CN103173441A - Amplification method, primer, sequencing method and mutation detection method of mitochondria whole genome DNA (Deoxyribonucleic Acid) - Google Patents
Amplification method, primer, sequencing method and mutation detection method of mitochondria whole genome DNA (Deoxyribonucleic Acid) Download PDFInfo
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Abstract
The invention discloses a primer pair and a kit for amplification of a mitochondria whole genome. The invention further discloses an amplification method, a sequencing method and a mutation detection method of the mitochondria whole genome DNA (Deoxyribonucleic Acid). According to the invention, the direct amplification of the given primer is combined with the direct sequencing method, so that a mitochondria whole genome sequence can be obtained; all mutations of the mitochondria genes can be detected by one step for being used for detecting the diseases caused by most of the mitochondria gene mutations.
Description
Technical field
The present invention relates to molecular biology, particularly relate to the primer pair group, test kit, amplification method, sequence measurement and the Mitochondrial DNA Mutation detection method that can be used for the amplification of plastosome complete genome DNA.
Background technology
Mitochondrial Genome Overview (mitochondrial DNA, mtDNA) is closed double-stranded circular structure, and length is 16569bp, is divided into coding region and non-coding region.The coding region is totally 37 genes, 22 coding transfer ribonucleic acids (tRNA), 2 coding rRNA (12S and 16S rRNA) arranged, 13 coded polypeptides in these 37 genes.The mtDNA sequence in the gene is tight, there is no intron between sequence.
The molecular structure characteristics of mtDNA determine that it has the hereditary property of following uniqueness:
(1) in general high copy number, only has two chromosomal DNA copies, and has 1000 in most cells matter to thousands of mtDNA copies in the somatic nucleus of the mankind;
(2) show as strict matrocliny characteristics, mtDNA passes to the offspring with the ovum of female parent;
(3) have higher mutation rate, studies show that in a large number the mtDNA mutation frequency is high more than 10 times than the nuclear gene group;
(4) heterogeneity, the mtDNA of sudden change and the mtDNA of wild-type coexist in a tenuigenin in varing proportions.
Due to the high mutation rate of mtDNA and the height evolution conservative of mtDNA, most mtDNA new mutants are all detrimental mutation, and the clinical phenotypes that the mtDNA sudden change causes is very extensive, almost relate to all tissues and organ, classical symptom does not appear in many patients, brought great difficulty to clinical diagnosis, a lot of mitochondrial diseases can't be made a definite diagnosis.Therefore the accurate detection of Mitochondrial Genome Overview has important Clinical significance of MG.
At present clinically for the detection of mtDNA, also only concentrate on minority common chondriogen site is suddenlyd change and lacks examination, as the examination to mitochondrial encephalomyopathy companion's hyperlactacidemia and palsy sample outbreak (MELAS) common mutations A3243G, positive rate is low, and Most patients is difficult to obtain etiological diagnosis accurately.In addition, the selection in these sites all is based on the experimental data of other ethnic groups (being mainly white people), and Chinese gene mutation site and European mutational site make a big difference on rule.The detection method of at present the most frequently used mtDNA is to use polymerase chain reaction (PCR) to amplify target DNA fragment, then adopts fluorescent mark sequenator (as the 3730DNA sequenator) that amplified production is directly checked order.Due to the restriction of fluorescent mark sequenator to the order-checking fragment length, need to even manyly carry out multiplex PCR to the primer pair Mitochondrial Genome Overview with 26 pairs, be divided into 26 sections even more the multi-disc section check order, operate too loaded down with trivial detailsly, and can't detect the very low variation of copy number.
Summary of the invention
The present invention is based on contriver's following discovery and completes:
The amplification Mitochondrial Genome Overview, can complete by designing minimum 2 pairs of PCR primers, but, the primer pair number is few high to the specification of quality of DNA profiling, genomic templates can not have too much degraded, and also higher to the requirement of Taq enzyme, and the Taq enzyme that needs to amplify the 10kb long segment carries out PCR, the oversize while of amplified fragments also can increase the mispairing rate of base, reduces the accuracy that detects.And the how right primer of the design Mitochondrial Genome Overview that increases, as 26 pairs, 43 pairs, PCR and follow-up purification process are loaded down with trivial details, and PCR product fragment is short, is applicable to chondriogen selection portion subregion and does the sanger order-checking.Select primer pair quantity moderate, as 8 couple of the present invention program, it is advantageous that, to template require lowly, PCR product mispairing rate is low, detection accuracy is high, more is applicable to follow-up high-flux sequence platform.
The objective of the invention is for the deficiencies in the prior art, a kind of primer pair group that can be used for the amplification of plastosome complete genome sequence is provided.
Another object of the present invention is to provide a kind of plastosome complete genome DNA amplification method.
Of the present inventionly be to provide again a kind of plastosome complete genomic sequence measurement an order.
An also purpose of the present invention is to provide a kind of sudden change detection method of Mitochondrial DNA.
A further object of the present invention is to provide a kind of test kit that can be used for the amplification of plastosome complete genome DNA.
For achieving the above object, the present invention has adopted following technical scheme:
The invention discloses the primer pair group for amplification plastosome whole genome sequence, described primer pair group comprises at least one pair of primer in following a-h, preferably contains all the eight pairs of primers in A-H,
a、seq?ID?No.1:5’-CACTCCCATACTACTAATCTC-3’,
seq?ID?No.2:5’-TAGCATGTACTGCTCGGAGGT-3’,
b、seq?ID?No.3:5’-GTCCTAAACTACCAAACCTGC-3’,
seq?ID?No.4:5’-GTGTTAGTCATGTTAGCTTG-3’,
c、seq?ID?No.5:5’-ATCTCTCCCTCACTAAACGTAAG-3’,
seq?ID?No.6:5’-ATGAGGGCGTGATCATGAAAGGT-3’,
d、seq?ID?No.7:5’-GCATACACCACATGAAACATC-3’,
seq?ID?No.8:5’-ATGCCGTCGGAAATGGTGAAG-3’,
e、seq?ID?No.9:5’-TCCCACTCCTAAACACATCC-3’,
seq?ID?No.10:5’-AAACCCGGTAATGATGTCGG-3’,
f、seq?ID?No.11:5’-GCCCACGGGCTTACATC-3’,
seq?ID?No.12:5’-GATTGTTAGCGGTGTGGTCG-3’,
g、seq?ID?No.13:5’-GCCTTCTTACGAGCCAAAACC-3’,
seq?ID?No.14:5’-TCCAGCGTCTCGCAATGCTAT-3’,
h、seq?ID?No.15:5’-TTCGCCTACACAATTCTCCG-3’,
seq?ID?No.16:5’-TTTATGGGGTGATGTGAGCC-3’。
The invention also discloses a kind of plastosome complete genome DNA amplification method, described method comprises, adopts above-mentioned primer pair group, take Mitochondrial DNA as template, carries out pcr amplification.
During described pcr amplification, the annealing temperature of each primer pair is 52-56 ℃, is preferably 56 ℃.
The present invention further discloses a kind of plastosome genome sequencing method, described method comprises, adopts above-mentioned method amplification to obtain the plastosome complete genome DNA, builds sequencing library, adopts the high-flux sequence platform to carry out genome sequencing.
Described high-flux sequence platform is selected from one of Illumina solexa/Hiseq, ABI SOLiD, Roche454 and single-molecule sequencing device; In one of the present invention preferred embodiment, described high-flux sequence platform is Illumina Miseq sequenator.
Particularly, described sequence measurement comprises, the product that pcr amplification is obtained respectively purifying and mix after, preparation DNA Paired-End PCR Free library, Insert Fragment is 100 ~ 1000bp, is preferably 400～600bp, more preferably 500bp, after the library quality inspection for preparing is qualified, direct Sequencing detection on the Miseq sequenator, read long 150bp.
In concrete embodiment of the present invention, preparation DNA Paired-End PCR Free library specifically comprises
I, the PCR product is interrupted, make and interrupt rear DNA master tape size and concentrate on the Insert Fragment size, reclaim the purpose fragment of required size;
Ii, the product of step I is carried out the end reparation;
Iii, the product of step I i is carried out 3 ' end add the A base;
Iv, the product fragment of step I ii is added sequence measuring joints;
V, selection are also reclaimed target DNA fragment, obtain described DNA library;
Randomly, described purpose fragment is 100 ~ 1000bp, is preferably 400～600bp, and more excellent is 500bp.
The library quality examination is preferably further carried out in the library that builds, and for example can using, Agilent2100Bioanalyzer and ABI StepOnerPlus Real-Time PCR System carry out the detection of Quality and yield.
The invention also discloses a kind of sudden change detection method of Mitochondrial DNA, described method comprises, adopts above-mentioned method to carry out the plastosome genome sequencing, obtains plastosome complete genome DNA sequence, and sequencing result and plastosome reference sequences are compared.
Described plastosome reference sequences is preferably revised Cambridge reference sequences NC_012920.1.
The invention also discloses a kind of test kit, described test kit contains above-mentioned primer pair group.
Described test kit is used for the amplification of plastosome complete genome DNA.
Owing to having adopted above technical scheme, the beneficial effect that the present invention possesses is:
The present invention utilize given primer directly amplification in conjunction with the method for direct Sequencing, can access the plastosome whole genome sequence, whole sudden changes that can disposable detection chondriogen can be applied to the detection of the disease that most of mitochondrial gene mutation cause.Wherein the Miseq sequenator is the new-generation sequencing instrument of having integrated amplification, order-checking and data analysis on single instrument, occupies advantage aspect simple, quick, flexible and low-cost, is specially adapted to the Mitochondrial Genome Overview order-checking.The present invention can be applicable to auxiliary line plastochondria disease clinical diagnosis, pathogenetic research, genetics research and different crowd haplotype somatotype.
Description of drawings
Fig. 1 is chondriogen PCR product electrophorogram of the present invention.
Annotate: 1: primer a PCR product; 2: primer b PCR product; 3: primer c PCR product; 4: primer d PCR product; 5: primer e PCR product; 6: primer f PCR product; 7: primer g PCR product; 8: primer h PCR product; M:DNA Marker III
Fig. 2 is sequencing library preparation flow figure of the present invention.
Fig. 3 is the reads figure in detection sample 3243 sites of the embodiment of the present invention.
Embodiment
Important variation intersperses among in the mtDNA genome clinically, and the full genome of mtDNA is difficult to be amplified.The present invention develops 8 pairs of primer multiplex PCRs of a kind of use and amplifies the complete genomic sequencing system of mtDNA, to address the above problem.
The present invention has synthesized 8 pairs of high specific PCR primers that can amplify the full genome base of plastosome.8 pairs of PCR primers of involved in the present invention this experiment proved that, can carry out specific amplification to the full genome of plastosome, and obtain true and reliable plastosome whole genome sequence data by the method for direct Sequencing.
The mitochondrion sequencing that the invention provides has covered the Mitochondrial Genome Overview total length with 8 pairs of PCR primers, and following table 1 has been listed this 8 pairs of PCR primer sequences.
Table 1 PCR primer sequence
Select above-mentioned plastosome genome sequencing with 8 pairs of PCR primers, the upstream and downstream primer is diluted to 10 μ mol/L, as working fluid.
The PCR reaction system: the system cumulative volume is 25 μ l, wherein contains DNA profiling 1 μ l(and contains 50ng complete genome DNA at least), each 1 μ l of the corresponding upstream and downstream primer of purpose fragment (10 μ mol/L), Taq enzyme (5U/ μ l) 0.25 μ l, 10 * PCR buffer(Mg
2+Plus) 2.5 μ l, dNTP Mixture(2.5mmol/L) 4 μ l, ddH
2O15.25 μ l.
The pcr amplification underlying parameter of primer is: 94 ℃ of sex change 45s, and definite annealing temperatures different from the PCR primer are carried out 30s, and 72 ℃ are extended 3min, amount to 35 circulations (table 2 is listed the pcr amplification condition).
Reclaim test kit purifying PCR reaction product with the DNA purifying.
PCR product after purifying mixes, and prepares DNA Paired-End PCR Free library, and Insert Fragment is 500bp, and the library preparation flow as shown in Figure 2.In addition, in actual mechanical process, in order to improve order-checking efficient, those skilled in the art know, and can be prepared into DNA Paired-End PCR Free Index library by increasing the index sequence, realize mixing machine with other samples libraries.
After the library quality inspection was qualified, direct Sequencing detected on the Miseq sequenator, reads long 150bp, the order-checking degree of depth 5000 * and, each Sample producing is the data volume of 83M approximately.
Sequencing data obtains plastosome whole genome sequence data through information analysis, with plastosome reference sequences (revised Cambridge sequence NC_012920.1) comparison, can obtain sample line plastochondria sequence variations information, comprises point mutation, micro-deleted and little repetition.
Utilize 8 pairs of primer pairs of the present invention can prepare test kit, be used for the amplification of plastosome whole genome sequence.
The present invention adopts 8 pairs of full genomes of primer pair plastosome to carry out the multi-PRC reaction amplification, and the PCR product adopts the Miseq sequenator to carry out high-flux sequence through building the PCR-free library after purifying at last.Order-checking flexible operation, simple, quick, the degree of depth can reach several thousand up to ten thousand taking advantage of even, taking advantage aspect the sudden change of detection mtDNA low frequency, can detect any point sudden change and micro-deleted, little repetition on mtDNA, assist clinicians improves the diagnosis rate of mitochondriopathy suspected patient.Except the application aspect gene test, also can be applicable to anthropology, genetics and the fields such as mtDNA sudden change and disease-related Journal of Sex Research.The full genome Miseq order-checking of plastosome detects the mtDNA variation for individuality is clinical with population genetic study an especially suitable platform is provided, not only can pay close attention to the key variation of mtDNA, shorten analysis time and improve flow, equally also can focus onto the arbitrary unknown pathogenic mutation on the mtDNA that detects.Order-checking is compared with tradition, and this sequence measurement shows considerable advantage when detecting the thousands of mtDNA base of analysis.
By reference to the accompanying drawings the present invention is described in further detail below by embodiment.
Embodiment:
Plastosome genome sequencing of the present invention sees Table 1 with the PCR primer.
The present invention is based on the method that described plastosome genome sequencing increases with PCR primer multiplex PCR, utilize 8 pairs of PCR primer amplification purpose fragments that cover the Mitochondrial Genome Overview total length, then constructed dna Paired-End PCR Free Index library, Miseq order-checking detection line plastochondria whole genome sequence obtains sample line plastochondria sequence variations information through the data analysis comparison.
Pcr amplification
Select the plastosome genome sequencing with 8 pairs of PCR primers (table 1), the upstream and downstream primer is diluted to 10 μ mol/L, as working fluid.PCR reaction system cumulative volume 25 μ l wherein contain DNA profiling 1 μ l(and contain 50ng complete genome DNA at least), this DNA profiling is from the poba gene group of a bit line plastochondria brain patients with myopathy.Each 1 μ l of the corresponding upstream and downstream primer of purpose fragment (10 μ mol/L), Taq enzyme (5U/ μ l) 0.25 μ l, 10 * PCR buffer(Mg
2+Plus) 2.5 μ l, dNTP Mixture(2.5mmol/L) 4 μ l, ddH2O 15.25 μ l.
This experiment PCR reaction agents useful for same is common reagent, and conventional method is adopted in the detection of PCR product.The pcr amplification parameter sees Table 2.
Table 2 pcr amplification condition
| The primer title | Denaturation temperature/time | Annealing temperature/time | Elongating temperature/time | Cycle index |
| a | 94℃?45s | 56℃?30s | 72℃?3min | 35 |
| b | 94℃?45s | 56℃?30s | 72℃?3min | 35 |
| c | 94℃?45s | 56℃?30s | 72℃?3min | 35 |
| d | 94℃?45s | 56℃?30s | 72℃?3min | 35 |
| e | 94℃?45s | 56℃?30s | 72℃?3min | 35 |
| f | 94℃?45s | 56℃?30s | 72℃?3min | 35 |
| g | 94℃?45s | 56℃?30s | 72℃?3min | 35 |
| h | 94℃?45s | 56℃?30s | 72℃?3min | 35 |
The pcr amplification product agarose gel electrophoresis detects
Pcr amplification product 1.5%q agarose gel electrophoresis detects, and guarantees that the amplification success of PCR product and band are single, and specificity is good, electrophoresis qualification result such as Fig. 1.
The purifying of pcr amplification product
Use the universal DNA purifying of TIANGEN to reclaim test kit (centrifugal column type) purifying pcr amplification product.
The preparation of DNA Paired-End PCR Free Index library
PCR product after purifying is mixed, prepare DNA Paired-End PCR Free Index library, Insert Fragment is 500bp, and the library preparation flow is seen Fig. 2.
Sample interrupts: with Covaris S2 or Covaris E210, the PCR product is interrupted, require to interrupt rear DNA master tape size and concentrate on the 500bp left and right.The DNA that has no progeny of air exercise detects by agarose gel electrophoresis, and the qualified product that interrupts is with QIAquick Gel Extraction Kit(QIAGEN) test kit carries out the purifying recovery.
End is repaired: configuration 200 μ l ends are repaired reaction system: DNA sample 60 μ l, T4 PNK buffer 20 μ l, dNTP Mixture(10mMOL/L) 8 μ l, T4 DNA polymerase 10 μ l, Klenow Fragment 2 μ l, T4 PNK 10 μ l, ddH2O 90 μ l.20 ℃ of reaction 30min in Thermo mixer.Repair product and carry out the purifying recovery with test kit.
3 ' end adds the A base: configuration 100 μ l3 ' ends add A base reaction system: DNA sample 64 μ l, 10 * blue buffer, 10 μ l, 1mM dATP 20 μ l, Klenow exo (3 ' to5 ' exo minus) 6 μ l.37 ℃ of reaction 30min in Thermo mixer.Add the A product and carry out the purifying recovery with test kit.
Fragment adds the solexa joint: configuration 100 μ l joint ligation systems: DNA sample 20 μ l, 2 * DNAligase buffer, 50 μ l, PE Index adapter oligo mix 20 μ l, T4 DNA ligase 10 μ l.16 ℃ of reactions are spent the night in Thermo mixer.Connect product and carry out the purifying recovery with test kit.
In the application, those skilled in the art know, and can according to reality check order platform or other correlated conditions, select suitable sequence measuring joints.In the present embodiment, shown in the following Seq ID of the sequence measuring joints sequence No.17 that adds:
5’-GATCGGAAGAGCACACGTCTGAACTCCAGTCAC
ATCTCGTATGCCGTCTTCTGCTTC-3’
The square frame position is the Index sequence label, in actual application, also can use as required other 5-11 base instead and substitute.
The sequence measuring joints that adds will form the not exclusively complementary Y type tree fork shape structure of two following chains:
The DNA fragmentation size is selected: the connection product to previous step passes through 2% sepharose 100V voltage electrophoresis 2h, carries out the selection of DNA fragmentation size.It is the 630bp left and right that the DNA fragmentation two ends add respectively top connection rear panel segment length, but because of adding joint be tree fork shape joint (two chains not exclusively complementary), that runs than general 630bp in electrophoresis process is slow, roughly in the position of 700bp marker, therefore select the electrophoretic band of 700bp size to cut the glue recovery, the blob of viscose of cutting-out carries out purifying with test kit and reclaims.
The library quality examination: the library that builds uses Agilent 2100 Bioanalyzer and ABI StepOnerPlus Real-Time PCR System to carry out the detection of Quality and yield.
The Miseq order-checking
After the library quality inspection was qualified, direct Sequencing detected on the Miseq sequenator, reads long 150bp, and the degree of depth that requires to check order reaches 5000 *, each Sample producing is the data volume of 83M approximately.
Information analysis
Sequencing data obtains plastosome whole genome sequence data through information analysis, with plastosome reference sequences (revised Cambridge sequence NC_012920.1) comparison, can obtain sample line plastochondria sequence variations information, comprises SNP and Indel.
The information analysis result is as follows:
Sequence and reference sequences that order-checking obtains are compared, and coverage reaches 100%, 34 SNP and 5 Indel detected altogether.Comprising the pathogenic mutation (Fig. 3) of 3243A → G, the information analysis in 3243 sites the results are shown in Table 3.
Table 3 3243 site information analytical resultss
Above content is in conjunction with concrete embodiment further description made for the present invention, can not assert that concrete enforcement of the present invention is confined to these explanations.For the general technical staff of the technical field of the invention, without departing from the inventive concept of the premise, can also make some simple deduction or replace, all should be considered as belonging to protection scope of the present invention.
Claims (11)
1. be used for the primer pair group of amplification plastosome whole genome sequence, it is characterized in that: described primer pair group comprises at least one pair of primer in following A-H, preferably contains all the eight pairs of primers in A-H,
a、seq?ID?No.1:5’-CACTCCCATACTACTAATCTC-3’,
seq?ID?No.2:5’-TAGCATGTACTGCTCGGAGGT-3’,
b、seq?ID?No.3:5’-GTCCTAAACTACCAAACCTGC-3’,
seq?ID?No.4:5’-GTGTTAGTCATGTTAGCTTG-3’,
c、seq?ID?No.5:5’-ATCTCTCCCTCACTAAACGTAAG-3’,
seq?ID?No.6:5’-ATGAGGGCGTGATCATGAAAGGT-3’,
d、seq?ID?No.7:5’-GCATACACCACATGAAACATC-3’,
seq?ID?No.8:5’-ATGCCGTCGGAAATGGTGAAG-3’,
e、seq?ID?No.9:5’-TCCCACTCCTAAACACATCC-3’,
seq?ID?No.10:5’-AAACCCGGTAATGATGTCGG-3’,
f、seq?ID?No.11:5’-GCCCACGGGCTTACATC-3’,
seq?ID?No.12:5’-GATTGTTAGCGGTGTGGTCG-3’,
g、seq?ID?No.13:5’-GCCTTCTTACGAGCCAAAACC-3’,
seq?ID?No.14:5’-TCCAGCGTCTCGCAATGCTAT-3’,
h、seq?ID?No.15:5’-TTCGCCTACACAATTCTCCG-3’,
seq?ID?No.16:5’-TTTATGGGGTGATGTGAGCC-3’。
2. plastosome complete genome DNA amplification method, described method comprises, adopts primer pair group claimed in claim 1, take Mitochondrial DNA as template, carries out pcr amplification.
3. plastosome complete genome DNA amplification method according to claim 2, it is characterized in that: during described pcr amplification, the annealing temperature of each primer pair is 52-56 ℃, is preferably 56 ℃.
4. plastosome genome sequencing method, described method comprise, adopt the described method amplification of claim 2 or 3 to obtain the plastosome complete genome DNA, build sequencing library, adopt the high-flux sequence platform to carry out genome sequencing.
5. plastosome genome sequencing method according to claim 4, it is characterized in that: described high-flux sequence platform is the Miseq sequenator.
6. plastosome genome sequencing method according to claim 5, it is characterized in that: described sequence measurement comprises, the product that pcr amplification is obtained respectively purifying and mix after, preparation DNAPaired-End PCR Free library, direct Sequencing detection on the Miseq sequenator after the library quality inspection for preparing is qualified.
7. sequence measurement according to claim 6 is characterized in that: described preparation DNAPaired-End PCR Free library specifically comprises,
I, described PCR product is interrupted, make and interrupt rear DNA master tape and concentrate on purpose clip size scope;
Ii, the product of step I is carried out the end reparation;
Iii, the product of step I i is carried out 3 ' end add the A base;
Iv, the product fragment of step I ii is added sequence measuring joints;
V, selection are also reclaimed target DNA fragment, obtain described DNA library;
Randomly, described purpose fragment is 100～1000bp, is preferably 400～600bp, and more excellent is 500bp.
8. the sudden change detection method of a Mitochondrial DNA, described method comprise, adopt the described method of claim 4-6 any one to carry out the plastosome genome sequencing, obtain plastosome complete genome DNA sequence, and sequencing result and plastosome reference sequences are compared.
9. sudden change detection method according to claim 8, it is characterized in that: described plastosome reference sequences is revised Cambridge reference sequences NC_012920.1.
10. test kit, described test kit contains primer pair group claimed in claim 1.
11. test kit according to claim 10 is characterized in that: described test kit is used for the amplification of plastosome complete genome DNA.
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2013
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