, databases have shown more than 200 different mutations, mainly missense mutations, in the PSEN1 gene. Mutations in approximately 70% of the PSEN1 gene occurred in exons 5, 6, 7, 8, 12.
PSEN1 is located at 14q24.3, 12 exons exist, the product presenilin 1 protein belongs to transmembrane protein, consists of 467 amino acid residues, can form a compound with APP in cells and participate in the transportation and post-synthesis processing of the APP, wild-type presenilin proteins 1 and 2 have anti-apoptosis effect, are easy to be cracked by caspase and can increase A β in neurons, PSEN1 mutation can reduce the stability of β chain protein, apoptosis-related gene par-4 is highly expressed, and neurons are easy to be apoptotic, meanwhile, presenilin protein is used as a catalytic subunit of gamma-secretase, the defect of the gene can influence the transportation and enzyme digestion processing of the APP, and the pathogenic mechanism of the gene is probably that the activity of the gamma-secretase is changed by the mutant presenilin protein, so that the generation ratio of A β 42 is increased.
PSEN1 has been increasingly discovered because of its high complete penetrance and early age of onset. The identification of the mutation of the PSEN1 gene in one family is not only beneficial to prenatal diagnosis and clinical genetic diagnosis and the diagnosis of PSEN1 carriers, but also can enrich the epigenetics of the PSEN1 gene and further promote the research of pathogenic mechanisms and the research and development of targeted drugs. So far, more than 200 mutations have been reported in PSEN1, the mutation range is wide, and the mutation records exist in different exon regions, so that the detection of the whole exon of the PSEN1 gene is necessary.
Disclosure of Invention
The invention aims to provide a primer for detecting PSEN1 gene mutation, which can be used for rapidly detecting the PSEN1 gene exon mutation in an Alzheimer disease patient by adopting a PCR (polymerase chain reaction) technology.
The invention provides a primer for detecting PSEN1 gene mutation, which comprises the following components: at least one pair of primers for amplifying PSEN1 gene exons, wherein the at least one pair of primers is selected from the group consisting of PSEN1-1F/PSEN1-1R, PSEN1-2-3F/PSEN1-2-3R, PSEN1-4F/PSEN1-4R, PSEN1-5F/PSEN1-5R, PSEN1-6F/PSEN1-6R, PSEN1-7F/PSEN1-7R, PSEN1-8F/PSEN1-8R, PSEN1-9F/PSEN1-9R, PSEN1-10F/PSEN1-10 1-11F/PSEN1-11 1-12-1F/PSEN1-12-1 1-12-2F/PSEN1-12-2 1-12-3F/PSEN1-12-3R 1-12, PSEN1-12-4F/PSEN1-12-4R, PSEN1-12-5F/PSEN1-12-5R, PSEN1-12-6F/PSEN1-12-6R, PSEN1-12-7F/PSEN1-12-7R, PSEN1-12-8F/PSEN1-12-8R, PSEN1-12-9F/PSEN1-12-9R or PSEN1-12-10F/PSEN1-12-10R, and the base sequence is as follows:
PSEN1-1F：TGTAAAACGACGGCCAGTGTGGAGCTCTGGGTTCTCC；
PSEN1-1R：AACAGCTATGACCATGTCATCTTTTAGGACACCGGC；
PSEN1-2-3F：TGTAAAACGACGGCCAGTTGATTGGTTTAAATGAATTTACTAGG；
PSEN1-2-3R：AACAGCTATGACCATGCTCAGAGGTGAGGGGAGATG；
PSEN1-4F：TGTAAAACGACGGCCAGTCCGTTACCTTGATTCTGCTG；
PSEN1-4R：AACAGCTATGACCATGAGGCCTTCAAGGTGATGATG；
PSEN1-5F：TGTAAAACGACGGCCAGTTGTTGGAGGTGGTAATGTGG；
PSEN1-5R：AACAGCTATGACCATGCTGTGACAAGAATACCCAACC；
PSEN1-6F：TGTAAAACGACGGCCAGTATTCTGTTGGCCTTTGCC；
PSEN1-6R：AACAGCTATGACCATGAATTCATCAACAAAGTACATGGC；
PSEN1-7F：TGTAAAACGACGGCCAGTTTGGGAGCCATCACATTATTC；
PSEN1-7R：AACAGCTATGACCATGAGAAAACACTCCAGTGGGGC；
PSEN1-8F：TGTAAAACGACGGCCAGTCCTCCCTACCACCCATTTAC；
PSEN1-8R：AACAGCTATGACCATGTTACATGTGCTTCAGTTCCG；
PSEN1-9F：TGTAAAACGACGGCCAGTAGGAAGACTGGCGATTTGTG；
PSEN1-9R：AACAGCTATGACCATGGGAGTCTATGACCAAAGAAAGACG；
PSEN1-10F：TGTAAAACGACGGCCAGTCTACAGCCCATGCTTTGTGG；
PSEN1-10R：AACAGCTATGACCATGGGAATCCATGACTTTGCAACAT；
PSEN1-11F：TGTAAAACGACGGCCAGTCATTGTGGGGTTGAGTAGGG；
PSEN1-11R：AACAGCTATGACCATGCCCACCTGGGGTTAAAACAG；
PSEN1-12-1F：TGTAAAACGACGGCCAGTAAGACTTGTGATTGAGTTTTGCC；
PSEN1-12-1R：AACAGCTATGACCATGTCGCAGTGTCAGTGAAATCG；
PSEN1-12-2F：TGTAAAACGACGGCCAGTATTTGAGGGACGAGGTCAAG；
PSEN1-12-2R：AACAGCTATGACCATGTCCAAATTTGACTTCAACATGATAG；
PSEN1-12-3F：TGTAAAACGACGGCCAGTATTTCTTCCCAAGGCCAGTC；
PSEN1-12-3R：AACAGCTATGACCATGCCAAGTTAAATATGGCTTTCCC；
PSEN1-12-4F：TGTAAAACGACGGCCAGTTGAAGCACTTTCTGTCCTGG；
PSEN1-12-4R：AACAGCTATGACCATGTTGGGAGAGAAAACAGCAGG；
PSEN1-12-5F：TGTAAAACGACGGCCAGTGCTGATCAGACCGTAAGCG；
PSEN1-12-5R：AACAGCTATGACCATGACACCCCACGTACCCTCTTC；
PSEN1-12-6F：TGTAAAACGACGGCCAGTTTGGAAGCATTCTTGAGGG；
PSEN1-12-6R：AACAGCTATGACCATGCTCTACTGCTGGTTTTGCCC；
PSEN1-12-7F：TGTAAAACGACGGCCAGTTCCAAATGCTAGTCTTCCACC；
PSEN1-12-7R：AACAGCTATGACCATGATACACTGTCTGAGGCCACG；
PSEN1-12-8F：TGTAAAACGACGGCCAGTAGAAGCAACAACTGAAGAGGC；
PSEN1-12-8R：AACAGCTATGACCATGAGGAGAAGGGCAGGTCAAAC；
PSEN1-12-9F：TGTAAAACGACGGCCAGTTTTTCTGGTATATGCCATCTTTC；
PSEN1-12-9R：AACAGCTATGACCATGATCCACTAAGAGGCACCCTG；
PSEN1-12-10F：TGTAAAACGACGGCCAGTTGTGATCATCTGCATAGTCCTC；
PSEN1-12-10R：
AACAGCTATGACCATGAAGTGCTAGACAGTTCTGTGAGTTAC。
further, the primers also comprise a pair of sequencing primers, and the base sequences of the sequencing primers are as follows:
M13F：TGTAAAACGACGGCCAGT；
M13R：AACAGCTATGACCATG。
further, PSEN1-1F, PSEN1-1R, PSEN1-2-3F, PSEN1-2-3R,
PSEN1-4F:PSEN1-4R、PSEN1-5F:PSEN1-5R、PSEN1-6F:PSEN1-6R、
PSEN1-7F:PSEN1-7R、PSEN1-8F:PSEN1-8R、PSEN1-9F:PSEN1-9R、
PSEN1-10F:PSEN1-10R、PSEN1-11F:PSEN1-11R、PSEN1-12-1F:PSEN1-12-1R、
PSEN1-12-2F:PSEN1-12-2R、PSEN1-12-3F:PSEN1-12-3R、
PSEN1-12-4F:PSEN1-12-4R、PSEN1-12-5F:PSEN1-12-5R、
PSEN1-12-6F:PSEN1-12-6R、PSEN1-12-7F:PSEN1-12-7R、
PSEN1-12-8F, PSEN1-12-8R, PSEN1-12-9F, PSEN1-12-9R and
PSEN1-12-10F, PSEN1-12-10R are all 1.
Further, M13F: the ratio of M13R is 1.
The primer provided by the invention is applied to the preparation of a kit for detecting PSEN1 gene mutation.
The invention also provides a method for detecting the PSEN1 gene mutation, which comprises the following steps:
(1) extracting genome DNA in a sample;
(2) amplifying the DNA in the step (1) by using at least one pair of amplification primers to obtain an amplification product;
(3) sequencing the amplification product in the step (2) by utilizing a pair of sequencing primers M13F and M13R to obtain a base sequence of the amplification product;
(4) comparing the base sequence in (3) with a PSEN1 gene wild type reference sequence to determine whether a mutation site exists; wherein the at least one pair of amplification primers is selected from the group consisting of PSEN1-1F/PSEN1-1R, PSEN1-2F/PSEN1-2R, PSEN1-3F/PSEN1-3R, PSEN1-4F/PSEN1-4R, PSEN1-5F/PSEN1-5R, PSEN1-6F/PSEN1-6R, PSEN1-7F/PSEN1-7R, PSEN1-8F/PSEN1-8R, PSEN1-9F/PSEN1-9R, PSEN1-10F/PSEN1-10R, PSEN1-11F/PSEN1-11R, PSEN 1-12F/PSEN 1-12-1R, PSEN1-12-2F/PSEN1-12-2R, PSEN1-12-3F/PSEN1-12-3R, PSEN1-12-4F/PSEN1-12-4R, PSEN1-12-5F/PSEN1-12-5R, PSEN1-12-6F/PSEN1-12-6R, PSEN1-12-7F/PSEN1-12-7R, PSEN1-12-8F/PSEN1-12-8R, PSEN1-12-9F/PSEN1-12-9R or PSEN1-12-10F/PSEN 1-12-10R.
The invention also provides a kit for detecting PSEN1 gene mutation, which comprises a detection system PCR amplification reaction solution and a sequencing system reaction solution, wherein the detection system PCR amplification reaction solution comprises at least one pair of amplification primers selected from PSEN1-1F/PSEN1-1R, PSEN1-2-3F/PSEN1-2-3R, PSEN1-4F/PSEN1-4R, PSEN1-5F/PSEN1-5R, PSEN1-6F/PSEN1-6R, PSEN1-7F/PSEN1-7R, PSEN1-8F/PSEN1-8R, PSEN1-9F/PSEN1-9R, PSEN1-10F/PSEN1-10R, PSEN1-11F/PSEN1-11R, PSEN1-12-1F/PSEN 1-1R, PSEN1-9, PSEN1-12-2F/PSEN1-12-2R, PSEN1-12-3F/PSEN1-12-3R, PSEN1-12-4F/PSEN1-12-4R, PSEN1-12-5F/PSEN1-12-5R, PSEN1-12-6F/PSEN1-12-6R, PSEN1-12-7F/PSEN1-12-7R, PSEN1-12-8F/PSEN1-12-8R, PSEN1-12-9F/PSEN1-12-9R or PSEN1-12-10F/PSEN1-12-10R, and the sequencing reaction solution comprises a pair of sequencing primers M13F and M13R.
Further, the PCR amplification reaction solution of the detection system also comprises 2 XPCR Buffer, dNTPs and KOD FXDNA Polymerase.
Furthermore, the reaction solution of the sequencing system also comprises EDTA, absolute ethyl alcohol, 75% ethyl alcohol, HIDI and BigdyeTerminator V3.1.
Further, the sequencing system reaction solution also comprises a sequencing purification solution, and the sequencing purification solution comprises exonuclease I and bovine small intestine alkaline phosphatase.
Has the advantages that: (1) the M13 joints are added to all the designed amplification primers, so that the PCR products of all 20 pairs of primers can be sequenced by using one pair of sequencing primers, the sequencing steps are greatly simplified, and the complexity of detection is reduced; (2) the 20 pairs of amplification primers provided by the invention cover the whole 12 exons of the PSEN1 gene, and can ensure that the condition of missed detection can not occur no matter where the exons are mutated, especially for the 12 th exon, the primers have longer sequences, while the invention designs 10 pairs of primers, respectively amplify different parts of the 12 th exon and can cover the whole 12 th exon; (3) the primers designed by the invention have the advantages that the amplification efficiency is optimized by adjusting the concentration of the primers and the like, and all the primers can use consistent annealing temperature, so that the detection difficulty is reduced; (4) compared with the fluorescent quantitative PCR method which needs to design a plurality of probes aiming at a plurality of known mutation sites which are possible to generate mutation in all 12 exons of the PSEN1 gene, the method only needs 20 pairs of primers and low-cost sequencing to detect all known and unknown mutations, and the detection cost is obviously reduced.
Drawings
FIG. 1 is an electrophoresis chart of the amplification products of 20 pairs of primers. And Marker is DL 1000.
FIGS. 2-4 show the sequencing results of one sample tested according to the invention, and 3 point mutations were found in exon 12 of the PSEN1 gene.
Detailed Description
The invention will be further elucidated with reference to the specific embodiments and the accompanying drawings. It should be noted that the conventional conditions and methods not described in the examples are generally employed by those skilled in the art according to the routine procedures: such as the fourth edition of "Fine molecular biology laboratory Manual", eds by auspicious and Kingston, or according to the procedures and conditions suggested by the manufacturer.
Example 1
A primer for detecting PSEN1 gene full exon polymorphic mutation sites has a base sequence as follows:
PSEN1-1F：TGTAAAACGACGGCCAGTGTGGAGCTCTGGGTTCTCC；
PSEN1-1R：AACAGCTATGACCATGTCATCTTTTAGGACACCGGC；
PSEN1-2-3F：TGTAAAACGACGGCCAGTTGATTGGTTTAAATGAATTTACTAGG；
PSEN1-2-3R：AACAGCTATGACCATGCTCAGAGGTGAGGGGAGATG；
PSEN1-4F：TGTAAAACGACGGCCAGTCCGTTACCTTGATTCTGCTG；
PSEN1-4R：AACAGCTATGACCATGAGGCCTTCAAGGTGATGATG；
PSEN1-5F：TGTAAAACGACGGCCAGTTGTTGGAGGTGGTAATGTGG；
PSEN1-5R：AACAGCTATGACCATGCTGTGACAAGAATACCCAACC；
PSEN1-6F：TGTAAAACGACGGCCAGTATTCTGTTGGCCTTTGCC；
PSEN1-6R：AACAGCTATGACCATGAATTCATCAACAAAGTACATGGC；
PSEN1-7F：TGTAAAACGACGGCCAGTTTGGGAGCCATCACATTATTC；
PSEN1-7R：AACAGCTATGACCATGAGAAAACACTCCAGTGGGGC；
PSEN1-8F：TGTAAAACGACGGCCAGTCCTCCCTACCACCCATTTAC；
PSEN1-8R：AACAGCTATGACCATGTTACATGTGCTTCAGTTCCG；
PSEN1-9F：TGTAAAACGACGGCCAGTAGGAAGACTGGCGATTTGTG；
PSEN1-9R：AACAGCTATGACCATGGGAGTCTATGACCAAAGAAAGACG；
PSEN1-10F：TGTAAAACGACGGCCAGTCTACAGCCCATGCTTTGTGG；
PSEN1-10R：AACAGCTATGACCATGGGAATCCATGACTTTGCAACAT；
PSEN1-11F：TGTAAAACGACGGCCAGTCATTGTGGGGTTGAGTAGGG；
PSEN1-11R：AACAGCTATGACCATGCCCACCTGGGGTTAAAACAG；
PSEN1-12-1F：TGTAAAACGACGGCCAGTAAGACTTGTGATTGAGTTTTGCC；
PSEN1-12-1R：AACAGCTATGACCATGTCGCAGTGTCAGTGAAATCG；
PSEN1-12-2F：TGTAAAACGACGGCCAGTATTTGAGGGACGAGGTCAAG；
PSEN1-12-2R：AACAGCTATGACCATGTCCAAATTTGACTTCAACATGATAG；
PSEN1-12-3F：TGTAAAACGACGGCCAGTATTTCTTCCCAAGGCCAGTC；
PSEN1-12-3R：AACAGCTATGACCATGCCAAGTTAAATATGGCTTTCCC；
PSEN1-12-4F：TGTAAAACGACGGCCAGTTGAAGCACTTTCTGTCCTGG；
PSEN1-12-4R：AACAGCTATGACCATGTTGGGAGAGAAAACAGCAGG；
PSEN1-12-5F：TGTAAAACGACGGCCAGTGCTGATCAGACCGTAAGCG；
PSEN1-12-5R：AACAGCTATGACCATGACACCCCACGTACCCTCTTC；
PSEN1-12-6F：TGTAAAACGACGGCCAGTTTGGAAGCATTCTTGAGGG；
PSEN1-12-6R：AACAGCTATGACCATGCTCTACTGCTGGTTTTGCCC；
PSEN1-12-7F：TGTAAAACGACGGCCAGTTCCAAATGCTAGTCTTCCACC；
PSEN1-12-7R：AACAGCTATGACCATGATACACTGTCTGAGGCCACG；
PSEN1-12-8F：TGTAAAACGACGGCCAGTAGAAGCAACAACTGAAGAGGC；
PSEN1-12-8R：AACAGCTATGACCATGAGGAGAAGGGCAGGTCAAAC；
PSEN1-12-9F：TGTAAAACGACGGCCAGTTTTTCTGGTATATGCCATCTTTC；
PSEN1-12-9R：AACAGCTATGACCATGATCCACTAAGAGGCACCCTG；
PSEN1-12-10F：TGTAAAACGACGGCCAGTTGTGATCATCTGCATAGTCCTC；
PSEN1-12-10R：AACAGCTATGACCATGAAGTGCTAGACAGTTCTGTGAGTTAC。
the oligonucleotide also comprises a pair of sequencing primers M13F and M13R, and the base sequences are as follows:
M13F：TGTAAAACGACGGCCAGT；
M13R：AACAGCTATGACCATG。
a kit for detecting PSEN1 gene mutation comprises a detection system PCR amplification reaction solution and a sequencing system reaction solution, wherein,
the PCR amplification reaction solution of the detection system comprises: 2 XPCR Buffer (10.0. mu.L); dNTPs (2 mM); KOD FXDNA Polymerase (1U/. mu.l); primers (10. mu.M) for the 12 exons of the PSEN1 gene.
The sequencing system reaction solution comprises: sequencing purified solution (exonuclease I:0.6U, bovine small intestine alkaline phosphatase: 1.2U); EDTA (125 mM); absolute ethyl alcohol; 75% ethanol; HIDI (highly deionized formamide); a pair of sequencing primers M13F (3.2. mu.M), M13R (3.2. mu.M); bigdye Terminator V3.1 (purchased from Applied Biosystems, usa).
The kit can also comprise a blood DNA extraction reagent.
The kit may also include a positive control, a negative control, and a blank control. The positive control is a solution containing human PSEN1 gene exon DNA, and repeated freezing and thawing is forbidden. Negative control is ddH2And O. Blank control was 2. mu.l of physiological saline or no substance added.
EXAMPLE 2 blood sample DNA extraction
Blood sample DNA extraction (according to Tiangen biological blood/cell/tissue gene DNA extraction kit instructions): the method for extracting human blood sample DNA comprises the following steps:
(1) mu.l of blood was taken and added to 900. mu.l of erythrocyte lysate, mixed by inversion, left at room temperature for 5 minutes, and mixed by inversion several times in the meantime. Centrifuging at 12000rpm for 1min, removing supernatant, collecting leukocyte precipitate, adding 200 μ l buffer solution GA, and shaking to thoroughly mix;
(2) adding 20 mul proteinase K solution, and mixing;
(3) adding 200 μ l buffer solution GB, fully reversing and mixing, standing at 70 deg.C for 10 min, cleaning the solution, and centrifuging briefly to remove water droplets on the inner wall of the tube cover;
(4) adding 200 μ l of anhydrous ethanol, shaking thoroughly, mixing for 15s, wherein flocculent precipitate may appear, and centrifuging briefly to remove water drops on the inner wall of the tube cover;
(5) adding the solution and flocculent precipitate obtained in the previous step into an adsorption column CB3 (the adsorption column is placed into a collecting pipe), centrifuging at 12000rpm for 30 s, pouring off waste liquid, and placing the adsorption column CB3 back into the collecting pipe;
(6) adding 500 μ l buffer GD (before use, whether absolute ethyl alcohol is added or not is checked), centrifuging at 12000rpm for 30 s, pouring off waste liquid, and putting adsorption column CB3 into a collection tube;
(7) adding 700 μ l of rinsing solution PW (checking whether anhydrous ethanol is added before use) into adsorption column CB3, centrifuging at 12000rpm for 30 s, pouring off waste liquid, and placing adsorption column CB3 into a collection tube;
(8) adding 500 μ l of rinsing solution PW into adsorption column CB3, centrifuging at 12000rpm for 30 s, and pouring off waste liquid;
(9) putting the adsorption column CB3 back into the collecting pipe, centrifuging at 12000rpm for 2 minutes, pouring the waste liquid, and placing the adsorption column CB3 at room temperature for a plurality of minutes to completely dry the residual rinsing liquid in the adsorption material;
(10) transferring the adsorption column CB3 into a clean centrifugal tube, dripping 100 mu l of elution buffer TE into the middle part of the adsorption membrane in a hanging manner, standing at room temperature for 2-5 minutes, centrifuging at 12000rpm for 2 minutes, and collecting the solution into the centrifugal tube.
EXAMPLE 3 sample DNA amplification
The blood sample DNA extracted according to example 2 was amplified from the exon of PSEN1 gene using the amplification primers in example 1 to obtain an amplification product. Amplification is carried out on a conventional PCR apparatus, and usable apparatuses include ABI veriti (Applied Biosystems, USA) and the like. The details are as follows:
(i) taking a PCR amplification reaction solution of a detection system according to the number n of samples (the number of samples to be detected, the number of negative controls 1, the number of positive controls 1 and the number of blank controls 1), and distributing 19 mu l of the PCR amplification reaction solution into reaction tubes;
(ii) and (3) respectively adding 1 mu l of the processed sample to be detected, the negative control substance and the positive control substance into the reaction tube, uniformly mixing, centrifuging at a low speed for several seconds, and carrying out PCR amplification to obtain an amplification product. The preparation method of the PCR amplification reaction solution of the test system is shown in Table 1. The PCR amplification reaction conditions are shown in Table 2. Details of each pair of amplification primers are shown in Table 3.
TABLE 1 test System PCR amplification reaction solution preparation
Note: PrimerF and PrimerR in the table were selected from the upstream and downstream primers of PSEN 1.
TABLE 2 PCR amplification reaction conditions
TABLE 3 information for each pair of amplification primers
Example 4 Sanger sequencing
Mu.l of the amplification product of example 3 and 2. mu.l of the purified sequencing solution of example 1 were purified according to the procedure described in Table 4, to thereby obtain purified products.
TABLE 4 purification procedure
Mu.l of the purified product obtained was mixed with the sequencing primers M13F (3.2. mu.M) and M13R (3.2. mu.M) of example 1, respectively, in accordance with the system in Table 5, and then sequenced in accordance with the sequencing reaction procedure in Table 6.
TABLE 5
TABLE 6 sequencing reaction procedure
And (3) a precipitation link:
adding 2 μ l of 125mM EDTA to the product after completion of the sequencing reaction, and standing for 5 min; adding 15 mul of absolute ethyl alcohol, and mixing evenly by vortex; centrifuging at 3700rpm for 30 min; inverting and centrifuging for 15sec, adding 50 μ l 70% ethanol, and mixing by vortex; centrifuging at 3700rpm for 15 min; inverting and centrifuging for 15sec, and placing on a metal bath at 95 ℃; denaturation assays were performed after the addition of 10. mu.l HIDI. The denaturation test comprises the following steps: 95 ℃ for 5 min; followed by 2min at-30 ℃ and then storage at 4 ℃.
After the denaturation procedure was completed, sequencing was performed using a sequencer (ABI 3730).
And (5) judging a result:
the sequencing results were aligned with the PSEN1 wild-type reference sequence (Genbank: NG-007386.2), respectively, and the results were reported based on the actual mutation.
Example 5 clinical sample testing
Clinical blood samples were taken and reagents were prepared, sample DNA extracted, amplified (amplification products obtained) and sequenced according to example 1, example 2, example 3 and example 4. 1. mu.l of sample was added to the PCR reaction solution of the detection system.
And carrying out DNA electrophoresis by using the obtained amplification product, wherein the electrophoresis conditions are 1.2% agarose gel electrophoresis, 110V and 25min, and the gel imaging system is used for observation. The electrophoresis result is shown in FIG. 1, wherein 1-12 are respectively electropherograms of amplification products of 12 exons, and M is Marker DL1000, and analysis of the electropherograms shows that the 20 pairs of primers are effective in amplification and have single bands.
FIGS. 2-4 show the sequencing result of exon 12 of PSEN1 gene in a sample, and the comparison shows that there are 3 point mutations in exon 12 in this sample.
As can be seen from the detection results, the primer of the invention includes the whole of 12 exon sequences of the PSEN1 gene, and can detect the mutation condition of the whole exon. According to the detection result, the PSEN1 gene of the sample has 3 point mutations in exon 12, but does not cause amino acid change, and belongs to nonsense mutation. In addition, the detection of the PSEN1 gene exon mutation positive control also shows that the primers, the method and the kit can detect the mutation condition of the PSEN1 gene exon.
Sequence listing
<110> Adekang medical laboratory Co., Ltd, Fuzhou
<120> primers and method for detecting PSEN1 gene mutation and application thereof
<160>42
<170>SIPOSequenceListing 1.0
<210>1
<211>37
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>1
tgtaaaacga cggccagtgt ggagctctgg gttctcc 37
<210>2
<211>36
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>2
aacagctatg accatgtcat cttttaggac accggc 36
<210>3
<211>44
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>3
tgtaaaacga cggccagttg attggtttaa atgaatttac tagg 44
<210>4
<211>36
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>4
aacagctatg accatgctca gaggtgaggg gagatg 36
<210>5
<211>38
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>5
tgtaaaacga cggccagtcc gttaccttga ttctgctg 38
<210>6
<211>36
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>6
aacagctatg accatgaggc cttcaaggtg atgatg 36
<210>7
<211>38
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>7
tgtaaaacga cggccagttg ttggaggtgg taatgtgg 38
<210>8
<211>37
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>8
aacagctatg accatgctgt gacaagaata cccaacc 37
<210>9
<211>36
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>9
tgtaaaacga cggccagtat tctgttggcc tttgcc 36
<210>10
<211>39
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>10
aacagctatg accatgaatt catcaacaaa gtacatggc 39
<210>11
<211>39
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>11
tgtaaaacga cggccagttt gggagccatc acattattc 39
<210>12
<211>36
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>12
aacagctatg accatgagaa aacactccag tggggc 36
<210>13
<211>38
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>13
tgtaaaacga cggccagtcc tccctaccac ccatttac 38
<210>14
<211>36
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>14
aacagctatg accatgttac atgtgcttca gttccg 36
<210>15
<211>38
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>15
tgtaaaacga cggccagtag gaagactggc gatttgtg 38
<210>16
<211>40
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>16
aacagctatg accatgggag tctatgacca aagaaagacg 40
<210>17
<211>38
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>17
tgtaaaacga cggccagtct acagcccatg ctttgtgg 38
<210>18
<211>38
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>18
aacagctatg accatgggaa tccatgactt tgcaacat 38
<210>19
<211>38
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>19
tgtaaaacga cggccagtca ttgtggggtt gagtaggg 38
<210>20
<211>36
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>20
aacagctatg accatgccca cctggggtta aaacag 36
<210>21
<211>41
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>21
tgtaaaacga cggccagtaa gacttgtgat tgagttttgc c 41
<210>22
<211>36
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>22
aacagctatg accatgtcgc agtgtcagtg aaatcg 36
<210>23
<211>38
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>23
tgtaaaacga cggccagtat ttgagggacg aggtcaag 38
<210>24
<211>41
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>24
aacagctatg accatgtcca aatttgactt caacatgata g 41
<210>25
<211>38
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>25
tgtaaaacga cggccagtat ttcttcccaa ggccagtc 38
<210>26
<211>38
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>26
aacagctatg accatgccaa gttaaatatg gctttccc 38
<210>27
<211>38
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>27
tgtaaaacga cggccagttg aagcactttc tgtcctgg 38
<210>28
<211>36
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>28
aacagctatg accatgttgg gagagaaaac agcagg 36
<210>29
<211>37
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>29
tgtaaaacga cggccagtgc tgatcagacc gtaagcg 37
<210>30
<211>36
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>30
aacagctatg accatgacac cccacgtacc ctcttc 36
<210>31
<211>37
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>31
tgtaaaacga cggccagttt ggaagcattc ttgaggg 37
<210>32
<211>36
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>32
aacagctatg accatgctct actgctggtt ttgccc 36
<210>33
<211>39
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>33
tgtaaaacga cggccagttc caaatgctag tcttccacc 39
<210>34
<211>36
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>34
aacagctatg accatgatac actgtctgag gccacg 36
<210>35
<211>39
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>35
tgtaaaacga cggccagtag aagcaacaac tgaagaggc 39
<210>36
<211>36
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>36
aacagctatg accatgagga gaagggcagg tcaaac 36
<210>37
<211>41
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>37
tgtaaaacga cggccagttt ttctggtata tgccatcttt c 41
<210>38
<211>36
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>38
aacagctatg accatgatcc actaagaggc accctg 36
<210>39
<211>40
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>39
tgtaaaacga cggccagttg tgatcatctg catagtcctc 40
<210>40
<211>42
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>40
aacagctatg accatgaagt gctagacagt tctgtgagtt ac 42
<210>41
<211>18
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>41
tgtaaaacga cggccagt 18
<210>42
<211>16
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>42
aacagctatg accatg 16
Claims (6)
1. Primer for detecting PSEN1 gene mutation, which is characterized by comprising the following components: at least one pair of primers for amplifying exons of the PSEN1 gene, wherein the at least one pair of primers is selected from the group consisting of PSEN1-1F/PSEN1-1R,
PSEN1-2-3F/PSEN1-2-3R、PSEN1-4F/PSEN1-4R、PSEN1-5F/PSEN1-5R、
PSEN1-6F/PSEN1-6R、PSEN1-7F/PSEN1-7R、PSEN1-8F/PSEN1-8R、
PSEN1-9F/PSEN1-9R、PSEN1-10F/PSEN1-10R、PSEN1-11F/PSEN1-11R、
PSEN1-12-1F/PSEN1-12-1R、PSEN1-12-2F/PSEN1-12-2R、
PSEN1-12-3F/PSEN1-12-3R、PSEN1-12-4F/PSEN1-12-4R、
PSEN1-12-5F/PSEN1-12-5R、PSEN1-12-6F/PSEN1-12-6R、
PSEN1-12-7F/PSEN1-12-7R、PSEN1-12-8F/PSEN1-12-8R、
PSEN1-12-9F/PSEN1-12-9R or PSEN1-12-10F/PSEN1-12-10R, and the base sequence is as follows:
PSEN-1F：TGTAAAACGACGGCCAGTGTGGAGCTCTGGGTTCTCC；
PSEN-1R：AACAGCTATGACCATGTCATCTTTTAGGACACCGGC；
PSEN-2-3F：TGTAAAACGACGGCCAGTTGATTGGTTTAAATGAATTTACTAGG；
PSEN-2-3R：AACAGCTATGACCATGCTCAGAGGTGAGGGGAGATG；
PSEN1-4F：TGTAAAACGACGGCCAGTCCGTTACCTTGATTCTGCTG；
PSEN1-4R：AACAGCTATGACCATGAGGCCTTCAAGGTGATGATG；
PSEN1-5F：TGTAAAACGACGGCCAGTTGTTGGAGGTGGTAATGTGG；
PSEN1-5R：AACAGCTATGACCATGCTGTGACAAGAATACCCAACC；
PSEN1-6F：TGTAAAACGACGGCCAGTATTCTGTTGGCCTTTGCC；
PSEN1-6R：AACAGCTATGACCATGAATTCATCAACAAAGTACATGGC；
PSEN1-7F：TGTAAAACGACGGCCAGTTTGGGAGCCATCACATTATTC；
PSEN1-7R：AACAGCTATGACCATGAGAAAACACTCCAGTGGGGC；
PSEN1-8F：TGTAAAACGACGGCCAGTCCTCCCTACCACCCATTTAC；
PSEN1-8R：AACAGCTATGACCATGTTACATGTGCTTCAGTTCCG；
PSEN1-9F：TGTAAAACGACGGCCAGTAGGAAGACTGGCGATTTGTG；
PSEN1-9R：AACAGCTATGACCATGGGAGTCTATGACCAAAGAAAGACG；
PSEN1-10F：TGTAAAACGACGGCCAGTCTACAGCCCATGCTTTGTGG；
PSEN1-10R：AACAGCTATGACCATGGGAATCCATGACTTTGCAACAT；
PSEN1-11F：TGTAAAACGACGGCCAGTCATTGTGGGGTTGAGTAGGG；
PSEN1-11R：AACAGCTATGACCATGCCCACCTGGGGTTAAAACAG；
PSEN1-12-1F：TGTAAAACGACGGCCAGTAAGACTTGTGATTGAGTTTTGCC；
PSEN1-12-1R：AACAGCTATGACCATGTCGCAGTGTCAGTGAAATCG；
PSEN1-12-2F：TGTAAAACGACGGCCAGTATTTGAGGGACGAGGTCAAG；
PSEN1-12-2R：AACAGCTATGACCATGTCCAAATTTGACTTCAACATGATAG；
PSEN1-12-3F：TGTAAAACGACGGCCAGTATTTCTTCCCAAGGCCAGTC；
PSEN1-12-3R：AACAGCTATGACCATGCCAAGTTAAATATGGCTTTCCC；
PSEN1-12-4F：TGTAAAACGACGGCCAGTTGAAGCACTTTCTGTCCTGG；
PSEN1-12-4R：AACAGCTATGACCATGTTGGGAGAGAAAACAGCAGG；
PSEN1-12-5F：TGTAAAACGACGGCCAGTGCTGATCAGACCGTAAGCG；
PSEN1-12-5R：AACAGCTATGACCATGACACCCCACGTACCCTCTTC；
PSEN1-12-6F：TGTAAAACGACGGCCAGTTTGGAAGCATTCTTGAGGG；
PSEN1-12-6R：AACAGCTATGACCATGCTCTACTGCTGGTTTTGCCC；
PSEN1-12-7F：TGTAAAACGACGGCCAGTTCCAAATGCTAGTCTTCCACC；
PSEN1-12-7R：AACAGCTATGACCATGATACACTGTCTGAGGCCACG；
PSEN1-12-8F：TGTAAAACGACGGCCAGTAGAAGCAACAACTGAAGAGGC；
PSEN1-12-8R：AACAGCTATGACCATGAGGAGAAGGGCAGGTCAAAC；
PSEN1-12-9F：TGTAAAACGACGGCCAGTTTTTCTGGTATATGCCATCTTTC；
PSEN1-12-9R：AACAGCTATGACCATGATCCACTAAGAGGCACCCTG；
PSEN1-12-10F：TGTAAAACGACGGCCAGTTGTGATCATCTGCATAGTCCTC；
PSEN1-12-10R：
AACAGCTATGACCATGAAGTGCTAGACAGTTCTGTGAGTTAC。
2. the primer of claim 1, further comprising a pair of sequencing primers having the base sequence:
M13F：TGTAAAACGACGGCCAGT；
M13R：AACAGCTATGACCATG。
3. the primer of claim 1, wherein PSEN1-1F is PSEN1-1R,
PSEN1-2-3F:PSEN1-2-3R、PSEN1-4F:PSEN1-4R、PSEN1-5F:PSEN1-5R、
PSEN1-6F:PSEN1-6R、PSEN1-7F:PSEN1-7R、PSEN1-8F:PSEN1-8R、
PSEN1-9F:PSEN1-9R、PSEN1-10F:PSEN1-10R、PSEN1-11F:PSEN1-11R、
PSEN1-12-1F:PSEN1-12-1R、PSEN1-12-2F:PSEN1-12-2R、
PSEN1-12-3F:PSEN1-12-3R、PSEN1-12-4F:PSEN1-12-4R、
PSEN1-12-5F:PSEN1-12-5R、PSEN1-12-6F:PSEN1-12-6R、
PSEN1-12-7F:PSEN1-12-7R、PSEN1-12-8F:PSEN1-12-8R、
The ratios of PSEN1-12-9F, PSEN1-12-9R and PSEN1-12-10F, PSEN1-12-10R were all 1.
4. The primer of claim 2, wherein M13F: the ratio of M13R is 1.
5. Use of the primer according to any one of claims 1 to 4 for preparing a kit for detecting PSEN1 gene mutation.
6. A method for detecting a mutation in the PSEN1 gene, comprising the steps of:
(1) extracting genome DNA in a sample;
(2) amplifying the DNA in the at least one pair of amplification primers (1) according to claim 1 to obtain an amplification product;
(3) sequencing the amplification product in the pair of sequencing primers (2) according to claim 2 to obtain a base sequence of the amplification product;
(4) comparing the base sequence in (3) with a wild-type reference sequence of the PSEN1 gene to determine whether a mutation site exists.
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