CN106497927A - A kind of DNA Marker and its preparation technology - Google Patents
A kind of DNA Marker and its preparation technology Download PDFInfo
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- CN106497927A CN106497927A CN201610982566.8A CN201610982566A CN106497927A CN 106497927 A CN106497927 A CN 106497927A CN 201610982566 A CN201610982566 A CN 201610982566A CN 106497927 A CN106497927 A CN 106497927A
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Abstract
The invention discloses a kind of electrophoretic band for indicating DNA molecular amount size clearly DNA Marker and its preparation technology, belong to biological technical field.The present invention designs a upstream primer according to carrier S EQ No.1, and amplification length is respectively 100bp, 250bp, 500bp, 750bp, 1000bp, 7 downstream primers of 1500bp, 2000bp, enter performing PCR amplification with carrier S EQ No.1 as template to target DNA fragment, determine amplified production content and carry out DNA fragmentation formulation optimization, obtain containing 7 DNA fragmentation 100bp, 250bp, 500bp, 750bp, 1000bp, the DL2000 DNA Marker of 1500bp, 2000bp.DL2000 DNA Marker disclosed by the invention devise the band of a 1500bp compared with commercially available DL2000 DNA Marker more, and the method is suitable for preparing the DNA Marker containing expanding the different size fragment for arriving according to their needs.
Description
Technical field
The present invention relates to biological technical field, more particularly to a kind of DNA Marker for indicating nucleic acid molecular weight size
And its preparation technology.
Background technology
Late 1970s to the beginning of the eighties, due to the continuous development of neontology, molecular biology, gene work
The appearance of journey, DNA recombinant techniques etc., based on bio-genetic material DNA based on, explain vital movement rule from microcosmic angle
Research and development are carried out like a raging fire.In these R＆D works for being related to nucleic acid substances DNA, DNA Marker are most widely used
One of reagent, it can judge and indicate nucleic acid molecular weight size, and it is fixed to carry out as nucleic acid molecular weight standard and to nucleic acid fragment
Amount analysis.
DNA Marker contain the DNA fragmentation of different molecular weight, and which is mainly used for the agarose gel electrophoresis in nucleic acid
When, it is used to refer to the molecular size range of unknown fragment in nucleic acid electrophoresis.It is involved by prepared by DNA Marker according to DNA molecular amount standard
And technology can be divided into digestion with restriction enzyme plasmid and phage DNA and round pcr amplification, the former main deficiency is
Restriction enzyme site is excessive or very few so that digestion products DNA sizes get too close to or too wide in the gap, it is impossible to according to target DNA fragment
The DNA fragmentation that size design is consistent, can not specify the molecular mass of DNA in specifically testing well；As PCR is expanded
There is the ability of powerful amplification of DNA fragments, there is no technical problem using fabrication techniques DNA Marker.Existing scholar's base
In the DNA fragmentation that round pcr amplifies 100-1000bp, make DL1000 Marker, DL1000 prepared using multiplex PCR
DNA Marker.The key that DNA molecular amount standard is prepared using round pcr is the choosing of the design of target DNA fragment size, template
Select, design of primers, PCR reaction conditions are groped, the DL2000 DNA Marker products prepared by the present invention set than conventional Chang Pin more
The DNA fragmentation that a size is 1500bp has been counted, the instruction that molecular weight is not higher than 2000bp target DNA fragments has been suitable for, special
It is not using prokaryotic micro-organisms 16S rDNA（About 1500bp）Gene pairs its carry out in the test of Species estimation.Of the invention used
Amplification template sequence is SEQ NO1, and size is about 5900bp, can be used for below 5kb, can expand the target sizes DNA fragmentation for arriving
Amplification.
Content of the invention
It is an object of the invention to provide a kind of DNA Marker and its preparation technology for indicating nucleic acid molecular weight size
DL2000 DNA Marker provided by the present invention be by 2000 bp, 1500bp, 1000 bp, 750 bp, 500 bp,
Totally 7 double chain DNA fragments are constituted for 250 bp and 100 bp, are mixed with sample-loading buffer, be can be directly used for gel electrophoresis,
5 μ l of each loading；For ease of observing after electrophoresis, 1000bp bands are most bright, about 100ng, and other are about 50ng；It is used as to indicate core
The molecular size range of unknown size DNA fragmentation in sour electrophoresis.
The preparation technology of DL2000 DNA Marker provided by the present invention is the design with plasmid SEQ No.1 as template
One upstream primer and 7 downstream primers, using PCR amplification size be respectively 2000 bp, 1500bp, 1000 bp, 750 bp,
The DNA fragmentation of 500 bp, 250 bp and 100 bp, determines amplified production content and carries out DNA fragmentation formulation optimization, finally
Obtain that designing according to target DNA stripe size, band is clear after electrophoresis and dyeing, the DL2000 DNA of appropriate density
Marker.
The design and synthesis of primer
By referring to the sequence/DNA coding sequence of plasmid SEQ No.1, in conjunction with 1 upstream of Ediseq and PrimerSelect Software for Design
Primer and 7 downstream primers, and send biotech firm to be synthesized.Its sequence is shown in Table 1：
Table 1 prepares DNA Marker the primer sequences.
The activation of bacterial classification and plasmid extraction
The activation of bacterial classification：JM109 (SEQ No.1) glycerol stock is connected in LB fluid nutrient mediums, in 37 DEG C of 140rpm overnight shakings
Culture 8-10 hours.It is uniformly coated on the LB solid mediums containing ampicillin after culture 12h with the bacterial classification for having activated, is chosen
Single bacterium colony is taken on fluid nutrient medium, plasmid overnight after shaking table, is extracted.The extraction of plasmid：Using alkaline lysis method of extracting plasmid DNA.
The PCR amplifications of target DNA fragment and reaction condition optimization
Using the PCR reaction systems of 50 μ l, including：ddH239 μ l of O, the 1 μ l of dNTPs of 10 × PCR Buffer 5 μ l, 10mM,
10 μM of 1 μ l of primer all-F, 10 μM of 1 μ l of downstream primer-R, 1 μ l of DNA profiling, the 1 μ l of Taq archaeal dna polymerases of 2U/ μ l.
Amplification condition is：94 DEG C of denaturations 5min, 94 DEG C of denaturation 30s, annealing temperature 49-54 DEG C 30s, 72 DEG C of extension 1-3min, common 30-
35 circulation, 72 DEG C re-extend 10min.DNA purpose bands are detected with 1% agarose gel electrophoresis.According to measurement result, carry out
The optimization of PCR conditions, such as changes the amount of the dNTPs in PCR reaction systems, the annealing temperature, circulation revolution in reaction condition with
And extension of time, make the purpose band of amplification clear and single.
The optimal PCR reaction conditions of 2 each DNA fragmentation of table.
DNA Marker are designed
In the Marker, the DNA fragmentation final concentration design of 1000bp is about 20ng/ μ l, and remaining each DNA fragmentation final concentration is designed about
For 10ng/ μ l.
DNA fragmentation assay：DNA fragmentation by low-molecular-weight in amplified production（100bp, 250bp）Quickly pure with DNA
Changing kit carries out determining nucleic acid content with ultramicron nucleic acid-protein concentration mensuration instrument after purification；Remaining each fragment directly uses ultra micro
Amount nucleic acid-protein concentration mensuration instrument carries out assay.
A small amount of DNA Marker formulas grope and system is amplified：By prefabricated Marker volumes calculate each DNA fragmentation content and
Sample volume is answered, the PCR primer mixing of the above DNA fragmentation of respective volume 500bp is taken, is added the 100bp for having purified according to quantity,
The DNA fragmentation of 250bp, detects Marker effects with agarose gel electrophoresis, according to the concentration that electrophoretic effects finely tune each fragment, determines
The proportioning of each amplified production.Reaction system is amplified to 400 μ l.For increasing the identification effect of DNA Marker, prominent bar
Band, marks bright 1000bp.Ratio in 2.5% adds the glycerine of Loading Buffer and 7.5%, uses Ago-Gel after mixing
Electrophoresis detection DNA Marker effects, according to the concentration that Ago-Gel image finely tunes each fragment, obtain good results, band clear
The highlighted DL2000 DNA Marker of clear, 1000bp.
Description of the drawings
The each fragment PCR amplifications of Fig. 1 DL2000 DNA Marker
Lane 1 ~ 7 is respectively 100bp, the amplified production of 250bp, 500bp, 750bp, 1000bp, 1500bp, 2000bp.
The agarose gel electrophoresis of Fig. 2 DL2000 DNA Marker
Lane 1~4 DL2000 DNA Marker.
Specific embodiment
DNA Marker of the present invention and its preparation technology implementation content are described with embodiment.But present disclosure not office
It is limited to following examples.
Embodiment：Prepare 400 μ l DL2000 DNA Marker.
（1）The design of primer
SEQ No.1 plasmid sequences are found in NCBI（GenBank：CS054777.1）DNAStar-Lasergene software is used afterwards
It is designed primer, primer sequence such as table 1.
（2）The recovery of bacterium
From ultra-low temperature storage case（-86℃）Middle taking-up e. coli jm109 (SEQ No.1）, 50 μ l are taken on aseptic operating platform to be added
To 5ml plus 5 μ l ampicillins（Amp）In LB fluid nutrient medium test tubes, it is positioned in gas bath shaking table with 37 DEG C, 140r/
The temperature of min and rotating speed culture 10-12h.
（3）The purifying of bacterium
Take JM109 (the SEQ No.1 of activation）100 μ l are coated on the solid medium containing ampicillin, in 37 DEG C of illumination
Culture 16-18h is inverted in constant incubator.Single bacterium colony is chosen with sterilized toothpick, the LB liquid containing ampicillin is put into
In culture medium, put in temperature control gas bath shaking table with 37 DEG C, 140r/min culture 10-12h standby.
（4）The extraction of DNA：With alkaline lysis method of extracting plasmid DNA, comprise the following steps that：
A. bacterium solution is proceeded in the Eppendorf pipes of 1.5ml, bacterial precipitation is eliminated to ttom of pipe with 8000r/min rotating speed 1min
Upper strata culture medium；
B. the solution I of 100 μ l precoolings is added, the abundant suspended bacterial of vortex oscillation instrument is placed, adds 4 μ l RNase, room temperature to place
2min.The solution II of 200 μ l is added, quick reverse, gentle mixing, ice bath 5min.150 μ l on ice precoolings are added thereto to again
Solution III, gentle mixing, ice bath 5min；
C.12000r/min rotating speed 5min, precipitate white floccule, supernatant liquor is gone in another 1.5mlEppendorf pipes.
D. the absolute ethyl alcohol of 2 times of supernatant liquor volumes and precooling is added thereto to, is mixed, placed and -20 DEG C of refrigeration 20min；
E. 12000r/min rotating speed 5min after taking out, eliminate supernatant liquor.70% ethanol purge of 500 μ l is added to precipitate, with
3000r/min rotating speeds 1min precipitates DNA, air-dries, and thoroughly volatilization removes ethanol；
F. 40 μ l distilled water dissolving DNAs are added, and remaining is positioned over -20 DEG C of refrigerations, stand-by.
(5) PCR amplifications and the optimization of reaction condition
PCR reaction systems using 50 μ l：ddH239 μ l of O, the 1 μ l of dNTPs of 10 × PCR Buffer 5 μ l, 10mM, 10 μM
1 μ l of primer all-F, 10 μM of 1 μ l of primer-R, 1 μ l of DNA profiling, the 1 μ l of Taq archaeal dna polymerases of 2U/ μ l.PCR reacts
DNTPs, template DNA in system,TaqDNA Polymerase consumptions, primer and Mg2+Concentration according to experimental result
Different and be adjusted optimizations, by regulation dNTPs,TaqThe consumption of DNA Polymerase and other holding primary quantities.And
Initially drafting for annealing temperature is according to Tm=according to the content of A, T, C, G in designed primer（4*G/C+2*A/T-5）To divide
Do not calculate, the Tm values further according to upstream and downstream primer calculate T=（Tm1+Tm2）/ 2-5, is then calculating 2 DEG C -3 DEG C of gained temperature plus-minus
Carry out setting range, after the completion of grads PCR instrument 8 thermogrades can be provided according to setting range.By finely tuning in PCR conditions
The consumption of dNTPs and period realize the optimization of each band improving genes of interest concentration, obtain one objectively optimum
PCR conditions, finally determine that the optimal PCR reaction conditions of each DNA fragmentation are shown in Table two.
(6) each DNA fragmentation formulation optimization
In order to project indicating effect, the DNA fragmentation of 1000bp is set to highlighted, the final concentration in DNA Marker is about 20
Ng/μ l, remaining DNA fragmentation final concentration design is for about 10 ng/μ l；DNA fragmentation by low-molecular-weight（100bp, 250bp）With
Nucleic acid content is determined with ultramicron nucleic acid-protein concentration mensuration instrument after DNA fast purifying kits, remaining DNA fragmentation is direct
Nucleic acid content is determined with ultramicron nucleic acid-protein concentration mensuration instrument.
After expanding each segment DNA under optimum reaction condition, 2000 bp, 1500bp, 1000 bp, 750 bp, 500 bp
DNA fragmentation content be respectively after measured：89.68 ng/μl、89.68 ng/μl、86.87 ng/μl、80.65 ng/μl、
113.00 ng/μl.In addition, 250 bp, the PCR primer of 100 bp are concentrated after purification, content is 117.42 ng/ μ l.
Volume need to be added to be respectively according to DNA fragmentation cubage：44.6μl、44.6μl、92.1μl、49.6μl、35.4μl、
34.1μl、34.1μl.Respective amount mixing is taken by result of calculation, the ratio in 2.5% adds 20 μ l Loading Buffer
With the 7.5% i.e. glycerine of 30 μ l, with aqua sterilisa constant volume to 400 μ l are close to, after mixing, DNA is detected with agarose gel electrophoresis
Marker effects, according to the concentration that Ago-Gel image finely tunes each fragment, obtain that good results, band be clear, 1000bp is high
Bright DL2000 DNA Marker.
Claims (7)
1. a kind of DL2000 DNA Marker, including：100bp, 250bp, 500bp, 750bp, 1000bp, 1500bp,
7 different size DNA fragmentations such as 2000bp, after agarose gel electrophoresis and dyeing, each DNA bands clearly become clear, and may indicate that
DNA fragmentation molecular size range.
2. material is prepared according to the DNA Marker described in claims 1, it is characterised in that：The template of PCR reactions, amplification
The primer, the template that is reacted as PCR with carrier S EQ No.1.
3. upstream primer used by expanding：all-F： 5’-ACAATTCCCCTATAGTGAGTCGTATT-3’
Downstream primer 7 used by amplification：
2k-R: 5’-CGAAGACCATTCATGTTGTTGCT-3’
1.5k-R： 5’-TCACTGCCCGCTTTCCAGT-3’
1k-R：5’-GCGCCGAGACAGAACTTAATGG-3’
750b-R：5’-CACCGCCGCTTTACAGG-3’
500b-R：5’-CTGGCCTGGTTCACCACGCG-3’
250b-R：5’-AATGGTGCATGCAAGGAGATG-3’
100b-R：5’-CCTGTGGCGCCGGTGATGC-3’.
4. according to the preparation technology of the DNA Marker described in claims 1：It is characterized in that PCR reaction systems and amplification bar
Part, the proportioning of amplified production.
5. its preparation technology is as follows：
PCR reaction systems and amplification condition：Using the PCR reaction systems of 50 μ l, including：ddH2O 39μl、10×PCR Buffer
The 1 μ l of dNTPs of 5 μ l, 10mM, 10 μM of 1 μ l of primer-F, 10 μM of 1 μ l of primer-R, 1 μ l of DNA profiling, the Taq of 2U/ μ l
1 μ l of archaeal dna polymerase.
6. amplification condition is：94 DEG C of denaturations 5min, 94 DEG C of denaturation 30s, annealing temperature 49-54 DEG C 30s, 72 DEG C of extension 1-
3min, common 30-35 circulation, 72 DEG C re-extend 10min, with agarose gel electrophoresis detect expanding effect, use ultramicron nucleic acid
Determination of protein concentration instrument determines nucleic acid content.
7. the ratio optimization of amplified production：DNA fragmentation by low-molecular-weight（100bp, 250bp）With DNA fast purifying kits
Assay is carried out after purification；The DNA fragmentation of 1000bp is pressed final concentration and is about 20 μ g/ μ l, and remaining each DNA fragmentation is by dense eventually
Degree is about 10 μ g/ μ l and is prepared, and the ratio in 2.5% adds the glycerine of Loading Buffer and 7.5%, uses fine jade after mixing
Sepharose electrophoresis detection DNA Marker effects, according to the concentration that Ago-Gel image finely tunes each fragment, obtain effect full
Meaning, the DL2000 DNA Marker that band is clear, 1000bp is highlighted.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108103179A (en) * | 2017-12-29 | 2018-06-01 | 武汉艾德士生物科技有限公司 | A kind of DNAmarker of variable bin number and its preparation |
| CN109355302A (en) * | 2018-10-17 | 2019-02-19 | 广东石油化工学院 | A kind of simple preparation method of DNA-Maker |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102337284A (en) * | 2011-09-30 | 2012-02-01 | 生工生物工程(上海)有限公司 | Plasmid for preparing DNA Marker, and construction method and application thereof |
| CN103276004A (en) * | 2013-03-01 | 2013-09-04 | 生工生物工程（上海）股份有限公司 | DNA marker plasmid and preparation and application thereof |
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2016
- 2016-11-09 CN CN201610982566.8A patent/CN106497927A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102337284A (en) * | 2011-09-30 | 2012-02-01 | 生工生物工程(上海)有限公司 | Plasmid for preparing DNA Marker, and construction method and application thereof |
| CN103276004A (en) * | 2013-03-01 | 2013-09-04 | 生工生物工程（上海）股份有限公司 | DNA marker plasmid and preparation and application thereof |
Non-Patent Citations (2)
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| 吕金浮 等: "基于ＰＣＲ技术快速制备ＤＮＡ Ｍａｒｋｅｒ的研究", 《河南农业科学》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108103179A (en) * | 2017-12-29 | 2018-06-01 | 武汉艾德士生物科技有限公司 | A kind of DNAmarker of variable bin number and its preparation |
| WO2019129172A1 (en) * | 2017-12-29 | 2019-07-04 | 武汉艾德士生物科技有限公司 | Dna marker with variable number of bands and preparation thereof |
| CN109355302A (en) * | 2018-10-17 | 2019-02-19 | 广东石油化工学院 | A kind of simple preparation method of DNA-Maker |
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