CN104498605A - Sanger sequencing reaction optimization agent, sequencing reaction system and sequencing method employing optimization agent - Google Patents
Sanger sequencing reaction optimization agent, sequencing reaction system and sequencing method employing optimization agent Download PDFInfo
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- CN104498605A CN104498605A CN201410778652.8A CN201410778652A CN104498605A CN 104498605 A CN104498605 A CN 104498605A CN 201410778652 A CN201410778652 A CN 201410778652A CN 104498605 A CN104498605 A CN 104498605A
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- 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 invention relates to the technical field of molecular biology and discloses a Sanger sequencing reaction optimization agent, as well as a sequencing reaction system and a sequencing method employing theoptimization agent. According to the volume ratio, the Sanger sequencing reaction optimization agent provided by the invention comprises the following components: 1 part of BigDye, 0.2-1 part of dGTP-BigDye, 0.1-1 part of dGTP and 0.1-1 part of dCTP. The optimization agent is added into the Sanger sequencing reaction and is applied to sequencing of common special DNA structures such as a hairpin structure, a high-GC sequence or a repetitive sequence, and the problems that the sequencing signal is interrupted in advance in the sequencing process, the effective sequencing sequence is short due to fast signal weakening and the sequence is inaccurate in the sequencing process can be effectively solved, so that a reliable and accurate sequencing result relative to the complex structure can be provided.
Description
Technical field
The present invention relates to technical field of molecular biology, particularly a kind of Sanger sequencing reaction optimizes agent, the sequencing reaction system applying this optimization agent and sequence measurement.
Background technology
In molecular biology research, the sequential analysis of DNA is the basis of research further and transformation goal gene.The Sanger dideoxy chain termination (Chain TerminationMethod) that Frederick Sanger invents is a kind of conventional DNA sequencing method.Sanger sequencing starts at a certain fixing point according to Nucleotide, the ddNTP that random introducing is marked respectively by 4 kinds of different colours fluorescence is to stop extension, material is thus formed extension products that a large amount of end is fluorescently labeled, different in size, then these extension products are separated with high-resolution capillary gel electrophoresis again, by the differentiation to extension products end 4 kinds of different fluorescence colors, software for calculation can automatic " reading " DNA sequence dna.
Sanger sequencing has been widely used in the sequencing technologies carried out bacterium (bacterium liquid, puncture bacterium, dull and stereotyped bacterium), the dissimilar sample such as plasmid, PCR primer (stoste, purifying), the designs of plasmid extraction, PCR purifying, sequencing primer, the master data analysis of DNA sequence dna is provided with this, and the bioinformatic analysis service such as sequence assembly after long segment walking order-checking.
Due to the impact of secondary structure or other internal factor, complex construction is huge challenge for DNA sequencing.Common special DNA structure has hairpin structure, high GC content sequence, tumor-necrosis factor glycoproteins etc., and order-checking signal premature discontinuation often appears in this class formation in the sanger sequence measurement of routine, signal weakening comparatively fast causes the problems such as order-checking ordered sequence is short, sequence is inaccurate.
Summary of the invention
One object of the present invention is to provide a kind of Sanger sequencing reaction to optimize agent, and this optimization agent is applied in Sanger sequencing reaction, can improve reliability and the accuracy of the sequencing result with complex construction DNA sample.
Another object of the present invention is to provide application this kind of Sanger sequencing reaction to optimize sequencing reaction system and the sequence measurement of agent.
For solving the problems of the technologies described above, embodiments of the present invention provide a kind of Sanger sequencing reaction and optimize agent, count by volume, and this kind is optimized agent and comprised following component:
BigDye 1 part,
DGTP-BigDye 0.2 ~ 1 part,
The dGTP of 10 μMs 0.1 ~ 1 part,
With the dCTP 0.1 ~ 1 part of 10 μMs.
The composition and ratio that this kind optimizes agent is determined according to needing the GC content of sequenced fragments, and GC content is higher, and the ratio of BDG, dGTP and dCTP is higher.In Sanger sequencing reaction, add this kind optimize agent, be applied to common special DNA structure, such as, in the order-checking of hairpin structure, high GC content sequence or tumor-necrosis factor glycoproteins etc., effectively can avoid occurring order-checking signal premature discontinuation in sequencing procedure, signal weakening comparatively fast causes the problems such as order-checking ordered sequence is short, sequence is inaccurate, thus reliable, the sequencing result accurately that are right against complex construction DNA can be provided.In embodiments of the present invention, respectively the optimization agent of said ratio is used for the Sanger of hair clip DNA sequence dna order-checking and in the Sanger order-checking of high GC content sequence, to sequencing procedure carry out smoothly and sequencing result accurately draw all have unusual effect.
Embodiments of the present invention also provide a kind of Sanger sequencing reaction system, every 10 μ l this kind of reaction system comprises following component: 5 × sequencing buffer 2 μ l, 3.2 μMs of sequencing primer 2 μ l, the sequencing template 1 μ l of 100 ~ 200ng/ μ l, Sanger sequencing reaction optimizes agent 1 μ l, adds water and supplies 10 μ l; Wherein, Sanger sequencing reaction optimizes agent, counts by volume, comprises following component: BigDye 1 part, dGTP-BigDye 0.2 ~ 1 part, the dGTP 0.1 ~ 1 part of 10 μMs and the dCTP 0.1 ~ 1 part of 10 μMs.
In addition, embodiments of the present invention also provide the application sequence measurement that above-mentioned Sanger sequencing reaction system is carried out, and the method comprises following step:
(1) denaturation of order-checking sample DNA, then ice water quenching; (2) the PCR circulation of sequencing reaction; (3) purifying is carried out to the product of sequencing reaction; (4) sex change is carried out to the product after purifying, then ice water quenching; (5) DNA sequencer is adopted to check order.
Preferably, in embodiments of the present invention, the reaction conditions carrying out denaturation in the step (1) of above-mentioned sequence measurement is: at 98 DEG C, carry out denaturation 8 minutes.For the template having hairpin structure or high GC content, general double-strand combines tightr, sequencing procedure needs to produce strand, conventional 95 DEG C can not make double-strand unwind completely and cause the follow-up single stranded DNA content that can be used for sequencing reaction low, therefore in the sequence measurement that embodiments of the present invention provide, by improving denaturation temperature to 98 DEG C and increasing denaturation time to 8 minute, ensure that double-strand is unwind completely.
Preferably, in embodiments of the present invention, the response procedures carrying out PCR circulation in the step (2) of above-mentioned sequence measurement is: within 4 minutes, be a circulation with 98 DEG C 10 seconds, 50 DEG C 1 minute, 60 DEG C, carries out 40 circulations.
Preferably, in embodiments of the present invention, the method of carrying out purifying to the product of sequencing reaction in the step (3) of above-mentioned sequence measurement is ethanol/sodium-acetate method, by this purification step, can remove the impurity such as raw material unnecessary in reaction system, enzyme and ion.The concrete operations of purification step are as follows:
(1) often pipe adds at the bottom of 1 μ l 125mM EDTA to pipe, and often pipe adds at the bottom of 1 μ l 3M NaAc to pipe; (2) often pipe adds 25 μ l 100% alcohol, mixing, and lucifuge condition room temperature leaves standstill 15min; (3) the centrifugal 30min of 3700rmp, is inverted 96 orifice plates, the centrifugal 1min of 800rmp at once; (4) often pipe adds 35 μ l 70% alcohol, concussion, and the centrifugal 30min of 3700rmp is inverted 96 orifice plates, the centrifugal 1min of 800rmp at once; (5) room temperature is volatilized clean alcohol.
Preferably, in embodiments of the present invention, be: sex change 8 minutes at 98 DEG C, makes double-stranded DNA fully unwind in the step (4) of above-mentioned sequence measurement to the method that the product after purifying carries out sex change, when checking order, strand is only effectively afterwards.
Preferably, in embodiments of the present invention, before sex change being carried out to the product after purifying in the step (4) of above-mentioned sequence measurement, add 10 μ l methane amides, and fully mix, add methane amide and contribute to DNA sample maintenance denatured state.
Preferably, in embodiments of the present invention, the DNA sequencer adopted in the step (5) of above-mentioned sequence measurement is ABI sequenator, such as 3730XL.
Accompanying drawing explanation
Fig. 1 is the sequencing result adopting sequence measurement provided by the invention to record hair clip DNA sequence dna in embodiment 1;
Fig. 2 adopts the conventional sequencing result recorded hair clip DNA sequence dna that checks order in the ratio test example of embodiment 2;
Fig. 3 is the sequencing result adopting sequence measurement provided by the invention to record high GC content sequence in embodiment 3;
Fig. 4 adopts the conventional sequencing result recorded high GC content sequence that checks order in the comparative experimental example of embodiment 4.
Embodiment
For making the object, technical solutions and advantages of the present invention clearly, below in conjunction with accompanying drawing, the embodiments of the present invention are explained in detail.But, persons of ordinary skill in the art may appreciate that in each embodiment of the present invention, proposing many ins and outs to make reader understand the application better.But, even without these ins and outs with based on the many variations of following embodiment and amendment, each claim of the application technical scheme required for protection also can be realized.
(in present specification, to Bigdye also referred to as BDT, to dGTP-BigDye also referred to as BDG)
Embodiment 1 adopts sequence measurement of the present invention to the order-checking case of hair clip DNA sequence dna (ShDNA sequence, exists hairpin structure)
1, in the present embodiment to hair clip DNA sample the PCR reaction system that checks order composed as follows:
5×sequencing buffer：2μl
Template (150ng/ μ l): 1 μ l
Primer (3.2 μMs): 2 μ l
Sanger sequencing reaction optimizes agent 1 μ l (wherein by volume: Bigdye 1 part, dGTP-BigDye0.2 part, the dGTP 0.2 part of 10 μMs and the dCTP 0.2 part of 10 μMs)
Water: 4 μ l.
2, the PCR response procedures checked order to hair clip DNA sample in the present embodiment is as follows:
98 DEG C of denaturations 8 minutes; Ice water quenching;
Within 4 minutes, be a circulation with 98 DEG C 10 seconds, 50 DEG C 1 minute, 60 DEG C, carry out 40 circulations;
4 DEG C of preservations.
3, PCR reaction product is carried out to the step of ethanol/sodium-acetate method purifying:
(1) often pipe adds at the bottom of 1 μ l 125mM EDTA to pipe, and often pipe adds at the bottom of 1 μ l 3M NaAc to pipe;
(2) often pipe adds 25 μ l 100% alcohol, mixing, and lucifuge condition room temperature leaves standstill 15min;
(3) the centrifugal 30min of 3700rmp, is inverted 96 orifice plates, the centrifugal 1min of 800rmp at once;
(4) often pipe adds 35 μ l 70% alcohol, concussion, and the centrifugal 30min of 3700rmp is inverted 96 orifice plates, the centrifugal 1min of 800rmp at once;
(5) room temperature is volatilized clean alcohol.
4, upper machine order-checking
To purified product often pipe add 10 μ l methane amides, fully short centrifugal after concussion, collect product at the bottom of pipe, sex change direct ice water quenching after 8 minutes at 98 DEG C; Products therefrom carries out capillary electrophoresis (ABI 3730XL sequenator).Sequencing result is shown in shown in accompanying drawing 1.
Embodiment 2 comparative experimental example: adopt conventional sequence measurement to contrast the order-checking of hair clip DNA sequence dna (ShDNA sequence, exists hairpin structure)
The present embodiment is the comparative experimental example of embodiment 1, namely adopts ordinary method to check order to hair clip DNA sequence dna.
1, the PCR reaction system of ordinary method is adopted:
5×sequencing buffer：2μl
Template (150ng/ μ l): 1 μ l
Primer (3.2 μMs): 2 μ l
BDT：1μl
Water: 4 μ l
2, the PCR program of ordinary method is adopted:
95 DEG C 5 minutes; Quenching;
Within 4 minutes, be a circulation with 95 DEG C 10 seconds, 50 DEG C 1 minute, 60 DEG C, carry out 40 circulations.
4 DEG C of preservations.
3, PCR reaction product is carried out to the step of purifying:
(1) often pipe adds at the bottom of 1 μ l 125mM EDTA to pipe, and often pipe adds at the bottom of 1 μ l 3M NaAc to pipe;
(2) often pipe adds 25 μ l 100% alcohol, mixing, and lucifuge condition room temperature leaves standstill 15min;
(3) the centrifugal 30min of 3700rmp, is inverted 96 orifice plates, the centrifugal 1min of 800rmp at once;
(4) often pipe adds 35 μ l 70% alcohol, concussion, and the centrifugal 30min of 3700rmp is inverted 96 orifice plates, the centrifugal 1min of 800rmp at once;
(5) room temperature is volatilized clean alcohol.
4, upper machine order-checking
To purified product often pipe add 10 μ l methane amides, fully short centrifugal after concussion, collect product at the bottom of pipe, sex change direct ice water quenching after 5 minutes at 95 DEG C; Products therefrom carries out capillary electrophoresis (ABI 3730XL sequenator).Sequencing result as shown in Figure 2.
The Comparative result of embodiment 1 and embodiment 2:
Comparing result illustrates: can see from the contrast of accompanying drawing 1 and accompanying drawing 2: this NDA sequence to be measured exists hairpin structure, adopts the conventional sequence measurement in embodiment 2, owing to not unwinding and the signal interruption that causes checking order completely in hairpin structure district, cannot continue order-checking; And after adopting the sequence measurement provided by the present invention in embodiment 1, unwind smoothly in hair clip region, measure correlated series smoothly.
Embodiment 3 adopts sequence measurement of the present invention to the order-checking case of high GC content sequence
1, in the present embodiment, the PCR reaction system that high GC content sample checks order is consisted of:
5×sequencing buffer：2μl
Template (150ng/ μ l): 1 μ l
Primer (3.2 μMs): 2 μ l
Sanger sequencing reaction optimizes agent 1 μ l (wherein by volume: Bigdye 1 part, dGTP-BigDye0.5 part, the dGTP 0.5 part of 10 μMs and the dCTP 0.5 part of 10 μMs)
Water: 4 μ l.
2, in the present embodiment to the PCR response procedures that high GC content sample checks order be:
98 DEG C of denaturations 8 minutes; Ice water quenching;
Within 4 minutes, be a circulation with 98 DEG C 10 seconds, 50 DEG C 1 minute, 60 DEG C, carry out 40 circulations;
4 DEG C of preservations.
3, PCR reaction product is carried out to the step of purifying:
(1) often pipe adds at the bottom of 1 μ l 125mM EDTA to pipe, and often pipe adds at the bottom of 1 μ l 3M NaAc to pipe;
(2) often pipe adds 25 μ l 100% alcohol, mixing, and lucifuge condition room temperature leaves standstill 15min;
(3) the centrifugal 30min of 3700rmp, is inverted 96 orifice plates, the centrifugal 1min of 800rmp at once;
(4) often pipe adds 35 μ l 70% alcohol, concussion, and the centrifugal 30min of 3700rmp is inverted 96 orifice plates, the centrifugal 1min of 800rmp at once;
(5) room temperature is volatilized clean alcohol.
4, upper machine order-checking
To purified product often pipe add 10 μ l methane amides, fully short centrifugal after concussion, collect product at the bottom of pipe, sex change direct ice water quenching after 8 minutes at 98 DEG C; Products therefrom carries out capillary electrophoresis (ABI 3730XL sequenator).Sequencing result as shown in Figure 3.
Embodiment 4, comparative experimental example: adopt conventional sequence measurement to contrast the order-checking of high GC content sequence
The present embodiment is the comparative experimental example of embodiment 3, namely adopts ordinary method to check order to high GC content sequence.
1, the PCR reaction system of ordinary method is adopted:
5×sequencing buffer：2μl
Template (150ng/ μ l): 1 μ l
Primer (3.2 μMs): 2 μ l
BDT：1μl
Water: 4 μ l
2, the PCR program of ordinary method is adopted:
95 DEG C 5 minutes; Quenching;
Within 4 minutes, be a circulation with 95 DEG C 10 seconds, 50 DEG C 1 minute, 60 DEG C, carry out 40 circulations.
4 DEG C of preservations.
3, PCR reaction product is carried out to the step of purifying:
(1) often pipe adds at the bottom of 1 μ l 125mM EDTA to pipe, and often pipe adds at the bottom of 1 μ l 3M NaAc to pipe;
(2) often pipe adds 25 μ l 100% alcohol, mixing, and lucifuge condition room temperature leaves standstill 15min;
(3) the centrifugal 30min of 3700rmp, is inverted 96 orifice plates, the centrifugal 1min of 800rmp at once;
(4) often pipe adds 35 μ l 70% alcohol, concussion, and the centrifugal 30min of 3700rmp is inverted 96 orifice plates, the centrifugal 1min of 800rmp at once;
(5) room temperature is volatilized clean alcohol.
4, upper machine order-checking
To purified product often pipe add 10 μ l methane amides, fully short centrifugal after concussion, collect product at the bottom of pipe, sex change direct ice water quenching after 5 minutes at 95 DEG C; Products therefrom carries out capillary electrophoresis (ABI 3730XL sequenator).Sequencing result as shown in Figure 4.
The Comparative result of embodiment 3 and embodiment 4:
Comparing result illustrates: the contrast as can be seen from accompanying drawing 2 and accompanying drawing 4: this sequence local to be measured GC content reaches 81.2%, in sequencing procedure, dNTP and ddNTP adopting ordinary method in embodiment 4 is equivalent, due to ddGTP, ddCTP, dGTP and dCTP etc. are excessive in partial spent, and system is uneven and cause signal to weaken in advance, therefore need extra supplementing.After adopting the sequence measurement provided by the present invention in embodiment 3, successful improves.
Persons of ordinary skill in the art may appreciate that the respective embodiments described above realize specific embodiments of the invention, and in actual applications, various change can be done to it in the form and details, and without departing from the spirit and scope of the present invention.
Claims (9)
1. Sanger sequencing reaction optimizes an agent, and it is characterized in that, count by volume, described optimization agent comprises following component:
BigDye 1 part,
DGTP-BigDye 0.2 ~ 1 part,
The dGTP of 10 μMs 0.1 ~ 1 part,
With the dCTP 0.1 ~ 1 part of 10 μMs.
2. a Sanger sequencing reaction system, it is characterized in that, described in every 10 μ l, reaction system comprises following component: 5 × sequencing buffer 2 μ l, the sequencing primer 2 μ l of 3.2 μMs, the sequencing template 1 μ l of 100 ~ 200ng/ μ l, Sanger sequencing reaction optimizes agent 1 μ l, adds water and supplies 10 μ l;
Wherein, described Sanger sequencing reaction optimizes agent, and meter comprises following component by volume: Bigdye1 part, dGTP-BigDye 0.2 ~ 1 part, the dGTP 0.1 ~ 1 part of 10 μMs and the dCTP 0.1 ~ 1 part of 10 μMs.
3. application rights requires to it is characterized in that the sequence measurement that the Sanger sequencing reaction system described in 2 is carried out, comprise following step:
(1) denaturation of order-checking sample DNA, then ice water quenching;
(2) the PCR circulation of sequencing reaction;
(3) purifying is carried out to the product of sequencing reaction;
(4) sex change is carried out to the product after purifying, then ice water quenching;
(5) DNA sequencer is adopted to check order.
4. sequence measurement according to claim 3, is characterized in that, the reaction conditions carrying out denaturation in described step (1) is: at 98 DEG C, carry out denaturation 8 minutes.
5. sequence measurement according to claim 3, is characterized in that, the PCR that carries out in described step (2) circulation is: within 4 minutes, be a circulation with 98 DEG C 10 seconds, 50 DEG C 1 minute, 60 DEG C, carries out 40 circulations.
6. sequence measurement according to claim 3, is characterized in that, the method for carrying out purifying to the product of sequencing reaction in step (3) is ethanol/sodium-acetate method.
7. sequence measurement according to claim 3, is characterized in that, is: sex change 8 minutes at 98 DEG C in step (4) to the method that the product after purifying carries out sex change.
8. sequence measurement according to claim 3, is characterized in that, before carrying out sex change, adds 10 μ l methane amides, and fully mixes in step (4) to the product after purifying.
9. sequence measurement according to claim 3, is characterized in that, the DNA sequencer adopted in step (5) is ABI 3730XL sequenator.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106591433A (en) * | 2016-11-09 | 2017-04-26 | 上海派森诺生物科技股份有限公司 | It is a kind of to extend the method that difficult template reads length |
| 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 |
-
2014
- 2014-12-15 CN CN201410778652.8A patent/CN104498605A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106591433A (en) * | 2016-11-09 | 2017-04-26 | 上海派森诺生物科技股份有限公司 | It is a kind of to extend the method that difficult template reads length |
| 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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