JP3521079B2 - DNA extraction and purification method and apparatus therefor - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a multi-specimen sample
The present invention relates to a method and an apparatus for extracting and purifying NA. More specifically, it relates to a method and apparatus for extracting and purifying plasmid DNA from a transformant transformed with Escherichia coli or the like.

[0002]

[Prior art]

DNA cloning is the most important method in molecular biology in which a specific DNA fragment is obtained in pure and large quantities necessary for analysis. Among them, cloning using a plasmid as a vector is the most popular technique and is used in various fields of molecular biology. Escherichia coli serving as a host for plasmid amplification contains genomic DNA and various RNAs as nucleic acids other than plasmids, and proteins and lipids as constituents of bacterial cells. As a method for extracting and purifying plasmid DNA from these contaminants, conventionally, the alkaline method (Birnboim, HC, Nu.
cleic Acid Res. vol7,1513 (1979) “Alkaline Lysis Me
thod ”) and Boyle method (Holdes, ds etc. Anal.Biochem.vol
114,193 (1981) “BOILING METHOD”) is known.

Although the alkaline method can obtain a plasmid having a relatively high purity, a dangerous reagent such as phenol or chloroform must be used. The boil method (boil method) is a method of extracting a plasmid by heating and has an advantage that the number of processing steps is smaller than that of the alkali method, but it is not suitable for sequences due to its disadvantage of poor purity. It is said that the alkaline method is mainly used in the genome analysis at present.

The reason why the Boyle method has not been used in the sequence is that the purity is inferior.
(1) There is a variation in the recovery amount depending on the heat treatment time,
(2) Decrease in sequence reaction efficiency due to lack of deproteinization. However, in recent years, due to improvements in reagents for DNA sequencing, it has become possible to measure with a sensitivity that is more than ten times that of conventional methods, and the range of the amount of DNA used in the reaction expands.
The sensitivity of DNA sequencers has also been improved. As a result, the inventors of the present invention were able to (1) react with a recent reaction reagent even if the DNA has a purity according to the Boyle method, and (2) even if the reaction efficiency is lowered due to protein contamination. Current DN due to improved fluorescence intensity of analytical reagents
We considered that data acquisition was possible with the A sequencer, and started research on a DNA extraction method for sequencing by the Boyle method.

[0006] The outline of the conventional general boil method is as follows. The transformant culture medium containing the plasmid is transferred to a microtube, and the cells are pelleted by centrifugation to separate the supernatant, and then the cell pellet is lysed. Set the suspended microtubes on a boil rack and heat in a boiling pot to aggregate and filter genomic DNA, protein, etc.,
Further, the supernatant liquid is salted out to aggregate the plasmid, and RNA is removed by centrifugal precipitation and washing to obtain a purified plasmid DNA.

According to the boil method, it is possible to collect bacteria and collect the final purified DNA solution in one microtube, but in the conventional ordinary boil method, the microtube is set in a rack for boil. Since it is heated in a boiling pot, it is difficult to process multiple samples at the same time. Furthermore, the loss of the sample in the filtration process cannot be ignored.

Attempts to extract and purify DNA by the Boyle method with high throughput and low cost have been proposed (Marco A.
Marra et al. Nucleic Acids Research, 1999, Vol. 27,
No.24). This method is a method for simultaneously processing multiple specimens by microwave heating using a 96-well block, but the heating is not uniform because the block is thick and microwave heating is used. There is a problem that occurs.

[0009]

SUMMARY OF THE INVENTION The present invention is intended to solve the problems of the conventional method, and it is an object of the present invention to provide a novel method and apparatus for extracting and purifying DNA for multiple samples by the Boyle method. And More specifically, even if multiple samples are processed at the same time, there is no variation in heating, sample loss is small, and even if each sample amount is very small, high efficiency,
It is an object of the present invention to provide a simple method and device capable of extracting and purifying DNA with high purity.

[0010]

Means for Solving the Problems As a result of intensive investigations by the present inventors, means for using the Boyle method for a sequence, which has the advantages that the number of treatment steps is smaller than that of the alkali method and that it is not necessary to use a dangerous reagent The present invention has been reached.

In order to reduce variations in heating, the present invention is characterized by using a plate for PCR reaction (Polymerase chain reaction). The PCR reaction plate is a plate for multiple samples used in the PCR reaction and usually has 12 × 8 = 96 wells, but it has been conventionally attempted to use this plate in the DNA extraction step. Not not. The present inventors have focused on applying this PCR reaction plate having a large number of wells in developing a boiling method for simultaneously processing multiple samples for DNA extraction. The PCR reaction plate has a small thickness and excellent heat conductivity, and since the PCR heat block can be used for heating, it has an advantage that uniform heating is possible. Furthermore, this PC
The R reaction plate can also have an advantage of being less susceptible to contamination in the process of decanting and filtering because the upper part of the well projects from the plate.

That is, according to the method of the present invention, when the plasmid DNA is extracted and purified from the transformed plasmid by the Boyle method, the cells are cultured, collected, lysed, suspended, in the wells of the multi-sample plate for PCR reaction. The present invention relates to a method for extracting and purifying DNA, which comprises heating and performing precipitation filtration.

The present invention also relates to a method for extracting and purifying DNA, characterized in that heating is carried out using a heat block for PCR reaction.

Further, according to the present invention, the hydrophilic property of 2 is added to the filtration step.
It was possible to reduce the loss of the sample by centrifuging in a sealed state using a polyvinylidene fluoride (PVDF) filter.

Furthermore, as a result of detailed examination of the steps in the DNA extraction and purification method, the present inventors have found that STET
(NaCl, Tris-HCl, EDTA, TritonX-100) In a lysis step performed using a solution and a lysozyme solution, in the method of the present invention in which a PCR reaction plate is applied, the STET solution was added after the lysozyme solution was added. Better yield, and 1 mM Tris-HCl in STET solution, Tr
It was found that optimum conditions can be obtained by adjusting itonX-100 to 1% by weight and boiling for about 25 seconds.

Furthermore, the present invention also relates to an apparatus including at least a PCR plate and a PCR heat block for carrying out these DNA extraction and purification methods.

According to the method and apparatus for extracting and purifying DNA of the present invention, the plasmid DNA can be efficiently extracted with high purity from the transformant.

[0018]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described in detail below. The transformant to which the DNA extraction and purification method of the present invention is applied may be any known plasmid, and examples thereof include PUC18 and PME18SFL3.

As the PCR reaction plate used in the present invention, any plate can be used as long as it is a plate for multiple samples which is usually used for PCR reaction. For example,
An example is a 12 × 8 = 96 well plate manufactured by Robbins. This plate is as shown in FIG. FIG. 1 (a) shows an overall perspective view of the PCR reaction plate, and FIG. 1 (b) shows a partially broken side view. Well plate (1) is 78 mm long × 118 m wide
m, made of polypropylene with a wall thickness of 0.3 to 0.4 mm, and each well (2) has an inner diameter of 5.5 mm and a depth of 22 mm.
Has the dimensions of. The upper part of each well is a protrusion (3) protruding about 5 mm on the plate.

As a heating device used in the boiling process of the present invention, for example, Tl thermocycler manufactured by Biometra can be used. The thermocycler is a thermostatic chamber whose temperature can be adjusted variably, and in the present invention, P
It is used as a heating device with a built-in CR reaction heat block. FIG. 2 illustrates an example of a heat block for PCR reaction. FIG. 2A is a cross-sectional view showing a state where the PCR reaction plate (1) is fitted to the PCR reaction heat block (4). FIG. 2B shows a thermocycler having a heat block for PCR reaction installed.

The DNA extraction and purification method by the Boyle method generally comprises the following steps. After the transformant was dispensed into wells and cultured (culture step), the supernatant of the cells obtained by centrifugation was removed (bacteria collection step), and the cell pellet was lysed in suspension. To break the bacterial wall (suspension / lysis), boiling to separate huge DNA chains such as genomic DNA (boil process), precipitation filtration of impurities such as proteins and lipids (precipitation filtration process) ), A step of precipitating a plasmid by salting out the supernatant with salt and alcohol (salting out step), a washing / evaporating step, and an RNA extracting / removing step.

Test by Conventional Method The present inventors first investigated problems in the conventional boiling method by carrying out the boiling method by the following protocol. A 96-well deep well plate (manufactured by Corning) was used as the plate. A Corning 96-well deep well plate is shown in FIG. FIG. 3 (a) is an overall perspective view of the deep well plate, and FIG. 3 (b) is a partially cutaway side view thereof.
As seen in FIG. 3 (b), this well plate is thick.

Plasmid extraction followed the following steps. (Plasmid extraction) 1) Ampicillin (antibiotic for cell wall synthesis inhibition) 50μ
Liquid LB medium containing g / ml (bact per liter
o-tryptone 10g, bacto-yeast extract 5g, NaCl10g)
900 μl is dispensed into each well of a Corning 96-well deep plate. 2) Pick a colony in each well with a toothpick and inoculate. 3) Incubate with shaking at 37 ° C for 18 to 24 hours. 4) After culturing, the cells are collected by centrifugation at 1,500 G for 10 minutes. 5) The supernatant liquid, which is an impurity, is removed with an aspirator while washing with 70% ethanol. 6) STET solution (NaCl 0.1M, Tris-HCl 10mM, EDTA
Add 200 μl of 1 mM TritonX-1005%) to each well and stir. The bacterial wall is broken by Triton X-10 contained in the STET solution. 7) Lysozyme solution (lysozyme 10mg / ml, Tris-HCl 10
Add 14 μl of mM (pH 8.0)) and suspend. 8) Heat with hot water at 98 ° C for 40 seconds. 9) Remove the decomposed large proteins and lipids that precipitate as a precipitate on the bottom by centrifugation at 1,900 G for 10 minutes. 10) Turn the plate over and place it on another plate, and collect only the supernatant on the other plate. 11) Add 24 μl of 3M sodium acetate to the supernatant and stir. 12) Add 240 μl of isopropanol and stir. The polymer colloidal plasmid is aggregated (salting out). 13) Centrifuge at 1,900 G for 20 minutes to precipitate the plasmid. 14) Remove the supernatant and add 200 μl of 70% ethanol.
In addition, wash. 15) Centrifuge at 1,900 G for 5 minutes. 16) Remove the supernatant and evaporate the ethanol. 17) 6ml of TE (10mM Tris-HCl, 1mMEDT in each well)
Add 25 μl of RNase A (10 mg / ml) to A). RNaseA
To remove RNA.

(Analysis of Results) The yield of the plasmid obtained by the above protocol is checked by agarose gel electrophoresis and used for the next sequence reaction. As a result of repeated additional tests, it was found that the suspension could not be removed by centrifugation after boiling and that the yield of plasmids varied. FIG. 4 shows agarose electrophoresis photographs of 24 samples. M (lanes at both ends) in the figure indicates a molecular weight marker. As shown in FIG. 4, it was found that the peak corresponding to the plasmid DNA did not appear depending on the sample, and the plasmid yield varied. The causes of the variations are (1) the plate thickness is too thick to apply heat evenly to each well, and (2) 10) the plate is turned over during decantation, causing a loss to receive the sample. it was thought. In addition, instead of heating the heating step of 8) in hot water of 98 ° C. for 40 seconds, the heating step of
Similarly, when the microwave heating was performed twice for 0 seconds, the yields also varied, and the result was that the suspended matter could not be removed by centrifugation after boiling.

Protocol Using Plate for PCR Therefore, in order to improve the heating method, a plate of Robbins Co., which is commonly used in PCR method, was tried. Since this plate has a small thickness and is used in a sequence reaction or a PCR reaction, it can be uniformly heated by using the heating method used in the PCR method. However, it was necessary to study the optimum conditions for increasing the recovery rate at the same time because the culture amount was reduced by changing the plate.

(Heating step) If the heating time is short, it is considered that lysis is insufficient and the yield of plasmid is reduced. Further, if the time is too long, the plasmid will be cleaved and denatured.
The heating temperature was set to 99 ° C., and the Tl thermocycler heating time was evaluated in 5 stages of 60, 40, 30, 25, and 20 seconds. As a result, the yield was highest when heated for 25 seconds, and other contaminants could not be confirmed. From the above, it was found that the optimum heating time is 25 seconds.

(Suspension / Lysis Process) Since the PCR plate has a well-bottomed bottom, the cells are solidified at the time of collecting the cells, and the lysis may not be sufficient with stirring after adding the STET solution. Was a concern. When the yield of the plasmid was confirmed by agarose electrophoresis, it was found that the bacterial cells remained in the wells that could not be recovered. Therefore, instead of adding the lysozyme solution and then the STET solution, the process was changed to the step of adding the STET solution and then adding the lysozyme solution. Since the lysozyme solution has a higher solubility and a smaller volume than the STET solution, the lysozyme solution is easier to suspend. Therefore, the order in which the STET solution is added after pipetting (suspension) is changed. By changing this process, plasmids could be collected evenly.

(STET solution) In order to reduce the cost, the reduction of the amount of reagent in the STET solution was examined. STE
TE in the T solution is used for inhibiting the dissolution of Triton X-100 and the action of DNA degrading enzyme. In this operation,
Since the plasmid is extracted immediately after heating, the action of DNA degrading enzyme is small. From this, it was thought that the amount of TE added could be lowered, and a 10-fold dilution was performed. As a result, similar data could be obtained even when diluted 10-fold.

Triton X-100 in STET solution is
Avoid adding excessively as it may become a contaminant of the sequence reaction. Currently, TritonX- is usually used in STET solution.
We use 5% of 100, but this time, we examined it by reducing it to 3,1,0.5%. As a result, a sufficient plasmid could be confirmed by agarose electrophoresis at any concentration. However, since the growth amount of bacterial cells varies depending on the clones transformed into E. coli, the optimum condition was set to 1% so as to cope with any increase.

From the above, the amount of reagent in the STET solution can be adjusted to TE.
1/10 of Tris-HCl 1 mM, EDTA
It was found that, even if the amount of 0.1 mM Triton X-100 was reduced to 1/5, that is, one-fifth, it was possible to obtain the yield of the plasmid which was the same as that in the case where the conventional reagent amount was used.

Through the above experiments, the optimum protocol could be established. That is, the heating time is about 25 seconds, the reagent addition order is to add STET solution and then lysozyme, and the reagent amount in STET solution is Tris-H.
Cl 1 mM, EDTA 0.1 mM and Triton X-100 are 1%.

(Filtration Step) Further, the method of the present invention using the PCR reaction plate also has an advantage that contamination in the filtration step after the heating step can be reduced. That is, by centrifuging after the heating step, genomic DNA, proteins, lipids, etc., which have been separated by the heating step, are precipitated, and the supernatant in the wells is centrifugally filtered using a filter. At this time,
As a filter, it is advantageous to use a filter plate (7) having a filter (8) that fits into the wells of a PCR reaction plate as shown in FIG. Plate for PCR reaction having supernatant in well (1)
As shown in FIG. 6, the plate is turned over and set on a filter plate (7) having the same number of filters as the number of wells of the PCR reaction plate, and another PCR reaction plate ( 1 ') can be set. By centrifuging the assembly of the PCR reaction plate, the filter plate and the PCR reaction plate, the filtered supernatant can be collected in another PCR reaction plate. PC
Since each well of the R reaction plate has a protrusion (3) at the upper portion and can be fitted with a filter, cross contamination due to the crossing of the samples in each well during decantation can be avoided, and Also, it is possible to avoid sample loss due to liquid leakage in the centrifugal filtration step and contamination from the outside.

A hydrophilic filter medium is used for the filter for filtration. Hydrophilic polyvinylidene difluoride (PVDF)
Is preferred. If a glass filter is used, it will adhere and the yield will deteriorate. The use of PVDF can also reduce sample loss in the filtration process.

Further, in order to establish a method with less impurities, the salting-out step after boiling, the medium, and the reagents used such as a surfactant were also examined. (Salting out step) In the above-mentioned protocol, isopropanol is used in the salting out step. However, in order to further increase the recovery rate, application of polyethylene glycol was examined.
As a result, the yield of the plasmid was higher than that by salting out with isopropanol and no variation was observed, but many samples for which data could not be obtained by subsequent nucleotide sequencing were included. It is considered that this is because polyethylene glycol has a higher viscosity than that of isopropanol, so that polyethylene glycol cannot be completely removed in the step of removing the supernatant after salting out, and may have influenced as an inhibitor during the sequence reaction. Therefore, it was found that the use of polyethylene glycol for salting out is not usually effective, and can be used as a countermeasure for a sample in which the yield of plasmid due to the sample is small.

(Medium) Evaluation was carried out using 2 × YT medium having a high nutritional value. 2 x YT medium per liter
bacto-tryptone 16g, bacto-yeast extract10g, NaCl 5
This is a medium containing g. Although it was possible to increase the yield of the plasmid as compared with the LB medium, it was found that good data could not be obtained by the measurement using the DNA sequencer. It is speculated that the sequence reaction was inhibited because a large amount of Escherichia coli-derived protein was contained in the recovered solution, and the constituent components of the medium remained as impurities. Therefore, it was found that the LB medium was more optimal than the 2 × YT medium.

(Surfactant) As the surfactant used in the boiling method, TWEEN20 is used in addition to Triton X-100. TW
EEN20 has a lower viscosity than Triton X-100 and is easy to use. However, since this process omits the cleaning process, TWEEN2
0 remained as a contaminant and inhibited the sequence reaction. Therefore, it was found that TWEEN20 is not suitable for the method of the present invention.

[0037]

EXAMPLES Examples will be shown below, but the present invention is not limited to the examples. Plasmid extraction Plasmid extraction was performed by the following procedure . The target plasmid vector for the extraction and purification method is PUC18.

1) 96-well plate for PCR (Robbin
liquid LB medium containing 50 μg / ml of ampicillin (antibiotic for inhibiting cell wall synthesis) in each well of S.
Dispense 5 μl each. 2) Pick a colony formed by E. coli in each well with a toothpick and inoculate it. 3) Shake culture at 37 ° C. for 15 to 17 hours. 4) After culturing, the cells are collected by centrifugation at 1,500 G for 5 minutes. 5) The supernatant liquid, which is an impurity, is removed with an aspirator while washing with 70% ethanol. 6) Add 7 μl of lysozyme solution to the obtained bacterial cell pellet and pipette to suspend. 7) NaCl 0.1M, Tris-HCl 1mM, EDTA 0.1mM, Triton
Add 70 μl of STET solution containing 1% of X-100 to each well and stir. The bacterial wall is destroyed by Triton X-10 contained in the STET solution. 8) Heat with Tl thermocycler which is a heat block for PCR at 99 ° C for 25 seconds. 9) Precipitate the plasmid as a precipitate on the bottom by centrifugation at 1,900 G for 5 minutes. 10) Filter with a PVDF filter (pore size 0.2 μm manufactured by Corning) while centrifuging at 1,900 G for 3 minutes. 11) Add 8 μl of 3M sodium acetate to the supernatant and stir. 12) Add 80 μl of isopropanol and stir. 13) Centrifuge at 1,900 G for 20 minutes to precipitate the plasmid. 14) Remove the supernatant and add 200 μl of 70% ethanol to wash. 15) Centrifuge at 1,900 G for 5 minutes. 16) Remove the supernatant and evaporate the ethanol. 17) Add 50 μl of a solution prepared by dissolving 9 μl of RNase A in 6 ml of TE to each well. Remove RNA with RNase A.

Results The results obtained in the above examples are shown in Table 1 in comparison with the results obtained with commercially available automatic extractors (PI-200, PI-1100 and MG768). In addition, PI-200 which is a commercially available automatic extraction device
(Kurashiki Spinning Co., Ltd.), PI-1100 (Kurashiki Spinning Co., Ltd.)
Both MG768 and Hitachi Koki Co., Ltd. are based on the alkaline SDS method. The average extraction amount was measured by absorbance. The value of OD260 / 280 is 1.8.
A value of ˜2 means that proteins other than plasmid DNA are less contaminated and the purity is high (see “Introduction to Genetic Engineering”, Nanzandou, pp. 43-44 (1986)).

[0040]

[Table 1] Comparison between the present invention and a commercial automatic extraction device

As can be seen from the table, according to the method and apparatus of the present invention, the running cost is 1/5 of that of PI-200, and the cost is lower than other conventional methods. Moreover, since the method of the present invention does not require the use of a special device, the facility cost associated with the purchase of the device can be reduced. Further, the processing time can be shortened by a factor of two or more as compared with other conventional devices. In addition, plasmid recovery and OD2
The purity represented by 60/280 is equal to or higher than that of a commercially available automatic extraction device, and the present invention is a simple and easy method / device, but in terms of recovery rate and purity. You can see that it is excellent.

An electrophoretic photograph of the plasmid DNA obtained in the above example is shown in FIG. According to FIG.
According to the extraction method of the present invention, it can be seen that the plasmid DNA was obtained without any variation in any sample.

Similar results were obtained when the vector was replaced with PME18SFL3. Also, Whatma as a filter
Similar results were obtained using a filter plate manufactured by n company with a pore size of 0.45 μm. Further, according to the method and apparatus of the present invention, it is not necessary to handle a dangerous reagent such as phenol in the alkaline method, so that it has an effect of high safety.

[0044]

INDUSTRIAL APPLICABILITY As described above in detail, according to the present invention, a simple and low-cost plasmid DN which has no variation even if multiple samples are treated at the same time and has excellent purity / recovery rate.
It was possible to provide the extraction purification method and apparatus of A. INDUSTRIAL APPLICABILITY Since the present invention can improve efficiency and is low cost, it is an effective means in mass genome analysis.

[Brief description of drawings]

FIG. 1 shows an example of a PCR reaction plate used in the present invention.

FIG. 2 shows an example of a heat block for PCR reaction used in the heating step of the present invention.

FIG. 3 shows an example of a deep well plate in a conventional method.

FIG. 4 shows an electrophoresis photograph of a plasmid extracted by a conventional method.

FIG. 5 shows an example of a filter plate used in the present invention.

FIG. 6 shows a combination of a PCR reaction plate and a filter plate in the centrifugal filtration step of the present invention.

FIG. 7 shows an electrophoretic photograph of a plasmid extracted according to the present invention.

[Explanation of symbols]

1, 1 ': PCR reaction plate 2: Well 3: Projection 4: Heat block for PCR reaction 5: Thermocycler 6: Deep well plate 7: Filter plate 8: Filter

─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Tomoyuki Aizu 102-20 Sato Castle 23-20, Suga Castle, Tama-ku, Kawasaki City, Kanagawa Prefecture (56) References Nucleic Acids Res. , Vol. 27, No. 24 (1999), e37 BioTechniques, Vol. 16, No. 3 (1994), p. 376-380 BioTechniques, Vol. 13, No. 3 (1992), p. 366 (58) Fields surveyed (Int.Cl. ⁷ , DB name) C12N 15/00-15/90 C07H 1/08 JSTPlus (JOIS) BIOSIS / WPI (DIALOG) PubMed

Claims (6)

(57) [Claims] 1. A method for extracting and purifying plasmid DNA from a transformant by the Boyle method, comprising culturing cells, collecting cells, lysing, suspending, using a heat block for PCR reaction using a multi-sample plate for PCR reaction. A method for extracting and purifying DNA, which comprises heating and performing a precipitation filtration step. 2. A method according to claim 1 which comprises using a hydrophilic difluoride Poribinijiden (PVDF) filter to precipitate the filtration process. After harvested wherein cells, was suspended by addition of lysozyme solution, The method according to claim 1 or 2 perform lysis by the addition of STET solution. 4. Tris-HCl in STET solution at 1 mM, Tr
The method of any of claims 1-3, characterized in that adjusting the itonX-100 to 1% by weight. 5. A method according to any one of claims 1-4, characterized in that performing heating for about 25 seconds. 6. A DNA extraction and purification apparatus by the Boyle method, comprising a PCR reaction plate and a PCR reaction heat block.