CN110093410A - A kind of DNA sequencing reaction reagent and preparation method thereof - Google Patents
A kind of DNA sequencing reaction reagent and preparation method thereof Download PDFInfo
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- CN110093410A CN110093410A CN201910424643.1A CN201910424643A CN110093410A CN 110093410 A CN110093410 A CN 110093410A CN 201910424643 A CN201910424643 A CN 201910424643A CN 110093410 A CN110093410 A CN 110093410A
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6869—Methods for sequencing
Abstract
The present invention discloses a kind of DNA sequencing reaction reagent and preparation method thereof, which is prepared by BigDye v3.1,5X Buffer and coenzyme, wherein the volume ratio of BigDye v3.1, coenzyme and 5X Buffer are 1~1.2: 1~1.1: 7~8.2；The present invention is matched by the way that coenzyme is added in the reagent that adds in sequencing reaction and adjusts the reagent of addition, it is practical by the DNA sequencing reaction in Sanger sequencing reaction, it can be applied to common special DNA structure, such as in the sequencing of hairpin structure, high GC content sequence or repetitive sequence etc., it can effectively avoid and occur that sequencing signal interrupts in advance, signal weakens comparatively fast leads to sequencing ordered sequence is short, sequence is inaccurate in sequencing procedure, so as to provide reliable, the accurately sequencing result that are right against labyrinth DNA.
Description
Technical field
The invention belongs to field of biotechnology, specifically, being related to a kind of DNA sequencing reaction reagent and preparation method thereof.
Background technique
There are mainly two types of methods for DNA sequencing at present: the chemical method of Maxam and Gilbert and the double deoxidation core of Sanger
Thuja acid cessation method, wherein Sanger method is similar to 2 ', 3 '-dideoxyribonucleoside, three phosphorus of normal dNTP by using chain terminating agent-
Acid specifically terminates the DNA chain of extension, and the T7 that reaction can be often applied to a kind of process modification in sequencing procedure bites
Thallus DNA polymerase, i.e. Sequenase (sequenase).
The numerous characteristics of natural polymerization enzyme are not suitable for DNA sequencing, and the characteristic that Sequenase should have includes: that (1) is higher
Processivity: processivity refers to enzyme from the journey that chain persistently extends before disengaging on primer-template annealing compound
Degree；(2) ability that heat resistance: resisting inactivation under the high temperature conditions or decomposes is enzyme in Modern High-Tech's investment cycle sequencing reaction
In most important factor；(3) nucleotide analog is embedded in dye terminator: being embedded in the ability of analog to dideoxy chain termination
Play deciding factor；The efficiency of termination with each labeling dye end must be with the effect that avoids low-quality uneven peak data
Rate is similar.
The development of automatic sequencing has expedited the emergence of the pursuit to better Sequenase, but is applied to the enzyme efficiency of sequencing reaction at present
It is not high, need to improve the efficiency of polymerase, deletion form Taq enzyme eliminates 5 ' -3 ' 5 prime excision enzyme activities, continues into property and there was only 2 bases
Left and right, archaeal dna polymerase continue into property (Processivity), i.e., catalysis conjunction after each polymerase combination primer-template complex
At oligonucleotides quantity, this performance can by enhancing enzyme-nucleic acid complex stabilization come realize enhancing, in addition, sequencing
Enzyme, which is prematurely detached from template and will lead to, is unable to get reliable sequence information by sequencing reaction.
Summary of the invention
The purpose of the present invention is to provide a kind of DNA sequencing reaction reagents and preparation method thereof.
The technical problem to be solved in the invention are as follows:
It is inefficient applied to the enzyme of sequencing reaction at present, it needs to improve the efficiency of polymerase, causes in sequencing procedure
It is easy to appear that sequencing signal interrupts in advance, signal weakens comparatively fast leads to sequencing ordered sequence is short, sequence is inaccurate.
The purpose of the present invention can be achieved through the following technical solutions:
A kind of DNA sequencing reaction reagent, the reagent are prepared by BigDye v3.1,5X Buffer and coenzyme；
Wherein the volume ratio of BigDye v3.1, coenzyme and 5X Buffer are 1~1.2: 1~1.1: 7~8.2, will
BigDye v3.1, coenzyme and 5X Buffer after mixing, obtain DNA sequencing reaction reagent according to the proportion；
The BigDye v3.1 is that Thermo company produces product；
The coenzyme selects 0.5-1U Taq (F667Y) archaeal dna polymerase；
The concentration of the coenzyme is more than or equal to 95%；
The enzymatic activity of the coenzyme is more than or equal to 5U/ul；
The preparation of the coenzyme includes the following steps:
The corresponding target sequence of S1, gene chemical synthesis and it is loaded into corresponding purpose carrier, plasmid will be obtained and be transformed into accordingly
Expression bacterial strain；
S2, picking monoclonal carry out expression screening；
S3, expression is amplified by the bacterial strain that IPTG induction obtains screening；
S4, protein purification is carried out to the strain protein obtained after expression by Ni affinity column method；
S5, destination protein over-molecular sieve are purified again；
S6, Protein Detection concentration purity；
S7, protein active detection.
The method that destination protein over-molecular sieve is purified again in step S5 are as follows:
Beta-propiolactone is added into the expression albumen obtained after Ni affinity column method preliminary purification to the micro- life of cause of disease
Object is inactivated, and carries out sieve chromatography later, collects destination protein using ultrapure water elution；
It is concentrated by ultrafiltration using the film packet that aperture is 30K, makes the concentration of target protein not less than 5mg/mL, finally use
The target protein that 0.2 micron of membrane filtration degerming is purified again.
The molecular sieve is by 50-60 parts by weight glucose gel, 5-8 parts by weight nano zeolite, 8-12 parts by weight concave convex rod
Soil is prepared with 3-4 weight account polyethylene alcohol, and wherein glucose gel is one of G-25 and G-50, the system of the molecular sieve
Preparation Method are as follows:
Attapulgite is dehydrated in 300 DEG C of roasting temperature 1-2h, then by silane coupling agent to attapulgite into
Row surface treatment obtains attapulgite modified by milli-Q water and drying after processing；
By the uniform modification attapulgite of nano zeolite, core material is obtained；
After evenly mixing by core material and polyvinyl alcohol, add ultrapure water after polyvinyl alcohol is added thereto and carry out water-bath
Heating, bath temperature are 95-98 DEG C, and stirring is cooled to 35-50 DEG C after being completely dissolved to polyvinyl alcohol, and to obtain decorating liquid stand-by；
Glucose gel is immersed in the phosphate buffer of 0.02mol/L and be swollen, wherein the pH of phosphate buffer is
6.2-6.8 uses ultrapure water, and the glucose gel after flushing is added in decorating liquid after swelling, with revolving speed 60-
Dress column gel is obtained after 90r/min stirring 15-25min；
Dress column gel is deaerated in vacuum desiccator after 20-30min, dress column is carried out, obtains molecular sieve.
Molecular sieve of the present invention carries out detention by promoting the adsorption capacity to thallus, by part thallus, is promoted subsequent
By the effect of membrane filtration degerming, the quantity of thallus in the destination protein that reduces.
The step of carrying out DNA sequencing using the sequencing reaction reagent are as follows:
SS1, plasmid g1904229109 is extracted using pet carrier；
SS2,96 orifice plates are taken out from storeroom, defrosting 4-7 seconds, be then centrifuged 96 orifice plates 2 seconds with 1500rpm, by matter
Grain template is placed in 96 orifice plates；
SS3, the sequencing reaction agent 1ul by 16 times of sequencing reaction dilution agent, in the hole of 96 orifice plates after addition dilution；
SS4, the PCR that Plasmid samples are sequenced react: 96 DEG C of ice water quenchings after initial denaturation 3 minutes；
With 96 DEG C handle 10 seconds, 50 DEG C handle 5 seconds, 60 DEG C processing 150 seconds for one circulation, carry out 32 circulation after 4
It is saved at a temperature of DEG C；
SS5, purifying and the sequencing of upper machine are carried out to PCR product.
Beneficial effects of the present invention:
The present invention is realized by the way that coenzyme is added in the reagent that adds in sequencing reaction and adjusts the reagent proportion of addition
, by this prioritization scheme in Sanger sequencing reaction, can be applied to common special DNA structure, such as hair clip knot
In the sequencing of structure, high GC content sequence or repetitive sequence etc., can effectively avoid in sequencing procedure occur sequencing signal shift to an earlier date in
Disconnected, signal, which weakens, comparatively fast leads to be sequenced that ordered sequence is short, sequence is inaccurate, is right against labyrinth so as to provide
Reliable, the accurately sequencing result of DNA.
Detailed description of the invention
Present invention is further described in detail in the following with reference to the drawings and specific embodiments.
Fig. 1 is the result measured using sequencing reaction reagent in embodiment 1 to plasmid g1904229109；
Fig. 2 is using the result that tradition sequencing agent measures plasmid g1904229109 in comparative example 1.
Specific embodiment
The technical scheme in the embodiments of the invention will be clearly and completely described below, it is clear that described implementation
Example is only a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, this field is common
Technical staff's all other embodiment obtained without creative efforts belongs to the model that the present invention protects
It encloses.
Embodiment 1
A kind of DNA sequencing reaction reagent, is prepared by BigDye v3.1,5X Buffer and coenzyme；
Wherein the volume ratio of BigDye v3.1, coenzyme and 5X Burr be 1: 1: 7, by BigDye v3.1, coenzyme with
5X Buffer after mixing, obtains DNA sequencing reaction reagent according to the proportion；
The BigDye v3.1 is that Thermo company produces product；
The coenzyme selects 0.5-1U Taq (F667Y) archaeal dna polymerase；
The concentration of the coenzyme is 96.3%；
The enzymatic activity of the coenzyme is 5.1U/ul；
The preparation of the coenzyme includes the following steps:
The corresponding target sequence of S1, gene chemical synthesis and it is loaded into corresponding purpose carrier, plasmid will be obtained and be transformed into accordingly
Expression bacterial strain；
S2, picking monoclonal carry out expression screening；
S3, expression is amplified by the bacterial strain that IPTG induction obtains screening；
S4, protein purification is carried out to the albumen obtained after bacterial strain expression by Ni affinity column method；
S5, destination protein over-molecular sieve are purified again；
S6, Protein Detection concentration purity；
S7, protein active detection.
The method that destination protein over-molecular sieve is purified again in step S5 are as follows:
Beta-propiolactone is added into the expression albumen obtained after Ni affinity column method preliminary purification to the micro- life of cause of disease
Object is inactivated, and carries out sieve chromatography later, collects destination protein using ultrapure water elution；
It is concentrated by ultrafiltration using the film packet that aperture is 30K, makes the concentration of target protein not less than 5mg/mL, finally use
The target protein that 0.2 micron of membrane filtration degerming is purified again.
The molecular sieve is by 60 parts by weight glucose gels, 5 parts by weight nano zeolites, 10 parts by weight attapulgites and 3.5
Weight account polyethylene alcohol is prepared, wherein glucose gel be G-50, the molecular sieve the preparation method comprises the following steps:
Attapulgite is dehydrated after 300 DEG C of roasting temperature 2h, then by vinyltrimethoxysilane to recessed
Convex stick soil is surface-treated, and by milli-Q water and drying after processing, is obtained attapulgite modified；
By the uniform modification attapulgite of nano zeolite, core material is obtained；
After evenly mixing by core material and polyvinyl alcohol, add ultrapure water after polyvinyl alcohol is added thereto and carry out water-bath
Heating, bath temperature are 95 DEG C, and stirring is cooled to 40 DEG C after being completely dissolved to polyvinyl alcohol；
Glucose gel is immersed in the phosphate buffer of 0.02mol/L and be swollen, wherein the pH of phosphate buffer is
6.5, ultrapure water is used after swelling, and the glucose gel after flushing is added in decorating liquid, with revolving speed 90r/min
Dress column gel is obtained after stirring 20min；
Dress column gel is deaerated in vacuum desiccator after 30min, dress column is carried out, obtains molecular sieve.
Comparative example 1
DNA sequencing is carried out using tradition sequencing agent, the tradition sequencing agent by BigDye v3.1 and 5X buffer according to
Volume ratio 1: 4 is formulated；
The BigDye v3.1 is that Thermo company produces product.
Test is measured with result:
SS1, plasmid F44426 is extracted using pet carrier；
SS2,96 orifice plates are taken out from storeroom, them is allowed to thaw 5 seconds, then by 96 orifice plates with 1500rpm centrifugation 2
Second, plasmid template is individually placed to the hole A01, the hole A02 in same reaction plate；
SS3, the sequencing reaction reagent in the embodiment 1 that the hole A01 addition 1ul dilutes 16 times；
SS4, the tradition sequencing agent in the comparative example 1 that the hole the A02 addition 1ul dilutes 8 times；
SS5, the PCR that Plasmid samples are sequenced react: 96 DEG C of ice water quenchings after initial denaturation 3 minutes；
With 96 DEG C handle 10 seconds, 50 DEG C handle 5 seconds, 60 DEG C processing 150 seconds for one circulation, carry out 32 circulation after 4
It is saved at a temperature of DEG C；
SS6, purifying and the sequencing of upper machine are carried out to PCR product.
Sequencing result is referring to Fig. 1 and Fig. 2；
Wherein Fig. 1 is the result measured using sequencing reaction reagent in embodiment 1 to plasmid F44426；
Fig. 2 is using the result that tradition sequencing agent measures plasmid F44426 in comparative example 1.
Sequencing comparing result explanation: it can be seen that in terms of sequencing result that two kinds of sequencing agent can from Fig. 1 and Fig. 2 comparison
Meeting sequencing to require, but is added to the sequencing result of coenzyme, signal of the signal instead than 1: 4 sequencing agent is high by 50%, and reading is long more preferable,
Peak height after 750bp is more preferable, and cost reduces half instead.
Above content is only to structure of the invention example and explanation, affiliated those skilled in the art couple
Described specific embodiment does various modifications or additions or is substituted in a similar manner, without departing from invention
Structure or beyond the scope defined by this claim, is within the scope of protection of the invention.
Claims (10)
1. a kind of DNA sequencing reaction reagent, which is characterized in that the reagent is prepared by BigDye v3.1,5X Buffer and coenzyme
It forms；
Wherein the volume ratio of BigDye v3.1, coenzyme and 5X Buffer are 1~1.2: 1~1.1: 7~8.2, will
BigDye v3.1, coenzyme and 5X Buffer after mixing, obtain DNA sequencing reaction reagent according to the proportion；
The BigDye v3.1 is the production of Thermo company；
The coenzyme selects 0.5-1U Taq (F667Y) archaeal dna polymerase.
2. a kind of DNA sequencing reaction reagent according to claim 1, which is characterized in that the concentration of the coenzyme be greater than etc.
In 95%.
3. a kind of DNA sequencing reaction reagent according to claim 1, which is characterized in that the enzymatic activity of the coenzyme is greater than
Equal to 5U/ul.
4. a kind of DNA sequencing reaction reagent according to claim 1, which is characterized in that the DNA sequencing reaction reagent by
BigDye v3.1, coenzyme and the 5X Buffer that volume ratio is 1: 1: 7 are formulated.
5. a kind of DNA sequencing reaction reagent according to claim 1, which is characterized in that the preparation of the coenzyme includes such as
Lower step:
The corresponding target sequence of S1, gene chemical synthesis and it is loaded into corresponding purpose carrier, plasmid will be obtained and be transformed into corresponding table
Up to bacterial strain；
S2, picking monoclonal carry out expression screening；
S3, the bacterial strain obtained to screening amplify expression；
The strain protein obtained after S4, expression carries out protein purification；
S5, destination protein over-molecular sieve are purified again；
S6, Protein Detection concentration purity；
S7, protein active detection.
6. a kind of DNA sequencing reaction reagent according to claim 5, which is characterized in that pass through IPTG in the step S3
The bacterial strain obtained to screening is induced to amplify expression.
7. a kind of DNA sequencing reaction reagent according to claim 5, which is characterized in that pass through Ni parent in the step S4
Bacterial strain expression albumen is purified with chromatographic column method.
8. a kind of DNA sequencing reaction reagent according to claim 1, which is characterized in that using the sequencing reaction reagent into
The step of row DNA sequencing are as follows:
SS1, plasmid F44426 is extracted using pet carrier；
SS2,96 orifice plates are taken out from storeroom, defrosting 4-7 seconds, be then centrifuged 96 orifice plates 2 seconds with 1500rpm, by plasmid mould
Plate is placed in 96 orifice plates；
SS3, the sequencing reaction agent 1ul by 16 times of sequencing reaction dilution agent, in the hole of 96 orifice plates after addition dilution；
SS4, the PCR that Plasmid samples are sequenced react: 96 DEG C of ice water quenchings after initial denaturation 3 minutes；
With 96 DEG C handle 10 seconds, 50 DEG C handle 5 seconds, 60 DEG C processing 150 seconds for one circulation, carry out 32 circulation after in 4 DEG C of temperature
Degree is lower to be saved；
SS5, purifying and the sequencing of upper machine are carried out to PCR product.
9. a kind of DNA sequencing reaction reagent according to claim 5, which is characterized in that destination protein is excessive in step S5
The method that son sieve is purified again are as follows:
Into the expression albumen obtained after Ni affinity column method preliminary purification be added beta-propiolactone to pathogenic microorganism into
Row inactivation, carries out sieve chromatography later, collects destination protein using ultrapure water elution；
It is concentrated by ultrafiltration using the film packet that aperture is 30K, makes the concentration of target protein not less than 5mg/mL, finally use 0.2
The target protein that the membrane filtration degerming of micron is purified again.
10. a kind of DNA sequencing reaction reagent according to claim 9, which is characterized in that the molecular sieve is by 50-60 weight
Measure part glucose gel, it is prepared by 5-8 parts by weight nano zeolite, 8-12 parts by weight attapulgite and 3-4 weight account polyethylene alcohol and
At, wherein glucose gel be one of G-25 and G-50, the molecular sieve the preparation method comprises the following steps:
Attapulgite is dehydrated in 300 DEG C of roasting temperature 1-2h, then table is carried out to attapulgite by silane coupling agent
Surface treatment obtains attapulgite modified by milli-Q water and drying after processing；
By the uniform modification attapulgite of nano zeolite, core material is obtained；
After evenly mixing by core material and polyvinyl alcohol, add ultrapure water after addition polyvinyl alcohol thereto and carry out water-bath and add
Heat, bath temperature are 95-98 DEG C, and stirring is cooled to 35-50 DEG C after being completely dissolved to polyvinyl alcohol, and to obtain decorating liquid stand-by；
Glucose gel is immersed in the phosphate buffer of 0.02mol/L and be swollen, wherein the pH of phosphate buffer is 6.2-
6.8, ultrapure water is used after swelling, and the glucose gel after flushing is added in decorating liquid, with revolving speed 60-90r/
Dress column gel is obtained after min stirring 15-25min；
Dress column gel is deaerated in vacuum desiccator after 20-30min, dress column is carried out, obtains molecular sieve.
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