CN102146443B

CN102146443B - Specific primer for detecting Leber hereditary optic neuropathy mitochondrial DNA mutation G10680A

Info

Publication number: CN102146443B
Application number: CN 201110001713
Authority: CN
Inventors: 姚永刚; 张阿梅
Original assignee: Kunming Institute of Zoology of CAS
Current assignee: Kunming Institute of Zoology of CAS
Priority date: 2011-01-06
Filing date: 2011-01-06
Publication date: 2013-01-16
Anticipated expiration: 2031-01-06
Also published as: CN102146443A

Abstract

The invention relates to a quick detection method for Leber hereditary optic neuropathy (LHON) mitochondrial DNA mutation G10680A, and belongs to the technical field of LHON research. Based on the principle of allele specific-polymerase chain reaction (AS-PCR), the first site of a mutation specific primer 3' terminal is designed into an A base L10680A, simultaneously a mismatched base (the base Ais mutated into a base T to form T/T mismatch) is introduced into the second site of the primer 3' terminal, and a pair of internal reference primers L4887/H5442 are designed in a 4866-5461 area of amitochondrial DNA sequence. A DNA sample to be detected is amplified in a PCR by using the two pairs of primers (L10680A/H10972 and L4887/H5442). The PCR amplification product is subjected to agarose electrophoresis detection, and the G10680A mutant is subjected to genotyping according to a mutation specific electrophoresis pattern so as to realize quick detection of mutant genes. The method is simple, quick, economic and sensitive. The method has significance for hereditary research and consultation of LHON.

Description

A kind of Auele Specific Primer that detects Leber hereditary optic neuropathy Mitochondrial DNA Mutation G10680A

Technical field:

The present invention relates to the Auele Specific Primer of a kind of detection Leber hereditary optic neuropathy Mitochondrial DNA (mitochondrial DNA, mtDNA) sudden change G10680A, belong to Leber hereditary optic neuropathy studying technological domain.

Background technology:

Leber hereditary optic neuropathy (Leber hereditary optic neuropathy, LHON; MIM 535000) be classical Mitochordrial Genetic Diseases, should disease be the matrilinear inheritance disease namely, mainly the point mutation by mtDNA causes morbidity.LHON has two large clinical spies: 1, sex preference, and namely male sex person between twenty and fifty are the crowds that easily send out; 2, incomplete penetrance phenomenon is namely carried the individuality of mtDNA primary mutation and is not necessarily fallen ill.Carry out for the research of LHON at present more, but for the rarer report of this sick epidemiology survey, the bright LHON people's train frequency of European crowd's questionnaire is 1/50000-1/25000, and asian population particularly China have no epidemiological investigation data.

Surpass one of three primary mutation G11778A, G3460A that 95% LHON patient carries mtDNA, T14484C, small number of patients can be carried two primary mutations simultaneously.Clinically to the diagnosis of LHON except according to typical clinical symptom and the familial inheritance history, final make a definite diagnosis the detection that also comprises primary mutation.Report shows, in Europe and China LHON crowd, the distribution frequency of three common primary mutations has larger difference, the frequency of G11778A in compatriots LHON patient is up to 90%, only about 1%, T14484C accounts for about 9% patient to the frequency of G3460A, and in European LHON patient, the frequency of these three sudden changes is about respectively 57%, 20% and 23%.Except the LHON patient who makes a definite diagnosis, also have a large amount of doubtful LHON patients clinically, these patients have more typical LHON clinical symptom, have or lack family's matrilinear inheritance history, do not carry three common primary mutations.We show recent research, in 1626 examples (doubtful) the LHON patient who collects, there are 843 examples not carry three common primary mutations, simultaneously we G3635A that also finds to suddenly change is China LHON crowd's a rare primary mutation, and this mtDNA mutation map that also further specifies China LHON patient and European LHON crowd there are differences.Study for these doubtful LHON cases, be expected to further enrich the mutation spectrum of compatriots LHON patient mtDNA, for genetic counseling and the clinical diagnosis and treatment of China LHON disease provides foundation.2009, the people such as Yang have reported a large family of China LHON of carrying primary mutation T14484C, this family shows complete penetrance, this prompting is except T14484C, certain other influence factor that exists causes all maternal patients' of family morbidity, find that after to propositus mtDNA complete sequence analysis this family is carried sudden change G10680A simultaneously.This sudden change is positioned at plastosome ND4L gene, has higher conservative property in vertebrates, therefore should sudden change probably act synergistically with T14484C, has caused this LHON family fully clinical outer aobvious.We detect the existence of G10680A after to 10 compatriots LHON family researchs without common primary mutation in a family.By evolution medical analysis method, we have got rid of the possibility that other mtDNA variant sites is caused a disease, simultaneously not retrieval from population 5000 many cases individualities in find the existence of this sudden change, so we G10680A that proposes to suddenly change is the paathogenic factor of this LHON family morbidity.The data that we do not deliver show that sudden change G10680A occupies certain ratio (being about 0.5%) in China LHON patient, and these research promptings G10680A is compatriots LHON patient's a rare pathogenic mutation.

Because therefore the present effective treatment measure that lacks LHON carries out genetic counseling by Molecular genetic test to this disease and clinical prevention has extremely important effect.Therefore, detect carry out the G10680A sudden change without the LHON patient of primary mutation G11778A, G3460A, T14484C and G3635A/family, have great importance for this sick genetic research, genetic counseling and prevention undoubtedly.

The technological method that a lot of detection dna mutations are arranged at present is such as sequencing, single-strand conformation polymorphism analysis (SSCP), sex change high performance liquid chromatography (DHPLC), and allele specific pcr.Wherein, allele specific pcr (Allele-specific PCR, AS-PCR) method is easy and simple to handle, quick, and required experiment equipment is simple, experiment reagent economy, so the method is widely used in the detection of dna mutation or single nucleotide polymorphism (SNP).The principle of allele specific pcr is that 3 ' terminal bases with a primer is designed to special mutating alkali yl, cooperate suitable reverse primer, thereby there is the DNA of sudden change to obtain the PCR product that suddenlys change special because of 3 ' terminal the amplification with the primer pairing, and can not match with the mutation specific primer without the dna profiling of sudden change, therefore can not increase obtains the PCR product.Agarose electrophoresis by routine detects having or not of PCR product, and then judges whether detect sample DNA exists sudden change.

By literature search, have no with the present invention and detect the identical open report in mutational site.

Summary of the invention:

The object of the present invention is to provide the Auele Specific Primer of a kind of detection Leber hereditary optic neuropathy Mitochondrial DNA (mitochondrial DNA, mtDNA) sudden change G10680A.

The present invention is based on the principle of AS-PCR, the primer 3 that sudden change is special ' first at end is designed to the A base and (sees Table 1, L10680A), be used for the amplification mutant DNA; Introduce a base mismatch (base A is mutated into base T, forms the T/T mispairing) at primer 3 ' end second, strengthen primer to the specificity of mutant DNA template amplification; On the mtDNA sequence, away from the zone of G10680A sudden change, namely comprise the 4866-5461 scope, designed a pair of confidential reference items primer (see Table 1, L4887/H5442), be used for monitoring pcr amplification reaction system whether suitable and reaction whether normally increase.Adopt above-mentioned two pairs of primers (L10680A/H10972 and L4887/H5442), in a PCR reaction, treat survey specimen dna sample and increase.Whether the specimen dna that no matter detects contains the G10680A sudden change, and confidential reference items primer L4887/H5442 can amplify the PCR product, so the false-negative detected result of sudden change G10680A with regard to having avoided causing owing to the PCR failure.Pcr amplification product detects through agarose electrophoresis, according to the special electrophoretogram of sudden change G10680A is suddenlyd change and carries out gene type, realizes that the fast and easy of transgenation detects.

Because Mitochondrial DNA Mutation has heterogeneity, i.e. sudden change and phenomenon normal mtDNA coexistence if the insufficient sensitivity of detection method is high, then can not detects sudden change, thereby cause false negative result in the patient that the sudden change heterogeneity is arranged.The present invention carries out AS-PCR by the normal people is mixed by different ratios with the DNA sample that contains G10680A sudden change patient, detecting the sensitivity of present technique method, and provides the technical parameter of the mutant DNA of the minimum level that present technique detects.

The present invention utilizes allele specific pcr (Allele-specific PCR, AS-PCR) method to detect Leber hereditary optic neuropathy Mitochondrial DNA Mutation G10680A, and concrete steps are as follows:

(1) DNA extraction: use conventional phenol/chloroform method or test kit method from clinical sample, to extract genomic dna.

(2) genomic dna that extracts take step (1) carries out the PCR reaction as template.

PCR reacts primer: amplimer sequence and product size see Table 1

Table 1 AS-PCR the primer composition, sequence and product size

PCR reaction system: totally be 20 μ L, comprise the 30ng genomic dna, 10mM Tris-HCl (pH 8.3), 1.5mM MgCl ₂, 50mM KCl, 0.5U TaKaRa rTaq, 175 μ M dNTP, each 0.3 μ M of sudden change special primer L10680A and H10972 and confidential reference items primer L4887 and H5442.

The PCR response procedures: 94 ℃, denaturation 3min; 94 ℃ of sex change 20s, 61 ℃ of annealing 20s, 72 ℃ are extended 30s, repeat 30 circulations; 72 ℃ are extended 5min.

(3) the PCR product detects: get PCR product 3 μ L electrophoresis on 1.5% sepharose, electrophoretic buffer is 1 * TAE, 150V constant voltage electrophoresis 15min, and Ethidum Eremide dyeing 5min observes under the gel imaging system and takes pictures.

(4) sensitivity detects:

Can inspection to the concentration of mutant DNA of minimum level: take the DNA that contains the G10680A sudden change of 1ng, 2.5ng, 5ng, 10ng and 15ng as template, use the amplification of the PCR reaction system identical with (3) with step (2) and program and detection.

Can inspection to the sudden change of minimum level heterogeneous: with normal people's dna sample with contain the situation that patient's dna sample that G10680A suddenlys change is 30ng in total amount under mix by preset proportion, make the dna sample ratio of sudden change be respectively 5%, 10% and 20%, the dna profiling that mixes is increased and detection according to the PCR reaction system identical with (3) with step (2) and program.

The present invention compares with other prior art, has quick, simple, economic, accurate, sensitive characteristics, and plant and instrument is less demanding, is suitable for applying in large-scale clinical sample detects.The present invention has great importance for genetic research and the consulting of Leber hereditary optic neuropathy.

Description of drawings:

Fig. 1 is for carrying out the electrophoretogram that allele specific pcr detects LHON mitochondrial mutations G10680A with the present invention.

Fig. 2 is that sensitivity of the present invention detects electrophoretogram.

Specific embodiments:

1, design of primers

Suddenly change according to mtDNA G10680A, the primer 3 that sudden change is special ' first at end is designed to the A base and (sees Table 1, L10680A), be used for the amplification mutant DNA, introduce a base mismatch (base A is mutated into base T, forms the T/T mispairing) at primer 3 ' end second simultaneously, to strengthen primer for the specificity in mutational site, design another primer (table 1, H10972), and the mutation specific primer amplifies the special fragment of sudden change of a 312bp together in the downstream.Simultaneously, at mtDNA 4866-5461 section design a pair of confidential reference items primer (see Table 1, L4887/H5442), no matter have or not G10680A sudden change, the confidential reference items primer can both increase and obtain the fragment of a 596bp.Primer is given birth to worker's biotechnology company limited by Shanghai and is synthesized.Primer sequence and product size see Table 1.

2, sample collection

Gather 3 examples confirm to carry the G10680A sudden change through determined dna sequence dna sample and 3 routine normal people's whole blood samples.

3, sample gene group DNA extraction

Use phenol/chloroform method to extract genomic dna in the whole blood sample.

4, pcr amplification

5, the PCR product detects

The PCR product detects: get PCR product 3 μ L electrophoresis on 1.5% sepharose, electrophoretic buffer is 1 * TAE, 150V constant voltage electrophoresis 15min, and Ethidum Eremide dyeing 5min observes under the gel imaging system and takes pictures.

6, sensitivity detects

Can inspection to the mutant DNA concentration of minimum level: take the DNA that contains the G10680A sudden change of 1ng, 2.5ng, 5ng, 10ng and 15ng as template, use the amplification of the PCR reaction system identical with

step

4 and 5 and program and detect;

Can inspection to the sudden change of minimum level heterogeneous: be to mix by a certain percentage in the situation of 30ng template DNA in the maintenance total amount with normal people's dna sample and the patient's dna sample that contains the G10680A sudden change, make the dna sample ratio of sudden change be respectively 5%, 10% and 20%, with the dna profiling that mixes the PCR reaction system identical with

step

4 and 5 and program amplification and detection.

Carry out the electrophoresis result (seeing Fig. 1) that allele specific pcr detects LHON mitochondrial mutations G10680A with the inventive method.Wherein swimming lane 1 is not for adding the negative control that dna profiling carries out pcr amplification, swimming lane 2-4 is the normal people's dna sample amplification without sudden change G10680A, swimming lane 5-7 is LHON patient's dna sample amplification that the G10680A sudden change is arranged, and swimming lane M is the nucleic acid molecular weight standard of 2000bp.The band of 596bp is the confidential reference items band among the figure, and except negative control, all detection samples have this band, show that PCR system and condition are suitable, increase successfully; The band of 312bp just can amplify this band for sudden change specific band, the sample that only has the G10680A sudden change to exist.

The sensitivity detected result (seeing Fig. 2) of the inventive method.Wherein swimming lane 1 is not for adding the negative control that dna profiling carries out pcr amplification; Swimming lane 2-6 is respectively take 1ng, 2.5ng, 5ng, 10ng and 15ng and contains the result that the DNA of G10680A sudden change increases as template; Swimming lane 7-9 is to mix in the situation of 30ng template DNA for sudden change in total amount with normal DNA, makes the DNA ratio of sudden change be respectively 5%, 10% and 20%, the result who increases again; Swimming lane 10 is for containing LHON patient's dna sample amplification of G10680A sudden change; Swimming lane M is the nucleic acid molecular weight standard of 2000bp.As shown in the figure, minimum to adopt the DNA of 5ng be that template is carried out effective augmentation detection to experiment condition provided by the invention.Be in the situation of 30ng in the dna profiling total amount, the present invention can detect the heterogeneous level that is low to moderate 10% G10680A sudden change.

Claims

1. Auele Specific Primer for detection of Leber hereditary optic neuropathy Mitochondrial DNA Mutation G10680A, it is characterized in that this primer sequence sees the following form, wherein L10680A and H10972 are for detecting the Auele Specific Primer of Mitochondrial DNA Mutation G10680A, and L4887 and H5442 are the confidential reference items primer:

。