CN102146443A - Quick detection method for Leber hereditary optic neuropathy mitochondrial DNA mutation G10680A - Google Patents
Quick detection method for Leber hereditary optic neuropathy mitochondrial DNA mutation G10680A Download PDFInfo
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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 A is 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 a mitochondrial 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
Technical field:
(mitochondrial DNA, the mtDNA) method for quick of sudden change G10680A belongs to Leber hereditary optic neuropathy studying technological domain to the present invention relates to a kind of Leber hereditary optic neuropathy Mitochondrial DNA.
Background technology:
Leber hereditary optic neuropathy (Leber hereditary optic neuropathy, LHON; MIM 535000) be classical mitochondrial inheritance disease, should disease be the matrilinear inheritance disease promptly, mainly the point mutation by mtDNA causes morbidity.LHON has two big clinical spies: 1, sex preference, and promptly male sex person between twenty and fifty are the crowds that easily send out; 2, incomplete penetrance phenomenon is promptly carried the individuality of former sudden change of mtDNA and is not necessarily fallen ill.Carry out at present for the research of LHON 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 do not see epidemiological investigation data.
LHON patient above 95% carries three former suddenly change one of G11778A, G3460A, T14484C of mtDNA, and small number of patients can be carried two former sudden changes simultaneously.Clinically to the diagnosis of LHON except that according to typical clinical symptom and the familial inheritance history, final make a definite diagnosis the detection that also comprises former sudden change.Report shows, in Europe and China LHON crowd, the distribution frequency of three common former sudden changes has than big-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 57%, 20% and 23% respectively.Except that the LHON patient who makes a definite diagnosis, also have a large amount of doubtful LHON patients clinically, these patients have typical LHON clinical symptom, have or lack family's matrilinear inheritance history, do not carry three common former sudden changes.We show recent research, in 1626 examples (doubtful) the LHON patient who collects, there are 843 examples not carry three former common sudden changes, simultaneously we G3635A that also finds to suddenly change is China LHON crowd's a rare former sudden change, and this mtDNA mutation map that also further specifies China LHON patient and European LHON crowd there are differences.Study at these doubtful LHON cases, be expected to further enrich the mutation spectrum of compatriots LHON patient mtDNA, for the genetic counseling and the clinical diagnosis and treatment of China LHON disease provides foundation.2009, people such as Yang have reported a big family of China LHON of carrying former mutation T 14484C, this family shows complete penetrance, this prompting is except that 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 is somebody's turn to do sudden change and acts synergistically with T14484C probably, has caused this LHON family clinical fully outer apparent.We detect the existence of G10680A in a family after the compatriots LHON family research to 10 no common former sudden changes.By evolution medical analysis method, we have got rid of the morbific possibility of other mtDNA variant sites, simultaneously not retrieval from general crowd's 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 the molecular genetics detection to this disease and clinical prevention has important role.Therefore, LHON patient/family of not having former sudden change G11778A, G3460A, T14484C and G3635A is carried out the G10680A sudden change detect, 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 as sequencing, single-strand conformation polymorphism analysis (SSCP), sex change high performance liquid chromatography (DHPLC), and allele specific pcr.Wherein, and 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 this 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 the dna profiling that does not have sudden change can not match with the sudden change Auele Specific Primer, and 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, do not see 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 method for quick of a kind of quick, simple and easy, economic Leber hereditary optic neuropathy Mitochondrial DNA Mutation G10680A.
The present invention is based on the principle of AS-PCR, special primer 3 ' first at the end of sudden change is designed to the A base (sees Table 1, L10680A), be used to the mutant DNA that increases; Introduce a base mismatch (base A is mutated into base T, forms the T/T mispairing) at primer 3 ' second at end, strengthen the specificity of primer the mutant DNA template amplification; On the mtDNA sequence,, promptly comprise the 4866-5461 scope, designed a pair of confidential reference items primer and (seen Table 1, L4887/H5442), be used to monitor the whether suitable and reaction of pcr amplification reaction system and whether normally increase away from the zone of G10680A sudden change.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 the rapid detection that makes things convenient for of transgenation.
Because Mitochondrial DNA Mutation has heterogeneity, i.e. sudden change and phenomenon normal mtDNA coexistence are if the insufficient sensitivity height of detection method then can not detect sudden change, thereby cause false negative result in the heterogeneous patient of sudden change 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 detected minimum level of present technique.
The present invention utilize allele specific pcr (Allele-specific PCR, AS-PCR) method detects Leber hereditary optic neuropathy Mitochondrial DNA Mutation G10680A, 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 with step (1) is a template, carries out the PCR reaction.
PCR reacts primer: amplimer sequence and product size see Table 1
Table 1AS-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 ℃, pre-sex change 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 bromination second pyridine dyeing 5min, gel imaging system observe down and take pictures.
(4) sensitivity detects:
The concentration of the mutant DNA of the minimum level that can examine: the DNA that contains the G10680A sudden change with 1ng, 2.5ng, 5ng, 10ng and 15ng is a template, uses amplification of PCR reaction system identical with (3) with step (2) and program and detection.
The sudden change heterogeneity of the minimum level that can examine: be to mix by preset proportion under the situation of 30ng normal people's dna sample and patient's dna sample of containing G10680A sudden change in total amount, 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 PCR reaction system identical with (3) with step (2) and program.
The present invention compares with other prior art, has quick, simple, economical, accurate, sensitive characteristics, and plant and instrument is less demanding, is suitable for applying in large-scale clinical sample detection.The present invention has great importance for the 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 inventive method.
Fig. 2 is that the sensitivity of the inventive method detects electrophoretogram.
Specific embodiments:
1, design of primers
Suddenly change according to mtDNA G10680A, special primer 3 ' first at the end of sudden change is designed to the A base (sees Table 1, L10680A), be used to the mutant DNA that increases, introduce a base mismatch (base A is mutated into base T, forms the T/T mispairing) at primer 3 ' second at end simultaneously, to strengthen the specificity of primer for the mutational site, design in the downstream another primer (table 1, H10972) and the sudden change Auele Specific Primer amplify the special fragment of sudden change of a 312bp together.Simultaneously, (see Table 1, L4887/H5442), no matter have or not the G10680A sudden change, the confidential reference items primer can both increase and obtain the fragment of a 596bp to have designed a pair of confidential reference items primer at mtDNA 4866-5461 section.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
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 ℃, pre-sex change 3min; 94 ℃ of sex change 20s, 61 ℃ of annealing 20s, 72 ℃ are extended 30s, repeat 30 circulations; 72 ℃ are extended 5min.
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 bromination second pyridine dyeing 5min, gel imaging system observe down and take pictures.
6, sensitivity detects
The mutant DNA concentration of the minimum level that can examine: the DNA that contains the G10680A sudden change with 1ng, 2.5ng, 5ng, 10ng and 15ng is a template, uses amplification of PCR reaction system identical with step 4 and 5 and program and detection;
The sudden change heterogeneity of the minimum level that can examine: is to mix by a certain percentage under the situation of 30ng template DNA normal people's dna sample and patient's dna sample of containing G10680A sudden change keeping total amount, 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 and program amplification and detection with step 4 and 5.
Carry out the electrophoresis result (see figure 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 not for there being normal people's dna sample amplification of 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 all 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 has only the G10680A sudden change to exist.
The sensitivity detected result (see figure 2) of the inventive method.Wherein swimming lane 1 is not for adding the negative control that dna profiling carries out pcr amplification; It is the result that template increases that swimming lane 2-6 is respectively the DNA that contains G10680A sudden change with 1ng, 2.5ng, 5ng, 10ng and 15ng; Swimming lane 7-9 is to mix under 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.In the dna profiling total amount is under the situation of 30ng, and the present invention can detect the heterogeneous level that is low to moderate 10% G10680A sudden change.
Claims (1)
1. the method for quick of a Leber hereditary optic neuropathy Mitochondrial DNA Mutation G10680A comprises extracting genome DNA, design of primers, pcr amplification, electrophoresis detection step, it is characterized in that the concrete steps of this method for quick are as follows:
(1) use phenol/chloroform method or test kit extraction method from clinical sample, to extract genomic dna;
(2) genomic dna that extracts with step (1) is a template, carries out the PCR reaction, and PCR reaction primer is:
The PCR reaction system: totally be 20 μ L, comprise the 30ng genomic dna, pH is 8.3 10mMTris-HCl, 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 L4884 and H5442;
The PCR response procedures: 94 ℃, pre-sex change 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 bromination second pyridine dyeing 5min, gel imaging system observe down and take pictures;
(4) sensitivity detects:
The concentration of the mutant DNA of the minimum level that can examine: the DNA that contains the G10680A sudden change with 1ng, 2.5ng, 5ng, 10ng and 15ng is a template, uses amplification of PCR reaction system identical with (3) with step (2) and program and detection;
The sudden change heterogeneity of the minimum level that can examine: be to mix by preset proportion under the situation of 30ng normal people's dna sample and patient's dna sample of containing G10680A sudden change in total amount, 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 PCR reaction system identical with (3) with step (2) and program.
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| CN114250296A (en) * | 2022-02-09 | 2022-03-29 | 深圳市妇幼保健院 | Primer group for detecting Leber hereditary optic neuropathy based on nucleic acid mass spectrum |
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