CN102329856A - Sanger sequencing reaction optimizing agent as well as preparation method and application thereof - Google Patents
Sanger sequencing reaction optimizing agent as well as preparation method and application thereof Download PDFInfo
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- CN102329856A CN102329856A CN2011101658166A CN201110165816A CN102329856A CN 102329856 A CN102329856 A CN 102329856A CN 2011101658166 A CN2011101658166 A CN 2011101658166A CN 201110165816 A CN201110165816 A CN 201110165816A CN 102329856 A CN102329856 A CN 102329856A
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Abstract
The invention discloses a Sanger sequencing reaction optimizing agent, which comprises 2.7-5mol/L of Betaine, 3-10 mol/L of dithiothreitol (DTT), 3-8% of DMSO (dimethylsulfoxide) and 30-80 mol/L of BSA (Bovine Serum Albumin). The optimizing agent disclosed by the invention is composed of four ingredients, namely Betaine, DTT, DMSO and BSA. During Sanger sequencing reaction preparation, 1/5 volume of optimizing agent is added to build a new sequencing reaction preparation system, and basic group read length and accuracy are obviously improved. Compared with the condition of adding no optimizing agent, a small quantity of optimizing agent disclosed by the invention is added to obtain the effect of longer read length, better peak type, more accurate result, better repeatability and better stability, a difficult template with a special structure and high GC (Gas Chromatograph) content can be optimized, and a use value is provided for the molecular biology sequencing technology. The invention provides a preparation method of the Sanger sequencing reaction optimizing agent.
Description
Technical field
The present invention relates to a kind of Sanger sequencing reaction and optimize agent, specifically a kind of Sanger sequencing reaction system to order-checking peak figure, read long optimization agent with good optimization function, belong to technical field of molecular biology.
Background technology
The Sanger PCR sequencing PCR mainly comprises 5 steps of foundation, sequencing reaction purifying, fluoroscopic examination of specimen preparation, sample detection, sequencing reaction system; Wherein the foundation of sequencing reaction system is the marrow place of Sanger PCR sequencing PCR; Add in the reaction system and have 4 kinds of fluorescently-labeled ddNTP of different colours; Accomplish the stopping of reaction and fluorescent mark, thereby when machine detects, find the fluorescent mark of 4 kinds of different colours, read corresponding fluorescence color and obtain 4 kinds of base information.The 3730XL of ABI can obtain high quality peak figure and the sequence results of 500bp now, and the peak figure after the 500bp and the accuracy of sequence have decline significantly.In actual mechanical process, we often go for longer sequence and result more accurately, relevantly optimize agent and optimize the sequencing reaction system so we obtain through experiment, obtain better peak figure and sequence results.
Summary of the invention
In order to remedy the defective of prior art, the present invention proposes a kind of Sanger sequencing reaction and optimizes agent, can play significant effect to the sequencing reaction result, and it is longer that order-checking is read, and sequence is more accurate.
Another object of the present invention is to propose a kind of preparation method of above-mentioned optimization agent.
The 3rd purpose of the present invention is to propose a kind of recommendation method of use of above-mentioned optimization agent.
To achieve these goals, invention thinking of the present invention is:
Invention thinking of the present invention is:
Trimethyl-glycine, WR 34678, DMSO have the amplification regulating and controlling effect that well is directed against template complex; The preparation of optimizing all ingredients does not influence promptly that the interference to optical dye reaches set effect again in the order-checking system, we according to a large amount of experiments and rationally the concentration of preparation all ingredients reach best result.
The concrete technical scheme of the present invention is:
A kind of Sanger sequencing reaction is optimized agent, and this optimization agent comprises trimethyl-glycine (Betaine), WR 34678 (DTT), DMSO and BSA; Its proportioning is:
2.7～5mol/L?Betaine、3～10mmol/L?DTT、3％～8％DMSO、30～80mg/L?BSA。
The best proportioning of said each composition is:
2.7mol/L?Betaine、6.7mmol/L?DTT、6.7％DMSO、55mg/L?BSA。
A kind of described Sanger sequencing reaction is optimized the preparation method of agent, and concrete steps are following:
(1) according to above-mentioned trimethyl-glycine proportioning preparation trimethyl-glycine;
(2) in the trimethyl-glycine that configures, add DTT, DMSO and the BSA for preparing in the ratio of claim 1 or 2, mix;
(3) filtration sterilization ,-20 ℃ of preservations after the packing.
The application of above-mentioned optimization agent in the Sanger sequencing reaction.
Above-mentioned application wherein, adds the claim 1 or the 2 described optimization agent of 1/5 sequencing reaction system volume in Sanger sequencing reaction system.
Precaution:
(1) Betaine is a monohydrate, non-hydrochloride compound, and other composition is added in the back that is dissolved in water.
(2) with join at present, guaranteed reagent,G.R. is fresh at present for DTT, and-20 ℃ of storages are no more than three months.
(3) DMSO is a thick liquid, and sampling accurately.
(4) the BSA storage liquid is avoided multigelation.
Advantage of the present invention and benefit:
Optimization agent of the present invention is grouped into by Betaine, DTT, DMSO (DMSO 99.8MIN.), four kinds of one-tenth of BSA.The present invention adds 1/5 volume and optimizes agent when Sanger PCR sequencing PCR prepared in reaction, set up a kind of new sequencing reaction and prepare system, reads to be significantly increased on long and the accuracy in base.Optimization agent of the present invention is compared with not adding the optimization agent, and an a small amount of interpolation of need can obtain reading longer, and the peak type is more excellent; The result is more accurate, and repeatability is better, and stability is better; Difficult template to special construction and high GC content has optimization function, to the molecular biology sequencing technologies use value is provided.The invention provides the preparation method.
Description of drawings
Fig. 1 does not add the preceding 200bp place of the order-checking peak figure that optimizes agent, the background peak height, and the order-checking effect is undesirable.
Fig. 2 is for adding the preceding 200bp place of the order-checking peak figure that optimizes agent, and background peaks is low, and it is satisfactory for result to check order.
Fig. 3 does not add Figure 50 0-520bp place, order-checking peak that optimizes agent, and the peak figure of repeating structure place is disorderly and influence subsequent peak figure, and the order-checking effect is undesirable.
Fig. 4 is for adding Figure 50 0-520bp place, order-checking peak that optimizes agent, and repeating structure does not influence peak figure, and it is satisfactory for result to check order.
Fig. 5 does not add the order-checking peak figure that optimizes agent to stop at the 600bp place.
Fig. 6 appoints so effective peak figure for adding the order-checking peak figure that optimizes agent at the 800bp place.
Embodiment
The preparation of embodiment 1 the present invention optimization agent
Optimize the agent composition:
2.7mol/L?Betaine、6.7mmol/L?DTT、6.7％DMSO、55μg/mlBSA。
Concrete preparation method
(1) preparation 2.7mol/L Betaine (Sigma company >=98%):
In the 50ml container, add 18.2g Betaine, add 40ml dd H
2O fully dissolves.
(2) preparation 1mol/L DTT:
With 20ml 0.01mol/L sodium acetate (AR of Beijing chemical reagents corporation) solution (pH5.2) dissolving 3.09gDTT (BioUltra of Sigma company, for molecular biology, >=99.5% (RT)), be distributed into the 1ml aliquot after the filtration sterilization and be stored in-20 ℃.
(3) 1mol/L DTT is joined in the 2.7mol/L Betaine solution:
Add 3.35ml 1mol/L DTT dilution in the 50ml volume and be 6.7mmol/L DTT.
(4) add DMSO (AR of Beijing chemical reagents corporation):
Add 3.35ml 100%DMSO dilution in the 50ml volume and be 6.7%DMSO.
(5) add BSA:
Adding 275 μ l 10mg/ml standard BSA dilution in the 50ml volume is 55 μ g/ml.
(6) filtration sterilization:
Use 0.22 μ m strainer filtration sterilization, after the packing of 1ml/ pipe ,-20 ℃ of preservations.
The preparation of embodiment 2 the present invention optimization agent
Optimize the agent composition:
5mol/L?Betaine、3mmol/L?DTT、8％DMSO、80μg/mlBSA。
Adopt the art methods preparation according to the mentioned component proportioning.
The preparation of embodiment 3 the present invention optimization agent
Optimize the agent composition:
3mol/L?Betaine、10mmol/L?DTT、3％DMSO、30μg/mlBSA。
Adopt the art methods preparation according to the mentioned component proportioning.
The concrete application examples of embodiment 4 the present invention optimization agent
The present invention " optimizes agent based on the reaction of Sanger PCR sequencing PCR " through specialty analysis software analysis sequencing result, and contrast peak figure and sequence are read peak graph structure, base to increase significantly on the accuracy of long and base.
One, optimizes the experimental technique of agent based on the reaction of Sanger PCR sequencing PCR
The concrete operations of sequencing reaction optimization agent are following:
1, according to the operation of BigDye V3.1 test kit operation instruction, in 20 μ l systems, add 4 μ lBigDye, 2 μ l BigDye Buffer, primer 3.2pmol, Template is suitable, the optimization agent of embodiment 1 preparation of 1/5 volume, promptly 4 μ l replenish ddH
2O to 20 μ l
2, carry out the PCR reaction according to following procedure:
3, after reaction finishes, the alcohol precipitation purifying.
4, ABI3730XL checks order.
Select for use primer 5-CACTCCCTTACCCGCTATCA-3 to check order, sample source and addition are all identical, sequencing result that add to optimize agent shown in SEQ ID No.1,561 bases of length overall.
Interpolation is optimized the result of agent shown in SEQ ID No.2,1015 bases of length overall.
Select for use primer 5-CTGAATCCACACCACCTCCT-3 to check order, sample source and addition are all identical, sequencing result that add to optimize agent shown in SEQ ID No.3,217 bases of length overall.
Interpolation is optimized the result of agent shown in SEQ ID No.4,631 bases of length overall.
To shown in Figure 6, Fig. 1 does not add the preceding 200bp place of the order-checking peak figure that optimizes agent like Fig. 1, the background peak height, and the order-checking effect is undesirable.Fig. 2 is for adding the preceding 200bp place of the order-checking peak figure that optimizes agent, and background peaks is low, and it is satisfactory for result to check order.Fig. 3 does not add Figure 50 0-520bp place, order-checking peak that optimizes agent, and the peak figure of repeating structure place is disorderly and influence subsequent peak figure, and the order-checking effect is undesirable.Fig. 4 is for adding Figure 50 0-520bp place, order-checking peak that optimizes agent, and repeating structure does not influence peak figure, and it is satisfactory for result to check order.Fig. 5 does not add the order-checking peak figure that optimizes agent to stop at the 600bp place.Fig. 6 appoints so effective peak figure for adding the order-checking peak figure that optimizes agent at the 800bp place.
Claims (5)
1. a Sanger sequencing reaction is optimized agent, it is characterized in that this optimization agent comprises trimethyl-glycine (Betaine), WR 34678 (DTT), DMSO and BSA; Its proportioning is:
2.7～5mol/L?Betaine、3～10mmol/LDTT、3％～8％DMSO、30～80mg/L?BSA。
2. Sanger sequencing reaction according to claim 1 is optimized agent, it is characterized in that said each composition proportion is:
2.7mol/L?Betaine、6.7mmol/L?DTT、6.7％DMSO、55mg/LBSA。
3. claim 1 or 2 described Sanger sequencing reactions are optimized the preparation method of agent, it is characterized in that concrete steps are following:
(1) according to claim 1 or 2 described trimethyl-glycine proportioning preparation trimethyl-glycine;
(2) in the trimethyl-glycine that configures, add DTT, DMSO and the BSA for preparing in the ratio of claim 1 or 2, mix;
(3) filtration sterilization ,-20 ℃ of preservations after the packing.
4. claim 1 or 2 application of described optimization agent in the Sanger sequencing reaction.
5. application according to claim 4 is characterized in that, in Sanger sequencing reaction system, adds the claim 1 or the 2 described optimization agent of 1/5 sequencing reaction system volume.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104711251A (en) * | 2015-03-27 | 2015-06-17 | 凯杰（苏州）转化医学研究有限公司 | Helicase-dependent isothermal amplification additive and helicase-dependent isothermal amplification method for sputum nucleic acid |
| CN110093410A (en) * | 2019-05-21 | 2019-08-06 | 通用生物系统（安徽）有限公司 | A kind of DNA sequencing reaction reagent and preparation method thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1932033A (en) * | 2006-09-22 | 2007-03-21 | 东南大学 | Nucleic acid sequencing process based on micro array chip |
| CN101210243A (en) * | 2006-12-26 | 2008-07-02 | 河南农业大学 | Amino acid sequence and polynucleotide sequence for chicken intestinal canal beta alexin and extraction method thereof |
| CN101824412A (en) * | 2007-04-29 | 2010-09-08 | 北京华大基因研究中心 | Method for conducting connection-by-amplification aiming at one or more target genomic regions |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1932033A (en) * | 2006-09-22 | 2007-03-21 | 东南大学 | Nucleic acid sequencing process based on micro array chip |
| CN101210243A (en) * | 2006-12-26 | 2008-07-02 | 河南农业大学 | Amino acid sequence and polynucleotide sequence for chicken intestinal canal beta alexin and extraction method thereof |
| CN101824412A (en) * | 2007-04-29 | 2010-09-08 | 北京华大基因研究中心 | Method for conducting connection-by-amplification aiming at one or more target genomic regions |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104711251A (en) * | 2015-03-27 | 2015-06-17 | 凯杰（苏州）转化医学研究有限公司 | Helicase-dependent isothermal amplification additive and helicase-dependent isothermal amplification method for sputum nucleic acid |
| CN104711251B (en) * | 2015-03-27 | 2016-10-05 | 凯杰（苏州）转化医学研究有限公司 | Helicase-dependent isothermal amplification additive and helicase-dependent isothermal amplification method for sputum nucleic acid |
| CN110093410A (en) * | 2019-05-21 | 2019-08-06 | 通用生物系统（安徽）有限公司 | A kind of DNA sequencing reaction reagent and preparation method thereof |
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