CN1490415A - Testing method for matrilinear hereditary deaf mitochondria gene 1555 place A-G catastrophe and reagent boxes - Google Patents
Testing method for matrilinear hereditary deaf mitochondria gene 1555 place A-G catastrophe and reagent boxes Download PDFInfo
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Abstract
A process for detecting the A-G mutation of the metroclinal deafness mitochondrion gene at site No.1555 includes such steps as using Genetool Lite program to helf the design of improved primer, PCR reacting to introduce new restriction site HaeIII, and relative enzyme severing reacting to detect the A-G mutation. Its relative reagent kit is also disclosed. Its advantages are simple process, low cost and direct and correct result.
Description
Technical field
The present invention relates to gene engineering technology field, specifically relate to the detection method of 1555 A of matrilineal inherited deafness chondriogen → G sudden change.The invention still further relates to the test kit of preparation according to 1555 A of the vitro detection matrilineal inherited deafness chondriogen → G sudden change of this method, and the application of this test kit in detecting said A → G sudden change.
Background technology
Aminoglycosides antibiotics (Streptomycin sulphate, gentamicin, kantlex, tobramycin and micronomicin etc.) is widely used in controlling Gram-negative and positive bacteria infection clinically because of the anti-microbial effect and the cheap price of its broad-spectrum high efficacy, but this type of microbiotic has serious ototoxicity side effect, can make the patient that irreversible hearing loss takes place.Discover over past ten years, part aminoglycoside antibiotics-induced patient has the matrilinear inheritance family history, and (be designated hereinafter simply as chondriogen with Mitochondrial DNA 12SrRNA gene, or mtDNA) the 1555th relevant (PrezantTR etc. of A → G uniformity point mutation, Nat Genet, 1993,4:289-294; Yuan Huijun etc., Chinese hals,Nasen und Ohrenheilkunde magazine, 1998,33:67-70).The aminoglycosides medicinal application of single dose can cause carrying the severe hearing loss of this mutated individual.According to the present result of study that oneself has as can be known, serious or more serious hearing loss all takes place in the individuality that almost carries this sudden change behind the aminoglycosides antibiotics of using various dose, is the acquired deaf major reason that takes place of acquired character.The gene clear and definite in view of having of this deafness changes and risk factor, makes this deafness become a kind of disease that can prevent.
(mtDNA1555A → G) sudden change matrilineal inherited deafness family is own above 50 at the chondriogen 1555A → G of oneself report of China, each family number of the infected does not wait from 2-50 people, consider the still undiscovered report of a lot of similar familys is still arranged, and this sudden change occupies certain ratio in the mechanism of causing a disease of sporadic deafness, and the prevention of this deafness is become even more important.The key link of its prevention is by examination, detects the individuality of finding to carry mtDNA1555A → G sudden change, this type of individual contact aminoglycosides antibiotics of absolute prohibition, thus avoid serious ototoxicity reaction to take place.
From 1993 with fork-like farm tool used in ancient China, except direct order-checking, the most frequently used screening method is BsmA1 (Alw261) enzyme cutting method (Fische1-Ghodsian etc., AmJOtolaryngol, 1993,14:399-403 to the detection of this sudden change; Fischel-Ghodsian etc., AmJOtolaryngol, 1997b, 18:173-178; Yuan Huijun etc., Chinese hals,Nasen und Ohrenheilkunde magazine, 1998,33:67-70).At home and abroad, widespread usage Restriction Enzyme BsmAI (Alw26I) enzyme is cut and is cooperated the sequential analysis evaluation to have or not mtDNA1555A → G sudden change, it is convenient and swift that enzyme is cut evaluation, reliable results, but insufficient be that BsmAI (Alw26I) price is more expensive, compare with Restriction Enzyme commonly used, price variance reaches 50-100 doubly, therefore, press for a kind of simple, quick, accurate and low detection method of cost, to satisfy needs to mtDNA1555A → extensive examination is carried out in G sudden change.
Summary of the invention
The objective of the invention is the existing shortcoming of method, the method that provides a kind of detection matrilinear inheritance induced deafness mtDNA1555A → G simple, quick, that cost is low to suddenly change at present detection mtDNA1555A → G sudden change; Another object of the present invention provides the test kit that detects matrilinear inheritance induced deafness mtDNA1555A → G sudden change; A further object of the invention provides the application of test kit of the present invention in prevention matrilinear inheritance induced deafness.
Purpose of the present invention realizes by following scheme:
Use GenetoolLite program (software that U.S. Biotools company provides) to assist the design primer, pass through pcr amplification, on the pcr amplified fragment of tested sample, produce rite-directed mutagenesis, thereby new restriction enzyme site that comprises 1555 of 1555 contiguous introducings at this gene, with corresponding Restriction Enzyme digestion, detect the existence of this sudden change then.In the wherein designed primer, forward primer is the Nucleotide 1463-1482 of Mitochondrial DNA, and its sequence is the SEQIDNO:1 in the sequence table; Reverse primer is the Nucleotide 1575-1556 of Mitochondrial DNA, and wherein 1557 A is replaced into C, and its sequence is the SEQIDNO:2 in the sequence table; The new restriction enzyme sites of introducing is HaeIII, and its recognition sequence is positioned at the Nucleotide 1554-1557 of Mitochondrial DNA.The inventor also utilizes the primer of method of the present invention and design to be provided for the test kit of 1555 A of vitro detection matrilineal inherited deafness chondriogen → G sudden change, be used for 1555 A of vitro detection matrilineal inherited deafness chondriogen → G sudden change, this test kit contains:
1. extract the reagent of blood sample DNA;
2.PCR amplification reaction reagent comprises dNTP, 10XPCR damping fluid, Mg++, tri-distilled water, Taq enzyme;
3. described forward primer of equal proportion blended claim 2 and reverse primer;
4. Restriction Enzyme HaeIII and supporting damping fluid thereof;
5. positive sample contrast, the contrast of negative sample, and
6. working instructions.
In embodiments of the invention, the guiding theory of design of primers is that 2-4 the base contiguous in 1555 sites introduced a base replacement, causes the possible new restriction enzyme site that can differentiate mtDNA1555 wild-type (A) and mutant (G).Use GenetoolLite programmatic assistance design primer, principle of design is the sequence of the chondriogen known according to oneself, make 3 ' end of forward primer be positioned at 1554, reverse primer is at the about 120bp in 1555 downstream place, or the 3 ' end that makes reverse primer is positioned at 1556, forward primer is at the about 120bp in 1555 upstream place, thereby makes the product behind the pcr amplification comprise 1555 Nucleotide (with reference to Fig. 1); Introduce possible base substitution at 1552,1553 and 1557,1558 respectively simultaneously, make in the new restriction enzyme recognition sequence of formation to comprise 1555 Nucleotide, go out to differentiate the new Restriction Enzyme site of chondriogen 1555 wild-types (A) and 1555 mutants (G) then with the enetoolLite program search.
Use the GenetoolLite program all possible base replacement is carried out in 1553,1552 sites respectively, find to differentiate the newly-increased restriction enzyme site of 1555A → G mutant and wild-type; 1557,1558 sites are carried out all possible base respectively when replacing, when the GenetoolLite program finds that the A with 1557 sites replaces with C, in mtDNA1555A → G mutant molecules, cause the recognition sequence (GGCC) of Restriction Enzyme HaeIII, its point of contact and does not have this sequence (its corresponding sequence is GACC) in the mtDNA1555 wild type molecule between 1555 and 1556.Designing reverse primer R1 thus is nt1575-nt1556, and wherein 3 ' second base of end (1557) is the different base (T-G) of joining; Forward primer F1 is nt1463-1482.The long 113bp of F1R1 amplicon.
With the primers F 1R1 of above design, be template with the DNA of tested sample, by obtaining the 100bp band that is slightly larger than limpid in sight behind the pcr amplification, prove reverse primer second base of 3 ' end and the different efficient that does not influence PCR of joining of template.The F1R1PCR product comprises the mtDNA1555 site, if template is 1555A → G mutant molecules, then there is the HaeOII restriction enzyme site in the PCR product of 113bp, and the HaeOII enzyme is cut the back and obtained 93bp and two bands of 20bp; If template is the mtDNA wild type molecule, then there is not the HaeOII restriction enzyme site in the PCR product of 113bp, can not be cut by HaeOII, is still band of 113bp after enzyme is cut.Utilize this difference, can carry out simply 1555A → G mutant molecules, measure fast.
The primer sequence that the present inventor designs in also according to the present invention obtains primer by synthetic (synthetic by the automatic nucleic acid synthesizer by given sequence), and with the reagent that extracts blood sample DNA; Pcr amplification reaction reagent comprises dNTP, 10 * PCR damping fluid, Mg++, tri-distilled water, Taq enzyme; Restriction Enzyme HaeOII and supporting damping fluid thereof; Positive sample contrast, the contrast of negative sample, and working instructions combine, the test kit that is used for 1555 A of vitro detection matrilineal inherited deafness chondriogen → G sudden change is provided, its major function is all reagent is integrated in the capsule, and making DNA extraction, nucleic acid fragment amplification, enzyme cut evaluation can order finish on the reagent basis that test kit provides.According to the difference of getting blood mode or tested sample form, test kit of the present invention have three types can be for selecting for use, except the reagent that extracts blood sample DNA, all the other components are all identical in three types.The reagent of the extraction blood sample DNA of one type test kit is the solution 1 as dna cleavage liquid, and its main component is Chelex (an ABI company product), is applicable to Heel blood blood sheet tested sample; The reagent of the extraction blood sample DNA of two type test kits is peripheral blood DNA test kit (selecting the supporting use in commercially available prod for use), is applicable to the peripheral blood tested sample; Three type test kits do not contain the reagent that extracts blood sample DNA, are applicable to the tested sample that DNA directly is provided.
Detecting 1555 A of matrilineal inherited deafness chondriogen → G sudden change with method of the present invention cuts authentication method with prior art BsmAI enzyme and has compared following advantage:
1. it is basic identical that the operation steps of HaeIII enzyme cutting method of the present invention and required time and BsmAI enzyme are cut authentication method, but the BsmAI enzyme require is from foreign procurement, supply untimely, and cost an arm and a leg, and HaeOII Restriction Enzyme domestic supply source is abundant, cost is extremely low, its price only is the 1/50-1/100 of BsmAI, these characteristics are used the condition that preceding preventative detection is provided convenience for examination and the microbiotic of carrying out mtDNA1555A → G sudden change in China, thereby fundamentally reduce the chance to the individuality generation drug induced deafness of aminoglycosides antibiotics sensitivity.
2. method of the present invention is cut mutant molecules with the HaeOII enzyme, and A → G mutant molecules can be cut by HaeIII, and enzyme is cut the existence of positive direct representation mtDNA1555A → G sudden change; And BsmAI cuts is wild type molecule, and A → G mutant molecules can not be cut by BsmAI, in this case, tested sample except A → G sudden change may, do not get rid of the possibility of other sudden changes, therefore, method of the present invention is more direct, reliable.
Description of drawings
Fig. 1 is a design of primers scheme synoptic diagram of the present invention.
A: 3 ' end of forward primer is positioned at 1554, and reverse primer is at the about 120bp in 1555 downstream place; B: 3 ' end of reverse primer is positioned at 1556, and forward primer is at the about 120bp in 1555 upstream place; Among Fig. 1,1 expression mtDNA; 2 expression forward primers; 3 expression reverse primers; O represents nucleotide position.
Fig. 2 is the family tree of matrilineal inherited deafness family 13.represents male individual among the figure; O represents female individual; Black icon representative morbidity is individual;
Represent the propositus; The individuality that/representative is dead.
Fig. 3 is the 2% agarose gel electrophoresis figure that the HaeOII enzyme cutting method is identified mtDNA1555A → G sudden change.
Swimming lane 1-3, the 5-7:mtDNA1555A-G patient that suddenlys change; Swimming lane 4,8: normal individual; Swimming lane 9: molecular weight sign (100bp at interval).
Embodiment
The detection of embodiment one matrilineal inherited deafness family plastosome base 1555A → G sudden change
1. detection sample
Selection is 14 of the deaf familys of transfer characteristic with the matrilinear inheritance, total number of persons is 301 people, deafness patient totally 84 people wherein, among the person under inspection, deafness patient is 33 people, with irrelevant individual 11 people of normal good hearing of deaf family, from the tested individual periphery whole blood DNA (Yuan Huijun etc. that extract, China's hals,Nasen und Ohrenheilkunde magazine, 1998,33 (2): 67-70; Li Weimin etc., clinical hals,Nasen und Ohrenheilkunde magazine, 2001,15 (supplementary issues): 53-58), as detecting sample.14 family comprehensive conditions see Table 1, and wherein the family tree of family 13 is seen Fig. 2.The disease transfer mode of all the other familys is all similar with family 13; each family all has the patient to use aminoglycosides antibiotics; characteristics are female patient AmAn all fell ill early, deaf degree is heavy if use, if do not use the AmAn normal or deaf morbidity of hearing evening then, degree is light.
2. design of primers
Use GenetoolLite programmatic assistance design improved primer, according to oneself disclosed chondriogen Cambridge sequence (CambridgeSequence), the primer design scheme has 2 kinds:
(1) make 3 ' end of forward primer be positioned at 1554, reverse primer at the about 120bp in 1555 downstream place (with reference to Fig. 1, A);
(2) make 3 ' end of reverse primer be positioned at 1556, forward primer (with reference to Fig. 1, B), thereby makes the product behind the pcr amplification comprise 1555 Nucleotide at the about 120bp in 1555 upstream place.Introduce possible base substitution at 1552,1553 and 1557,1558 respectively simultaneously, make in the new restriction enzyme recognition sequence of formation to comprise 1555 Nucleotide, go out to differentiate the new restriction enzyme sites of chondriogen 1555 wild-types (A) and 1555 mutants (G) then with the GenetoolLite program search.Use the GenetoolLite program all possible base replacement is carried out in 1553,1552 sites respectively, find to differentiate the newly-increased restriction enzyme site of 1555A → G mutant and wild-type; All possible base is carried out in 1557,1558 sites respectively replaces, when the GenetoolLite program finds that the A with 1557 sites replaces with C, in mtDNA1555A → G mutant molecules, cause the recognition sequence (GGCC) of Restriction Enzyme HaeOII, its point of contact and does not have this sequence (its corresponding sequence is GACC) in the mtDNA1555 wild type molecule between 1555 and 1556.Designing reverse primer R1 thus is mtDNAnt1575-nt1556, promptly 5 '-ACTTACCATGTTACGACTGG-3 ', wherein 3 ' second base of end (1557) is the different base (T-G) of joining, this sequence is set at SEQIDNO:2; In addition, design forward primer F1 is mtDNAnt1463-1482, promptly 5 '-GGCCCTGAAGCGCGTACACA-3 ', this sequence is set at SEQIDNO:1, the long 113bp of F1R1 amplicon.
3.PCR amplified reaction
Primer sequence SEQIDNO:1 and SEQIDNO:2 according to above-mentioned design, obtain forward primer F1 and reverse primer R1 by synthetic (synthetic by the automatic nucleic acid synthesizer) by given sequence, and be template with the tested sample peripheral blood DNA of said extracted, carry out pcr amplification reaction:
Reaction system:
Template 50ng
10×dNTP????????5μl
10×Buffer??????5μl
Mg
++Final concentration 1.5mmol/L
Taq enzyme 1.0u
Add tri-distilled water to 50 μ l volume.
Reaction conditions:
95 sex change were followed 94 ℃ of sex change 30 seconds after 5 minutes, annealed 30 seconds for 50 ℃, and 72 ℃ were extended 30 seconds, carried out 30 circulations altogether, extended under 72 ℃ 7 minutes at last again.
Check product with 2% agarose gel electrophoresis.F1R1 can obtain the 100bp band that is slightly larger than limpid in sight behind the pcr amplification, proof reverse primer second base of 3 ' end and the different efficient that does not influence PCR of joining of template, the F1R1PCR product comprises the mtDNA1555 site, if template is 1555A → G mutant molecules, then there is the HaeIII restriction enzyme site in the PCR product of 113bp, and the HaeIII enzyme is cut the back and obtained 93bp and two bands of 20bp; If template is the mtDNA wild type molecule, then there is not the HaeIII restriction enzyme site in the PCR product of 113bp, can not be cut by HaeIII, is still band of 113bp after enzyme is cut.
4.HaeIII enzyme is cut detection
The pcr amplification product of tested sample detects 1555A → G sudden change by following condition.The endonuclease reaction system:
PCR product 12-18 μ l
10x damping fluid 3 μ l
HaeIII Restriction Enzyme 5-10u
BSA???????????????0.3μl
Tri-distilled water is supplied volume to 30 μ l, 35 ℃ of incubations 1 hour.The endonuclease reaction thing is with 2% sepharose inspection.
5. result
As shown in table 1, in 14 familys altogether, the F1R1 amplified production (113bp) of 33 deafness patients of 13 familys can be cut into 93bp and 20bp two bands by HaeIII, shows that there is mtDNA1555A-G sudden change (referring to swimming lane 1-3, the 5-7 of Fig. 2) in it.The F1R1 amplified production of a deafness patient in another family can not be cut by HaeIII, show and do not have mtDNA1555A-G sudden change (family 3 in the table 1) in this family, the F1R1 amplified production of individuality and normal control individuality all can not be cut (referring to the swimming lane 4,8 of Fig. 2) by HaeIII in 11 familys with patient's consanguinity-less relation, shows that there is not mtDNA1555A → G sudden change in it.
6.HaeIII the check of enzyme cutting method reliability
1555 sites of all detected individualities are all passed through BamAI (Alw261) enzyme and are cut check (Yuan Huijun etc., Chinese hals,Nasen und Ohrenheilkunde magazine, 1998,33 (2): 67-70; Li Weimin etc., clinical hals,Nasen und Ohrenheilkunde magazine, 2001,15 (supplementary issues): 53-58), as shown in table 1, its as a result identity basis HaeIII enzyme cut and show that it carries 33 deafness patients of 1555A → G sudden change, its amplified production that contains 1555 sites all can not be cut by BamAI; And the HaeIII enzyme is cut and is shown 11 normal individuals that do not carry 1555A → G sudden change, and its amplified production that contains 1555 sites all can be cut by BamAI.Family 3 also shows as the deaf family of matrilinear inheritance aminoglycosides sensitivity, and a women person under inspection HaeOII enzyme of this family is cut and shown that it does not carry the 1555A-G sudden change, further detects this patient and two younger sister through BamAI (Alw26I) enzyme cutting method
Table 1: matrilinear inheritance family situation and plastosome 1555 site detected results
| The family numbering | Algebraically | Total number of persons | Deafness patient | The disease transfer mode | ???AmAn 1Medication history | Examined the deafness patient number | 1555A → G detection that suddenlys change | ||
| Improvement primer HaeIII detects | Alw26 detects | Order-checking | |||||||
| ????1 ????2 ????3 ????4 ????5 ????6 ????7 ????8 ????9 ????10 ????11 ????12 ????13 ????14 | ????3 ????3 ????3 ????3 ????3 ????4 ????4 ????3 ????3 ????3 ????3 ????2 ????4 ????4 | ????26 ????18 ????17 ????22 ????15 ????11 ????24 ????14 ????15 ????8 ????14 ????3 ????25 ????89 | ????6 ????6 ????7 ????4 ????4 ????6 ????8 ????5 ????4 ????5 ????6 ????2 ????8 ????14 | The maternal vertical transmission of the maternal vertical transmission of the maternal vertical transmission of the maternal vertical transmission of the maternal vertical transmission of the maternal vertical transmission of the maternal vertical transmission of the maternal vertical transmission of the maternal vertical transmission of the maternal vertical transmission of the maternal vertical transmission of the maternal vertical transmission of the maternal vertical transmission of maternal vertical transmission | Have | ????2 ????3 ????1 ????4 ????2 ????2 ????3 ????1 ????1 ????1 ????3 ????2 ????4 ????4 | ????+ ????+ ????- ????+ ????+ ????+ ????+ ????+ ????+ ????+ ????+ ????+ ????+ ????+ | ????+ ????+ ????- ????+ ????+ ????+ ????+ ????+ ????+ ????+ ????+ ????+ ????+ ????+ | ????+ ????+ ????- ????+ ????+ ????+ ????+ ????+ ????+ ????+ ????+ ????+ ????+ ????+ |
1AmAn: the aminoglycosides antibiotics younger sister, and their three children show that all no 1555A-G suddenlys change.The identical rate of two kinds of enzyme blanking methods is 100%, and the reliability and stability of using the inventive method to detect 1555A → G sudden change are described.
In addition, each family all choose at least 1 routine patient through enzyme cut detect show have 1555A → the DNA sample of G sudden change carries out the direct order-checking inspection in 1555 sites, the result is except that family 3, sequencing result shows that all the replacement of A → G takes place in the mtDNA1555 site, and is in full accord with the result of HaeIII enzyme cutting method.
1555 of embodiment two external detection matrilineal inherited deafness chondriogens
One type test kit and application thereof of A → G sudden change
1. a type test kit (100 person-portion) that is applicable to the Heel blood sheet comprises following component:
(1) solution I (major ingredient is 5%Chelex) 25ml;
(2) PCR reagent (comprising 8mM dNTP 150 μ l, 10 * PCR damping fluid 1ml, 25mM Mg++ solution 1ml, tri-distilled water 1ml * 4 pipes, 5u/ μ l Taq enzyme 25 μ l);
(3) forward primer mtDNAnt1463-1482; Reverse primer mtDNAnt1575-1556, wherein mtDNAnt1557 bit base A sports G, and forward and reverse primer mixes (concentration is 10 μ M), totally 150 μ l by equal proportion;
(4) Restriction Enzyme HaeOII, 20u/ μ l, 110 μ l and supporting 10x damping fluid 1ml;
(5) positive control sample 125 μ l, negative control sample 125 μ l;
(6) working instructions.
2. detected object
Selecting family 13 (seeing Table 1) is tested family, and this family has 4 generations, 25 members, has 8 deafness patients, and premorbid all has the aminoglycosides antibiotics medication history.Present embodiment is selected wherein, and 4 deafness patients are detected object.
3. detection method and result
Working instructions according to this test kit, extract person under inspection's sufficient root blood blood sheet DNA with solution I (major ingredient is 5%Chelex), and get 50ng as template, adding primer mixture 100ng, 10 * dNTP, 5 μ l, 10x damping fluid 5 μ l, Mg++ final concentration are 1.5mmol/L, after adding tri-distilled water to 50 μ l, add 1.0u Taq enzyme, 95 ℃ of sex change are after 5 minutes, follow 94 ℃ of sex change 30 seconds, annealed 30 seconds for 50 ℃, 72 ℃ were extended 30 seconds, carried out 30 circulations altogether, extended under 72 ℃ 7 minutes at last again.By same PCR reaction conditions, do positive simultaneously and negative control PCR reaction with the 1555A that provides in the test kit → G positive template and negative template, and set up no template PCR blank pipe, amplified production is checked with 2% agarose gel electrophoresis in the reaction back, the result, no template contrast is not seen amplified production, and the PCR product of 4 patients and the positive, negative control sample is 113bp.The PCR product is cut detection with the HaeIII enzyme, and the endonuclease reaction system is 30 μ l, comprising:
10x damping fluid 3 μ l
100×BSA?????0.3μl
HaeIII???????20u
PCR product 12-18 μ l
Supply volume with tri-distilled water, 30 ℃ of incubations 1 hour.The endonuclease reaction thing is with 2% agarose gel electrophoresis inspection, the visible 93bp of PCR product and the 20bp band of 4 deafness patients and positive control, and it is positive to show that mtDNA1555A → G suddenlys change; The rarely seen 113bp band of negative control shows no mtDNA1555A → G sudden change.Further cut and check order and confirm that these 4 patients all carry mtDNA1555A → G sudden change with the Alw26 enzyme.
1555 of embodiment three external detection matrilineal inherited deafness chondriogens
Two type test kit and application thereof of A → G sudden change
3 detected objects by name in 6 deafness patients of present embodiment selection family 2 (referring to table 1).Except examined samples are peripheral blood, and the blood sample DNA extraction agent in the test kit is commercially available peripheral DNA extraction test kit, and extracts beyond peripheral blood DNA with this test kit, and other components are identical with embodiment two with detection method in the test kit.Detected result sees Table 1.
The embodiment limbs detect 1555 of matrilineal inherited deafness chondriogens outward
Three type test kit and application thereof of A → G sudden change
3 detected objects by name in 6 deafness patients of present embodiment selection family 11 (referring to table 1).Except examined samples are the DNA that extracts from blood sample, and do not contain in the test kit beyond the agent of blood sample DNA extraction, other components are identical with embodiment two with detection method in the test kit.Detected result sees Table 1.
The present invention is used for the sudden change of 1555 A → G of vitro detection matrilineal inherited deafness chondriogen.
Matrilineal inherited deafness chondriogen .ST25SEQUENCE LISTING＜110〉wear detection method and kit＜130 thereof of Piao＜120〉matrilineal inherited deafness chondriogen A-G sudden change〉Patentln3.1＜160〉2＜170〉Patentlnversion3.1＜210〉1＜211〉20＜212〉DNA＜213〉Homo sapiens＜400〉1ggccctgaag cgcgtacaca 20＜210〉2＜211〉20＜212〉DNA＜213〉Homo sapiens＜400〉2acttaccatg ttacgactgg 20
Claims (7)
1. the detection method of 1555 A of a matrilineal inherited deafness chondriogen → G sudden change, it is characterized in that this method use Genetool Lite programmatic assistance design primer, pass through pcr amplification, on the pcr amplified fragment of tested sample, produce rite-directed mutagenesis, thereby new Restriction Enzyme site that comprises 1555 of 1555 contiguous introducings at this gene, with corresponding Restriction Enzyme digestion, detect the existence of this sudden change then.
2. method according to claim 1 is characterized in that in the wherein designed primer that forward primer is the Nucleotide 1463-1482 of Mitochondrial DNA, and its sequence is the SEQIDNO:1 in the sequence table; Reverse primer is the Nucleotide 1575-1556 of Mitochondrial DNA, and wherein 1557 A is replaced into C, and its sequence is the SEQIDNO:2 in the sequence table.
3. method according to claim 2 is characterized in that the new Restriction Enzyme site of introducing behind the pcr amplification wherein is HaeIII.
4. preparation is characterized in that according to the test kit of 1555 A of the vitro detection matrilineal inherited deafness chondriogen → G sudden change of the described method of claim 2 this test kit contains:
(1) reagent of extraction blood sample DNA;
(2) pcr amplification reaction reagent comprises dNTP, 10 * PCR damping fluid, Mg++, tri-distilled water, Taq enzyme;
(3) described forward primer of equal proportion blended claim 2 and reverse primer;
(4) Restriction Enzyme HaeIII and supporting damping fluid thereof;
(5) positive sample contrast, the contrast of negative sample;
(6) working instructions.
5. test kit according to claim 4, the reagent that it is characterized in that wherein said extraction blood sample DNA is for extracting the solution I of sufficient root blood DNA, and its main component is Chelex.
6. test kit according to claim 4, the reagent that it is characterized in that wherein said extraction blood sample DNA are that peripheral blood DNA extracts test kit.
7. test kit according to claim 4 is characterized in that wherein comprising other compositions and working instructions except that the reagent of said extraction blood sample DNA.
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