CN101875966A - Improved mitochondrial genome complete sequence determination method - Google Patents
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Abstract
The invention discloses an improved mitochondrial genome complete sequence determination method based on the conventional PCR technology, which aims at providing a low-cost, fast and new mitochondrial genome complete sequence determination method having a simple experimental technology. The method is characterized by the screening of templates and the design of PCR primers, i.e determining the minimal homology of mitochondrion complete sequences of closely-related species which can be used as the templates; carrying out primer design in two batches, and introducing a nested-PCR method to the sequence amplification process; and presenting general rules for the design of the PCR primers. The method is mainly used for mitochondrial genome complete sequence determination, and serves basic scientific researches, such as genetics, molecular biology and the like.
Description
Technical field
The present invention relates to a kind of improved mitochondrial genome complete sequence determination method and the application in measuring mitochondrial genome complete sequence thereof.
Background technology
Plastosome is a kind of basic, the important organoid that is present in most eukaryotic cells. and it is the place that cell carries out oxidative phosphorylation. and people belong to the eukaryotic second genetic information system to this genetic information system of genetic material Mitochondrial Genome Overview (mtDNA) of plastosome itself, or extranuclear gene and expression system thereof. a large amount of different animals mitochondrial genome complete sequence determinations are shown the animal mitochondria gene is by the double-stranded cyclic DNA molecular composition that is roughly 15～20kb.Chondriogen about 22 tRNAs that encoding, 2 rRNAs of size and 13 hydrophobic protein polypeptide, these polypeptide have comprised the subunit of the combined enzyme agent that combines with mitochondrial inner membrane: the subunit of cytochrome b (Cytb), 2 ATP enzymes, 3 cytochrome cs (Cytc) (CO I of oxidasic subunit, II and III), (ND21 of subunit of 7 NADH reductase enzyme complex bodys, 22,23,24,24L, 25 and 26) (Lee and Kocher, Genetics., 1995,139:873-887; Gares, Genetics., 1998,118:649-663).Mitochondrial DNA is one of important topic of molecular biology research over nearly 20 years, and it causes that people's very big interest is:
1, the Mitochondrial Genome Overview good model being not only researching DNA structure and dna replication dna, transcribing, also be the most suitable model system (Liao Shunyao etc. of the general considerations of research eukaryotic cell nucleic acid and protein synthesis, biological chemistry and biophysics progress, 2000,27:508-512).
2, Mitochondrial Genome Overview and the nuclear gene group mutual relationship on genetic information is expressed is a very important problem.Mitochondrial Genome Overview has independently duplicated ability, but the realization Mitochondrial Genome Overview is duplicated and is expressed required many enzymes (as archaeal dna polymerase, RNA polymerase) but by nuclear gene group coding (Anderson, et al., Nature., 1981,290:457-465).In fact, the genomic nuclear gene quantity of coding line plastochondria has substantially exceeded the gene dosage that is present in mtDNA itself, and the genetic information capacity of mtDNA and little, its codifiability just examine the dna encoding capacity several ten thousand to hundreds of thousands of/one, the mechanism itself of setting up this independently mitochondrial inheritance system makes mitochondrial inheritance seem extremely meaningful.In addition, chondriogen inserts and duplicates to the swivel base of nuclear gene, as a model analyzing swivel base mechanism, virus transfection.The contrast of the homologous gene structure of Mitochondrial Genome Overview and nuclear gene group also be widely used in nuclear gene and the extranuclear gene Study on Evolution (Delarbre, et al., Genetics., 1998,150:331-344).
3, because animal mitochondria DNA (mtDNA) has simple in structure, strict matrilinear inheritance, recombinates hardly, rate of evolution is fast and the different zones rate of evolution such as there are differences at characteristics, make its kind differentiate and the research of classification position aspect and the research aspect application of different grades hierarchy growth very extensive.
At present, the research method of mitochondrial genome complete sequence determination mainly contains three kinds.
1, based on the method for physical sepn strategy
Before round pcr service wire mitochondrial DNA (being called for short mtDNA) separated, the mensuration for mitochondrial genome complete sequence mainly comprised the acquisition of (1) high purity mtDNA; (2) the mtDNA fragment changes into short dna fragment 2 partial contents that are suitable for cloning and sequencing.The acquisition of high purity mtDNA, mainly contain cesium chloride density gradient centrifugation (Lansman, et al., J Mol Evol., 1981,17:214-226) and differential centrifugation (Yan Hua is superfine, biotechnology communication, 2007,18:95-97; Tamura and Aotsuka, Biochem Genet., 1988,26:815-819).In addition, carry out the method for local optimization in addition on this basis, (Yan Hua is superfine, biotechnology communication, 2007,18:95-97 as alkaline denaturation of DNase method, alkaline denaturation and improvement etc.; Xia Yuling etc., the bombycology communication, 2002,22:24-29); The mtDNA fragmentation interrupts 2 kinds of methods at random with digestion with restriction enzyme or ultrasonic wave usually and the mtDNA fragment is turned to the short dna fragment that is fit to cloning and sequencing.But, needing expensive equipment, experimental period is longer, and is especially bigger to experiment material consumption.
2, based on the method for LA-PCR technology
The LA-PCR above dna fragmentation of 5kb that increases usually, to template, primer and polysaccharase all have comparatively strict requirement (Ye Weiping etc., the zoology magazine, 2003,38:105-109).With Mitochondrial Genome Overview amplification is that 2 or the big fragment of several overlapped DNA are adopted by numerous researchists, becomes a kind of ideal Mitochondrial Genome Overview and separates tactful.The amplified production of LA-PCR both can be used as the template of conventional PCR secondary amplification after separation and purification, utilize universal primer to increase into (the Zhou that checks order after the short dna fragment, et al., Genome., 2007,50:855-866), the template that also can be used as the primer walking order-checking checks order, even after can further carrying out the restriction enzyme cutting, carry out cloning and sequencing (Wu Xiaobing etc., Science Bulletin, 2003,48:1954-1958).But the LA-PCR technology is preserved sample and is had relatively high expectations, and amplification condition significant difference between different plant species.
3, based on the method for conventional round pcr
With reference to the Mitochondrial Genome Overview research universal primer (Simon that announces, et al., Ann Entomol Soc Am., 1994,87:651-701), or the mitochondrial genome complete sequence by nearly source species, design and to cover complete genomic primer, adopt conventional round pcr directly by among total DNA Mitochondrial Genome Overview being increased, form a series of dna fragmentations that are shorter in length than 5kb, and clone it into that plasmid vector checks order.Most conventional pcr amplification product length can directly be cloned into plasmid vector and check order, and also can carry out the PCR method by identical primer and directly check order.But this method is subjected to the interference of plastosome pseudogene easily
Summary of the invention
The present invention provides a kind of improved mitochondrial genome complete sequence determination and primer design method on the basis of conventional round pcr.
Content of the present invention comprises screening, design of primers, the pcr amplification of template, 4 parts of splicing of sequencing result:
1, the screening of template: login NCBI (
Http:// www.ncbi.nlm.nih.gov/), search and determinand kind belong to same section even same purpose species mitochondrial genome complete sequence, and homology is getting final product more than 75%, if there are a plurality of sequences can supply to utilize, compare by blast software, the sequence of the species that preferential selection and species sibship to be measured are nearest is advisable.
2, primer design thinking: with the sequence that obtains by screening is template, with the primer that PCR primer-design software Primer Premier5.0 is designed for pcr amplification, sees accompanying drawing I.Concrete thinking is as follows:
1) considers present determined dna sequence state of the art, a reaction can be measured about 800-1000bp, but the preceding 40-60bp of sequencing result does not detect or is insincere, must foreclose, to have between adjacent 2 sections amplified fragments one section overlapping, so that the splicing and the consideration of research cost aspect, the PCR product length between the every pair of primer is advisable with 1300-1500bp;
2) because PCR design of primers template is the sibling species of species to be measured,, obtain the logarithm of the primer of roughly required design with the length/1300-1500bp of template plastosome sequence.
3) design of primers divides 2 batches to carry out, first primer logarithm be total primer logarithmic half, the spacing distance between adjacent two pairs of primer amplification fragments is 1000-1200bp, sees accompanying drawing 1.
4) replace original sequence of corresponding site in the template sequence after relatively through blast software with the PCR product sequencing result of first primer and design of primers are template used, the sequence fragment that soon newly obtains is mounted in the template sequence, the sequence that obtains like this is only about half of be new be mitochondrial sequence to be measured.
5) with 4) in the sequence that is inlaid with new sequence that obtains be that template is carried out second batch of design of primers, and, in second batch of primer, each all must drop in the new sequence primer, and guarantee every section PCR product of second batch of primer and the new sequence in the template all one section overlapping.
6) if having certain to or several expanding effect to primer bad, can inlay pcr amplification (seeing accompanying drawing 2) to a pair of PCR of the inlaying primer of primer outside design at this, carrying out outside P CR amplification earlier, is to survey pcr amplification in template is carried out to guarantee the pcr amplification success with outside PCR product again.Must guarantee with adjacent PCR product fragment have overlapping, to guarantee final sequence assembly.
3, extraction and the pcr amplification of total DNA
1) extraction of total DNA: the extracting method of the total DNA of various species is different, can choose corresponding total DNA extraction method according to different situations.
2) pcr amplification is a template with total DNA, and the reaction system cumulative volume is 25 μ l, wherein, and distilled water 16.2 μ l, 10 * ExTaqbuffer, 2.5 μ l, dNTP 2.5 μ l, forward primer 1.3 μ l, reverse primer 1.3 μ l, total dna profiling 1 μ l, ExTaq archaeal dna polymerase 0.2 μ l.The PCR response procedures is determined according to the reality of primer.
Amplified production reclaims the test kit purifying with UNIQ-10 pillar PCR product after electrophoresis detection, send biotech company to finish order-checking.
4, sequencing result's splicing:
1) utilizes the online software of blast of Primer Premier 5.0 softwares and NCBI website will obtain 20-30 PCR sequencing fragment result and be spliced into a complete mtDNA.
2) utilize the DNAStar software package that sequence is carried out searching of open reading frame, the acquisition of sequence complementary strand; Utilize the ORFfinder software of NCBI website to finish the translation of albumen coded sequence and the investigation of amino acid coding situation; Compare with online software of blast and template sequence and to determine each chondriogen and the particular location of other non-coding sequence in sequence.
Advantage of the present invention and positively effect show:
(1) with based on the method for physical sepn strategy relatively, the pcr amplification template only needs total DNA, and does not need pure Mitochondrial Genome Overview DNA, has reduced the cost and the technical difficulty of experiment.
(2) compare with conventional round pcr, the present invention has determined technical parameters that some are relevant and has formed a kind of novel method of systemic and complete new amplification Mitochondrial DNA complete sequence, as: as the nearly edge species of design of primers template can be the Mitochondrial DNA complete sequence of any species in the same section on the taxonomy, and is getting final product more than 75% with mitochondrial dna sequence dna homology to be measured; The substantial distance 1300-1500bp of every one couple of PCR primer extension product; The necessary overlapping 50-200bp of adjacent 2 pairs of PCR primer extension products; The primer logarithm of the required usefulness of whole pcr amplification is the length/1300-1500bp of template plastosome sequence.
(3) compare with existing plastosome complete sequence determination technology, topmost characteristics of the present invention are the PCR design of primers, the conservative property of considering some position, primer place is not strong, and the present invention has introduced in these primer inboards and inlayed the PCR primer and inlay pcr amplification and guarantee pcr amplification success.
(4) the innovative point of the present invention the primer that exactly whole sequence increased divides 2 batches to design, first primer design is a template with the mtdna sequence of nearly edge species, and the second batch of PCR primer is that template designs with self mtdna sequence fully then.
Because the message structure of Mitochondrial DNA, status, effect and with the mutual relationship of nuclear gene; The necessity of independent heredity etc. was current mitochondrial molecule biological study focus during cell was evolved; Mitochondrial origin and differentiation, and the vital role of portion gene sequence in molecular system generation and molecular evolution research.It is low to the invention provides a kind of cost, and experimental technique is the mitochondrial genome complete sequence determination novel method simply and fast.
Description of drawings
Fig. 1 is first pcr amplification primer schema
Fig. 2 is second batch of pcr amplification primer schema, and wherein diagonal line hatches is partly for after the order-checking of first PCR product being mounted to sequence the result behind the template sequence.
Fig. 3 is for inlaying PCR design of primers figure, wherein thick arrow be the bad primer outside of pcr amplification effect inlay the PCR primer.
Fig. 4 is the agarose gel electrophoresis figure of first PCR primer extension product of ripple lip fishing line plastochondria genom sequence
Fig. 5 is the agarose gel electrophoresis figure of second batch of PCR primer extension product of ripple lip fishing line plastochondria genom sequence
Fig. 6 is that first PCR primer amplification effect is bad, inlays primer and the agarose behind pcr amplification and coagulates but add second pair
Gel electrophoresis figure
Fig. 7 is a ripple lip fishing line plastochondria genom sequence, and the sequence total length is 17173 bases
Embodiment
Embodiment 1: the mensuration of ripple lip fishing line plastochondria genom sequence
1, from the screening of NCBI query site, obtain the mitochondrial genome complete sequence of Labridae Halichoeres, be template with this sequence, carry out the PCR design of primers of ripple lip fishing line plastochondria genome complete sequence determination
2, utilize software Primer Premier 5.0 to carry out the PCR design of primers, the result is shown in following table 1 and 2:
First PCR primer of table 1 ripple lip fishing line plastochondria genome complete sequence determination and gene-amplification effect are bad and the second couple that add inlays primer
Second batch of PCR primer of table 2 ripple lip fishing line plastochondria genome complete sequence determination
Designed primer is all given birth to worker Bioisystech Co., Ltd by Shanghai and is synthesized.
3, extraction and the pcr amplification of total DNA:
1) total DNA extraction is slightly done change by the method for " molecular cloning experiment guide " (third edition, 2002).Get the tail fin or the muscle (or fresh blood) of dehydrated alcohol preservation, TE liquid soaked 1-2 days, and middle the replacing 2 times begins to fall before TE.If fresh and alive material is then cleaned with physiological saline.Shred, add the DNA extraction lysate, even SDS and the Proteinase K of adding of shake, 55 ℃ of digestion are fully.Use saturated phenol, phenol/chloroform/primary isoamyl alcohol (25: 24: 1), chloroform/primary isoamyl alcohol (24: 1) extracting then, the centrifuging and taking supernatant, add small amount of N aCl and greater than the dehydrated alcohol of 2 times of volumes precipitation, use 70% ethanol rinsing then, remove ethanol, add an amount of TE dissolving after the seasoning, 4 ℃ of preservations, prolonged preservation then after the packing-20 ℃ of preservations standby.Get the part dna profiling and on the nucleic acid-protein quantitative instrument, measure A260 and A280 calculating concentration and purity.
2) pcr amplification is a template with total DNA, and the reaction system cumulative volume is 25 μ l, wherein, and distilled water 16.2 μ l, 10 * ExTaqbuffer, 2.5 μ l, dNTP 2.5 μ l, forward primer 1.3 μ l, reverse primer 1.3 μ l, total dna profiling 1 μ l, ExTaq archaeal dna polymerase 0.2 μ l.The PCR response procedures is determined according to the reality of primer.The PCR product reclaims test kit (worker is given birth in Shanghai) purifying with UNIQ-10 pillar PCR product after 1% agarose gel electrophoresis detects, serve Hai Shenggong and finish order-checking.
3) to the unfavorable primer of pcr amplification product (the 3rd, 7,9 pair of primer) after testing, design a pair of primer again in its outside respectively, carry out pcr amplification and order-checking by above-mentioned identical method.
4) with the online software of blast of Primer Premier 5.0 softwares and NCBI website first PCR product sequencing result is analysed and compared, and it is embedded in the neutralize homologous fragment position of this tract correspondence of original template, obtain a long mixed sequence like this, wherein a part is the plastosome sequence of ripple lip fish, and another part is the plastosome sequence of Halichoeres.With this mixed sequence is that template is carried out second batch of PCR design of primers again, but second batch of PCR primer location must drop on the plastosome sequence inside of ripple lip fish; Then according to above-mentioned 2) and 3) method increase and check order.The agarose gel electrophoresis figure of first and second batch of PCR product as shown in Figure 4 and Figure 5.
4, the splicing of gained sequence results:
To obtain 21 PCR sequencing fragment results with the online software of blast of Primer Premier 5.0 softwares and NCBI website and be spliced into a complete mtDNA; Utilize the DNAStar software package that sequence is carried out searching of open reading frame again, the acquisition of sequence complementary strand; And the ORFfinder software that utilizes the NCBI website is finished the translation of albumen coded sequence and the investigation of amino acid coding situation; Compare with online software of blast and template sequence and to determine each chondriogen and the particular location of other non-coding sequence in sequence.The plastosome complete sequence that obtains ripple lip fish as shown in Figure 7.
Claims (6)
1. be based upon a kind of improved mitochondrial genome complete sequence determination method on the conventional round pcr basis, comprise the steps: 1) screening of template; 2) primer design; 3) total DNA extraction, pcr amplification and order-checking; 4) sequence assembly of sequencing result.
2. method according to claim 1 is characterized in that: the species mitochondrial genome complete sequence that sifting sort belongs to same section on learning is as template, and itself and sequence homology to be measured get final product more than 75%.
3. according to claim 1 and 2 described methods, it is characterized in that: utilize Primer Premier 5.0 softwares to carry out design of primers, software Primer Premier 5.0 is at the software net
Http:// www.premierbiosoft.com/Download, design of primers divides 2 batches to carry out, and first primer number is half of total primer number, and the spacing distance between adjacent two pairs of primer amplification fragments is 1000-1200bp.
4. according to claim 1 and 2 described methods, it is characterized in that: the PCR product sequencing result of first primer and design of primers are template used to replace original sequence of corresponding site in the template sequence after relatively through blast software.
5. method according to claim 4, it is characterized in that: with the sequence that is inlaid with new sequence that obtains is that template is carried out second batch of design of primers, and, in second batch of primer, each all must drop in the new sequence primer, and guarantees that every section PCR product of second batch of primer and the new sequence in the template all have one section 50-100bp overlapping.
6. according to claim 1 and 3 described methods, it is characterized in that: for the bad primer of pcr amplification effect, introduce one couple of PCR primers again, inlay pcr amplification in its outside.
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Cited By (8)
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| CN104480207A (en) * | 2014-12-17 | 2015-04-01 | 杭州吉洛生物医药科技有限公司 | Primer pair capable of detecting specificity of human mitochondrial genome |
| CN105331726A (en) * | 2015-12-01 | 2016-02-17 | 上海派森诺生物科技股份有限公司 | Method for sequencing human mitochondria genetic set based on sanger |
| CN105653899A (en) * | 2014-09-30 | 2016-06-08 | 深圳华大基因研究院 | Method and system for determining mitochondria genome sequence information of various samples at the same time |
| CN107974507A (en) * | 2017-11-20 | 2018-05-01 | 浙江海洋大学 | A kind of curved spot octopus mitochondrial genome complete sequence primer and design, phylogenetic analysis and amplification method |
| CN109321644A (en) * | 2018-10-23 | 2019-02-12 | 杭州贝英福生物科技有限公司 | It is a kind of expand Pseudococcidae mitochondrial genomes kit and its application |
| CN110106257A (en) * | 2019-05-08 | 2019-08-09 | 绵阳师范学院 | The construction method of kamao phylogenetic tree |
| CN111808970A (en) * | 2020-07-13 | 2020-10-23 | 深圳市血液中心(深圳市输血医学研究所) | Kit and method for amplifying full-length mRNA sequences of GYPA, GYPB and GYPE genes |
| CN112301098A (en) * | 2020-10-22 | 2021-02-02 | 深圳市卫生健康发展研究中心 | Method for amplifying human mitochondrial DNA complete sequence and kit for amplifying human mitochondrial DNA complete sequence |
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| CN105653899A (en) * | 2014-09-30 | 2016-06-08 | 深圳华大基因研究院 | Method and system for determining mitochondria genome sequence information of various samples at the same time |
| CN105653899B (en) * | 2014-09-30 | 2018-02-09 | 深圳华大基因研究院 | The method and system of the mitochondrial genomes sequence information of a variety of samples is determined simultaneously |
| CN104480207A (en) * | 2014-12-17 | 2015-04-01 | 杭州吉洛生物医药科技有限公司 | Primer pair capable of detecting specificity of human mitochondrial genome |
| CN105331726A (en) * | 2015-12-01 | 2016-02-17 | 上海派森诺生物科技股份有限公司 | Method for sequencing human mitochondria genetic set based on sanger |
| CN105331726B (en) * | 2015-12-01 | 2019-02-12 | 上海派森诺生物科技股份有限公司 | Method based on sanger sequencing human mitochondria gene group |
| CN107974507A (en) * | 2017-11-20 | 2018-05-01 | 浙江海洋大学 | A kind of curved spot octopus mitochondrial genome complete sequence primer and design, phylogenetic analysis and amplification method |
| CN109321644A (en) * | 2018-10-23 | 2019-02-12 | 杭州贝英福生物科技有限公司 | It is a kind of expand Pseudococcidae mitochondrial genomes kit and its application |
| CN110106257A (en) * | 2019-05-08 | 2019-08-09 | 绵阳师范学院 | The construction method of kamao phylogenetic tree |
| CN111808970A (en) * | 2020-07-13 | 2020-10-23 | 深圳市血液中心(深圳市输血医学研究所) | Kit and method for amplifying full-length mRNA sequences of GYPA, GYPB and GYPE genes |
| CN112301098A (en) * | 2020-10-22 | 2021-02-02 | 深圳市卫生健康发展研究中心 | Method for amplifying human mitochondrial DNA complete sequence and kit for amplifying human mitochondrial DNA complete sequence |
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Application publication date: 20101103 |