CN101575600A - Super plasmid for producing DNA molecular weight standard with odd 200 bp gradients and preparation method thereof - Google Patents
Super plasmid for producing DNA molecular weight standard with odd 200 bp gradients and preparation method thereof Download PDFInfo
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- CN101575600A CN101575600A CNA2008100157784A CN200810015778A CN101575600A CN 101575600 A CN101575600 A CN 101575600A CN A2008100157784 A CNA2008100157784 A CN A2008100157784A CN 200810015778 A CN200810015778 A CN 200810015778A CN 101575600 A CN101575600 A CN 101575600A
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Abstract
The invention relates to a super plasmid for producing a DNA molecular weight standard with odd 200 bp gradients and a preparation method thereof, belonging to the preparation field of common reagents of molecular biology. The tailing activity of a Taq DNA polymerase and the selective gel recycle of a connecting product are utilized to construct a brand-new plasmid pYO7200 for producing the DNA molecular weight standard. The plasmid contains a series of odd section gradients (300 bp, 500 bp, 700 bp, 900 bp, 1500 bp and 3000 bp) and releases sections contained in the plasmid by restricted enzyme digestion so as to form a 200 bp nucleic acid molecular weight standard gradient (DNA marker). The preparation method of the DNA molecular weight standard with 200 bp gradients are as follows: converting, fermenting and culturing production strains, preparing and purifying target plasmids on a large scale and processing the target plasmids by enzyme digestion. The invention is characterized in that the developed production technologies and the genetic engineering plasmids can be utilized to rapidly and accurately prepare the odd 200 bp gradients for the DNA molecular weight standard on the large scale.
Description
Technical field
The present invention relates to molecular biology reagent preparation and genetically engineered Application Areas, be a kind of super plasmid that makes up by genetic engineering means and preparation method thereof specifically, this super plasmid is used to use the method for digestion with restriction enzyme to prepare DNA molecular weight standard with odd 200 bp gradients.
Background technology
Synthesize in the genetic manipulation process that reaches variety of way at DNA, need monitor synthetic or the dna molecular of operating size variation, method commonly used is to carry out electrophoresis simultaneously by the DNA hybrid standard thing (molecular weight standard, DNA marker) with dna molecular to be measured and a kind of known molecular amount, and the migration distance in electrophoresis process is judged the size of DNA sample molecule by dna molecular more to be measured and dna molecular standard.Therefore, the standard DNA mixture that needs some known molecular amounts of preparation.
The existing several different methods of the method for preparation standard DNA in the prior art:
Chemical synthesis: the method by chemical reaction is examined picric acids with 4 kinds of different ribodesoses: VITAMIN B4 (dATP), guanine (dGTP), cytosine(Cyt) dCTP), thymus pyrimidine (dTTP) combines, the dna fragmentation of synthetic target sizes.This method operation is very loaded down with trivial details, and efficient is extremely low, and synthetic DNA length is subjected to serious restriction, but the length of general synthetic DNA is no more than 400bp, the dna fragmentation of macromolecule can't directly synthesize with chemical synthesis, and cost is too high, thereby the preparation of the routine of inapplicable dna molecular amount.
Fragment connection method: the dna molecular (as 100bp) that obtains small molecular weight earlier with PCR or synthetic method, with the enzymatic ligation small molecule DNA is linked in sequence one by one again, form different sizes dna moleculars (100bp, 200bp, 300bp, 400bp ...).Say the dna molecular that can prepare all size on this theoretical method, but operation is very loaded down with trivial details, reproducibility is poor, can't realize mass preparation.
The polymerase chain reaction method: the archaeal dna polymerase chain reaction has been widely used in the preparation as gene clone and the DNA sheet.Be widely used for preparing dna molecular amount standard substance at present both at home and abroad.But this method wastes time and energy, and its throughput is limited.
Enzyme cutting method: cut with the plasmid of one or both restriction enzymes combination, produce the dna fragmentations of different sizes, with as dna molecular amount standard to phage DNA or artificial design.The advantage of this method is a large amount of easily preparations, the productive rate height.But the shortcoming of this method is also very outstanding: the dna fragmentation size that enzyme is cut generation is subjected to the restriction of used Restriction Enzyme kind, and its restraining factors are design levels of artificial constructed plasmid.
For a long time, molecular biology reagent and genetically engineered Application Areas, need a kind of high-caliber artificial constructed super plasmid mentality of designing, cut the restriction that the dna fragmentation size of generation is subjected to used Restriction Enzyme kind to overcome enzyme, to produce DNA molecular weight standard with odd 200 bp gradients fast, accurately, in large quantities.
Summary of the invention
Through secular laboratory study, the present invention has exactly satisfied the demand, and its purpose is: provide a kind of that make up, brand-new by genetic engineering means, can be fast, accurately, enzyme cutting method is produced the super plasmid of 200bp odd number gradient dna molecular amount standard in large quantities.
The present invention can further purpose be: the preparation method that a kind of super plasmid of the 200bp of production odd number gradient dna molecular amount standard is provided.
To achieve these goals, the present invention has adopted following technical scheme:
Method by PCR is increased respectively 300bp, 500bp, 700bp, 900bp, 1500bp, a few bar segment of template with Lambda DNA, wherein 1500bp and 900bp increase by the pfu archaeal dna polymerase, and 300bp-A, 300bp-B, 500bp, 700bp increase with the Taq archaeal dna polymerase respectively.Utilize the tailing activity of Taq archaeal dna polymerase that 1500bp and 900bp two bar segment are added T then.Utilize the A/T complementary principle system that connects again in centrifuge tube, utilize the T4 ligase enzyme to make 500bp, 700bp, 900bp connect into the unit of a 500-700-900, same 300bp-A, 300bp-B and 1500bp connect into the unit of 300A-1500-300B.By T/A clone test kit, said two units is cloned into formation intermediate carrier T579 and T3153 in the T carrier respectively then.
Cut by Pst I enzyme the 300A-1500-300B unit is cut out from T3153, and utilize nucleic acid purification to reclaim test kit and reclaim and purifying.While T579 carrier is also cut holoenzyme by Pst I enzyme and cuts, and utilizes calf intestine alkaline phosphatase (CIAP) to carry out the carrier dephosphorization treatment, to prevent that carrier is from connecting.Set up suitable linked system, the 300-1500-300 unit is connected the into T579 carrier gained intermediate carrier called after pYO7200 of dephosphorization treatment.
The pYO7200 that is obtained is a kind of brand-new super dna molecular amount standard plasmid.Wherein contain 300bp, 500bp, 700bp, 900bp, 1500bp, 3,000 six bar segment, wherein 300bp is two copies, the fluorescence intensity when observing to strengthen it.Plasmid pYO7200 can obtain a 200bp odd number dna molecular amount normal gradients of being made up of 6 bar segment after EcoR I enzyme is cut.Have certain ratio row relations (6: 5: 7: 9: 15: 30) simultaneously between the concentration (quality) of each bar DNA band.
Beneficial effect of the present invention is: utilize sophisticated production technique and genetically engineered to make up a kind of artificial super plasmid, this plasmid is a kind of brand-new dna molecular amount standard plasmid, this plasmid is cut preparation 200bp odd number gradient dna molecular amount standard, the dna fragmentation size that has overcome traditional enzyme cutting method generation is subjected to the restriction of used Restriction Enzyme kind, a large amount of easily preparations, the productive rate height has been realized purpose of the present invention.
Description of drawings
Fig. 1 is the super plasmid pYO7200 collection of illustrative plates that is used to produce DNA molecular weight standard with odd 200 bp gradients.
Embodiment
Produce the super plasmid preparation method of DNA molecular weight standard with odd 200 bp gradients, specifically comprise the steps:
1. the method by PCR is increase respectively 300bp, 500bp, 700bp, 900bp, 1500bp, a few bar segment of template with Lambda DNA, wherein 1500bp and 900bp increase by the pfu archaeal dna polymerase, and 300bp-A, 300bp-B, 500bp, 700bp increase with the Taq archaeal dna polymerase respectively.
2. utilize the tailing activity of Taq archaeal dna polymerase that 1500bp and 900bp two bar segment are added T.
3. utilize the A/T complementary principle system that connects in centrifuge tube, utilize the T4 ligase enzyme to make 500bp, 700bp, 900bp connect into the unit of a 500-900-700, same 300bp-A, 300bp-B and 1500bp connect into the unit of 300A-1500-300B.
4. by T/A clone test kit, two unit of above-mentioned 500-900-700,300A-1500-300B are cloned into formation intermediate carrier T579 and T3153 in the T carrier respectively.
5. cut by Pst I enzyme the 300A-1500-300B unit is cut out from T3153, and utilize nucleic acid purification to reclaim test kit and reclaim and purifying.While T579 carrier is also cut holoenzyme by Pst I enzyme and cuts, and utilizes calf intestine alkaline phosphatase (CIAP) to carry out the carrier dephosphorization treatment, to prevent that carrier is from connecting.
6. connecting the 300-1500-300 unit into, the T579 carrier of dephosphorization treatment obtains plasmid, called after pYO7200.
The pYO7200 that is obtained is a kind of brand-new super dna molecular amount standard plasmid.Wherein contain 300bp, 500bp, 700bp, 900bp, 1500bp, 3,000 six bar segment, wherein 300bp is two copies, the fluorescence intensity when observing to strengthen it.Plasmid pYO7200 can obtain a 200bp odd number dna molecular amount normal gradients of being made up of 6 bar segment after EcoR I enzyme is cut.Have certain ratio row relations (6: 5: 7: 9: 15: 30) simultaneously between the concentration (quality) of each bar DNA band.
Claims (4)
1. produce the super plasmid of DNA molecular weight standard with odd 200 bp gradients, this plasmid is a kind of brand-new super dna molecular amount standard plasmid, it is characterized in that: wherein contain 300bp, 500bp, 700bp, 900bp, 1500bp, 3000bp six bar segment; 300bp is two copies; After cutting, EcoR I enzyme can obtain a 200bp odd number dna molecular amount normal gradients of forming by 6 bar segment; Have certain ratio row relations (6: 5: 7: 9: 15: 30) simultaneously between the concentration (quality) of each bar DNA band.
2. the preparation method of the super plasmid of production DNA molecular weight standard with odd 200 bp gradients according to claim 1, comprise that the method by PCR is increase respectively 300bp, 500bp, 700bp, 900bp, 1500bp, a few bar segment of template with Lambda DNA, it is characterized in that:
(1) 300bp-A, 300bp-B, 500bp, 700bp are increased by polysaccharase respectively;
(2) 1500bp, 900bp are increased by polysaccharase respectively;
(3) utilize the tailing activity of Taq archaeal dna polymerase that 1500bp and 900bp two bar segment are added T;
(4) utilize the A/T complementary principle system that in centrifuge tube, connects, utilize the T4 ligase enzyme to make 500bp, 700bp, 900bp connect into the unit of a 500-900-700, same 300bp-A, 300bp-B and 1500bp connect into the unit of 300A-1500-300B;
(5) by T/A clone test kit, two unit of above-mentioned 500-900-700,300A-1500-300B are cloned into formation intermediate carrier T579 and T3153 in the T carrier respectively;
(6) cut by Pst I enzyme the 300A-1500-300B unit is cut out from T3153, and utilize nucleic acid purification to reclaim test kit and reclaim and purifying; While T579 carrier is also cut holoenzyme by Pst I enzyme and cuts, and utilizes calf intestine alkaline phosphatase (CIAP) to carry out the carrier dephosphorization treatment, to prevent that carrier is from connecting;
(7) connecting the 300-1500-300 unit into, the T579 carrier of dephosphorization treatment obtains plasmid pYO7200.
3. the super plasmid preparation method of production even 200 bp gradient dna molecular amount standard according to claim 2, it is characterized in that: described 300bp-A, 300bp-B, 500bp, 700bp increase by polysaccharase respectively, specifically are meant with the Taq archaeal dna polymerase and increase.
4. the super plasmid preparation method of production even 200 bp gradient dna molecular amount standard according to claim 2, it is characterized in that: described 1500bp, 900bp increase by polysaccharase respectively, specifically are meant with the pfu archaeal dna polymerase and increase.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101880660A (en) * | 2010-06-11 | 2010-11-10 | 济南大学 | DNA small fragment preparation template p1251 in DL2000 based on PCR (Polymerase Chain Reaction) amplification and preparation system thereof |
| CN102304508A (en) * | 2011-07-29 | 2012-01-04 | 上海捷瑞生物工程有限公司 | Method for preparing DL2000 DNA molecular weight marker as well as product and applications thereof |
| 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 |
| CN107653240A (en) * | 2017-10-23 | 2018-02-02 | 福建省农业科学院农业质量标准与检测技术研究所 | A kind of DNA MarkerI molecular weight standards and preparation method and application |
-
2008
- 2008-05-07 CN CNA2008100157784A patent/CN101575600A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101880660A (en) * | 2010-06-11 | 2010-11-10 | 济南大学 | DNA small fragment preparation template p1251 in DL2000 based on PCR (Polymerase Chain Reaction) amplification and preparation system thereof |
| CN102304508A (en) * | 2011-07-29 | 2012-01-04 | 上海捷瑞生物工程有限公司 | Method for preparing DL2000 DNA molecular weight marker as well as product and applications thereof |
| CN102304508B (en) * | 2011-07-29 | 2013-05-01 | 上海捷瑞生物工程有限公司 | Method for preparing DL2000 DNA molecular weight marker as well as product and applications thereof |
| 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 |
| CN107653240A (en) * | 2017-10-23 | 2018-02-02 | 福建省农业科学院农业质量标准与检测技术研究所 | A kind of DNA MarkerI molecular weight standards and preparation method and application |
| CN107653240B (en) * | 2017-10-23 | 2019-12-03 | 福建省农业科学院农业质量标准与检测技术研究所 | A kind of DNA-MarkerI molecular weight standard and the preparation method and application thereof |
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