Method of isolating purified plasmid DNA using a nonionic detergent, solution

A method of isolating purified plasmid DNA includes the steps of providing cells containing plasmid DNA and lysing the cells with an alkyldimethylphosphine oxide (APO) detergent to produce a mixture, centrifuging the cell and APO mixture, reserving a supernatant containing purified plasmid DNA and isolating the purified plasmid DNA, from the supernatant. Isolation and purification occurs in the absence of hazardous materials such as phenol and chloroform. Prior to mixing, the APO detergent is preferably combined with a strong base to produce a stable solution.

TECHNICAL FIELD 
This invention generally relates to the isolation of purified plasmid 
deoxyribose nucleic acid (DNA). More particularly, the invention relates 
to a method of isolating purified plasmid DNA using an 
alkyldimethylphosphine oxide (APO) detergent, a solution containing the 
APO detergent, and a kit containing the APO detergent. 
BACKGROUND OF THE INVENTION 
Plasmid deoxyribose nucleic acid (DNA) is a circular or looped format of 
genetic material located within all living cells. Purified plasmid DNA is 
widely studied and exploited in molecular biology as a tool to determine 
gene structure and function. One use of the purified plasmid DNA is the 
mapping of the human genome. The purified plasmid DNA must be sufficiently 
pure so as to permit additional investigational manipulations involving 
restriction enzymes and sequencing. 
Two procedures to isolate plasmid DNA are alkaline lysis (Maniatis et al., 
Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor 
Laboratory, Cold Spring Harbor, N.Y. (1989)) and boiling (Akela et al, 
Rapid Isolation and Sequencing of Double Stranded Plasmid DNA, 
Biotechniques 14: 726-728 (1993) and Holmes et al., A Rapid Boiling Method 
for the Preparation of Bacterial Plasmids, Anal. Biochem. 114: 193-197 
(1981)). Many of these methods use hazardous chemicals such as 
phenol/chloroform, guanidine hydrochloride and the like. Detergents used 
in these procedures often contain a phenyl ring that interferes with 
ultraviolet (UV) spectrophotometric analysis of proteins or nucleic acids. 
Some detergents, such as sodium dodecylsulfate (SDS), leave a residue that 
inhibits cutting of the plasmid DNA with restriction enzymes. 
Reduction or elimination of phenol in the preparation of isolated plasmid 
DNA is highly desirable. Wang et al, Simplified Large-Scale Alkaline Lysis 
Preparation of Plasmid DNA With Minimal Use of Phenol, Biotechniques, Vol. 
17, No. 1, pp 27-28 (1994). 
Holmes et at. discloses a boiling method for plasmid isolation that uses 
Triton X-100. Triton X-100 has the following shortcomings: 
(a) Due to a phenyl ring in its structure, Triton X-100 interferes with 
direct, spectral estimations of protein and nucleic acid concentration. 
(b) Triton X-100 is not a definable, pure compound. Rather, it is a mixture 
containing various chain lengths of ethoxylated isooctylphenol. The use of 
undefined mixtures is not compatible with Good Manufacturing Practice 
(GMP) procedures often required for processing of pharmaceuticals. 
(c) Polyethoxylated nonionic detergents such as Triton X-100 frequently are 
contaminated or become contaminated with peroxides upon aging. This 
contaminant can be especially destructive to the biological integrity and 
activity of proteins and nucleic acids. 
(d) In order to maintain solutions of Triton X-100 above the cloud point of 
30.degree. C., external heat must be continuously applied which adds 
mechanical complexity and further increases the risk of damaging certain 
sensitive proteins and nucleic acids. 
During isolation of plasmid DNA from bacteria, a large number of double 
stranded DNA templates are routinely generated. Some of the DNA templates 
contain significant amounts of secondary structures that often terminate 
the sequencing reactions prematurely making it difficult to read and 
analyze the DNA. One way to overcome this problem is to sub-clone the DNA 
fragment into a single stranded vector such as M13. Alternately, high 
temperature sequencing can be performed using, e.g., Taq polymerase. 
However, high temperature sequencing has been shown to be prone to error. 
See, for example, Lee-Jackson et al., Artifactual Frame Shift p53 Mutation 
at Codon 249 Detected with Cyclyst.TM. DNA Sequencing Method, 
Biotechniques 15: 363-4 (1993). 
Many detergents such as SDS that include an ester undergo alkaline 
hydrolysis that makes them unstable in alkaline solutions such as those 
produced by mixing SDS and sodium hydroxide. Due to this lack of 
stability, the sodium hydroxide and SDS solution should be made fresh 
before every use which renders alkaline SDS solutions useless for 
inclusion in a kit to isolate purified plasmid DNA. See Wang et al. 
A method of isolating purified plasmid DNA, solution and kit that overcome 
at least some of the above-identified shortcomings is highly desirable. 
SUMMARY OF THE INVENTION 
A method of isolating purified plasmid deoxyribose nucleic acid (DNA) 
includes the steps of providing cells containing plasmid DNA and mixing 
the cells with an alkyldimethylphosphine oxide (APO) detergent. The method 
can also include the step of isolating purified plasmid DNA from the whole 
cell and APO detergent mixture. The method is performed in the absence of 
hazardous chemicals typically used in other, conventional isolation and 
purification methods. 
The APO detergent does not interfere with the UV spectrophotometric 
analysis of proteins or nucleic acids. This lack of interference is 
presently theorized to be due to the lack of a phenyl ring in the APO 
detergent. Residual APO detergent causes less inhibition of cutting of the 
DNA with restriction enzymes as compared to residual sodium dodecylsulfate 
(SDS). 
The APO detergent is a definable, substantially pure compound that makes it 
compatible with Good Manufacturing Practice (GMP) procedures often 
required for processing of pharmaceuticals. The APO detergent is not 
contaminated nor does it become contaminated with peroxides upon aging 
which avoids the problems associated with loss of biological integrity or 
activity often seen when contaminants are present in other detergents. 
External heat need not be continuously applied which minimizes mechanical 
complexity and reducing the risk of damaging certain sensitive proteins 
and nucleic acids. 
There is little or no premature termination during sequencing reactions 
using DNA isolated with APO detergent, which results in improved reading 
and analysis of DNA sequencing. The improved results are presently 
theorized to be due to the effect of APO detergent on secondary 
structures. The elimination of the problem with secondary structures means 
that there is no need to sub-clone the DNA fragment into a single stranded 
vector or use high temperature sequencing. 
The APO detergent is stable in an alkaline solution, and it does not 
undergo alkaline hydrolysis. This makes the APO detergent-containing 
alkaline solution suitable for use in a kit. A novel method of using the 
APO detergent includes the step of combining the APO detergent with whole 
cells containing plasmid DNA to lysis the cells. 
A stable solution includes the APO detergent and sodium hydroxide. 
A kit for isolating plasmid DNA includes the APO detergent and can include 
the APO detergent mixed with sodium hydroxide. 
Numerous other advantages and features of the present invention will become 
readily apparent from the following detailed description of the preferred 
embodiments and the appended claims.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT 
Although this invention is susceptible to embodiment in many different 
forms, there are described in detail herein, presently preferred 
embodiments of the invention. It should be understood, however, that the 
present disclosure is to be considered as an exemplification of the 
principles of this invention and is not intended to limit the invention to 
the embodiments described. 
A method of isolating purified plasmid deoxyribose nucleic acid (DNA) 
includes the steps of providing cells containing plasmid DNA and mixing 
the cells with an alkyldimethylphosphine oxide (APO) detergent. The method 
isolates and purifies DNA from the mixture in the absence of hazardous 
chemicals such as phenol and chloroform. A representative procedure for 
isolating and purifying is an alkaline lysis procedure, which is preferred 
and described in more detail herein below. Alternatively, lysis can be 
performed by a boiling procedure that uses a nonionic detergent, e.g., 
Triton X-100. In both procedures, the APO detergent is substituted for the 
conventional detergent. 
The APO detergent is a substituted aliphatic alkane nonionic detergent 
having the following formula: 
##STR1## 
wherein R is a straight chain saturated alkyl moiety having about 6 to 
about 24, preferably about 8 to about 14, carbon atoms. The most preferred 
APO detergents are decyldimethylphosphine oxide (APO-10) and 
dodecyldimethylphosphine oxide (APO-12). FIG. 1 illustrates the structure 
of APO-10. FIG. 2 illustrates the ultra-violet (UV) absorption spectrum of 
a 1.0 mM APO-10 solution (b) as compared to a 0.1 mM Triton.RTM. X-100 
solution (a) and water (c). The full scale of FIG. 2 corresponds to 0.2 
absorbance units. 
The APO detergents are preferably synthesized by the method described in 
Laughlin, Journal of Organic Chemistry, Vol. 30, pp 1322-1324 (1965) and 
Hays, Journal of Organic Chemistry, Vol. 33, pp. 3690-94 (1968). The APO 
detergent is also commercially available. 
The cell can be obtained from a bacteria, mammalian, yeast or similar 
source. Representative bacteria include Eschericheria coli, Bacillus 
subtilis, Agrobacterium tumefaciens and the like. Representative mammalian 
tissues or cells include those known to be useful for DNA research. A 
representative yeast cell is Saccharomyces cerevisiae. 
For the alkaline lysis procedure, the cells and APO detergent are mixed 
under conditions to lyse the cells. Preferably, the conditions include a 
temperature in the range of about 0.degree. to about 25.degree. C., a time 
period of about 10 to about 30 minutes and adequate agitation. 
The cells are grown in a suitable growth medium, harvested using a 
centrifuge and the resulting pellet subsequently resuspended in a buffer 
prior to mixing of the cells with the APO detergent. The APO detergent can 
be introduced into the resuspension as a solution or a solid. The mixture 
is neutralized and centrifuged to obtain a supernatant that is then 
precipitated. The precipitated supernatant is centrifuged to produce a 
pellet of plasmid DNA that is washed with an alcohol to effect extraction 
of the purified DNA and then dried to produce a pellet of isolated and 
purified DNA. Water or the buffer containing a hydrolytic agent can be 
used to remove any residual RNA from the DNA pellet. The resulting DNA is 
ready for either restriction digestion or double stranded DNA sequencing. 
The cell-containing growth medium, mixture and precipitated supernatant 
are centrifuged under conditions effective to obtain the pellet or 
supernatant. The growth medium, buffer and neutralizing agent are 
convention and are selected based on the cell being used as one of 
ordinary skill in the art recognizes. 
The buffer is preferably a solution containing 50 mM glucose, 10 mM 
ethylene-diaminotetra-acetic acid (EDTA) and 25 mM tris (hydroxymethyl) 
amino methanol (Tris). The buffer has a pH of about 8.0. 
The APO detergent is preferably used with a strong base, e.g., sodium 
hydroxide, potassium hydroxide or the like. The APO detergent is 
preferably present an amount in the range of about 0.5 to about 5 weight 
percent (wt %). The hydroxide is preferably present in an amount in the 
range of about 0.5 to about 5 wt %. The weight percents are based on the 
total weight of the mixture of the APO detergent, hydroxide and water. 
Alternatively, the APO detergent can be in a solid state mixed with the 
cells. 
The neutralizing agent is preferably a 3M potassium acetate solution having 
a pH of about 4.8. 
Preferably, the precipitating and extracting steps are performed using a 
lower alcohol, e.g., methanol, ethanol or propanol. The extracting step 
can be performed using a solution containing about 60 to about 80 volume 
percent of the alcohol and 20 to 40 volume percent of water or an 
appropriate buffer. 
The hydrolytic agent that is mixed with water or the buffer to remove any 
residual RNA is preferably RNase A. 
The APO detergent and sodium hydroxide solution is very stable for 
prolonged time periods, has a long shelf life and shows no signs of 
chemical decomposition when stored at room temperature for prolonged time 
periods. Ongoing testing of a 1% APO-10 in 0.2N sodium hydroxide solution 
at room temperature shows no change in the proton and phosphorus .sup.31 P 
NMR spectra after six weeks. The stability makes the APO detergent and the 
solution containing the APO detergent very desirable as part of a kit to 
isolate and purify plasmid DNA. The kit can also contain other reagents 
and the like used to isolate and purify the DNA. These other reagents 
include buffers, neutralizers, precipitating agents, washes, and the like. 
A method of using the APO detergent includes the step of combining the APO 
detergent with whole cells containing plasmid DNA to lyse the cells. 
Preferably, the APO detergent is combined with sodium hydroxide. 
The following examples are given by way of representation and not 
limitation. 
EXAMPLE 1: ISOLATION OF PURIFIED PLASMID DNA 
To isolate plasmid DNA, E. coli bacteria (DH5.alpha.) was briefly spun down 
(1 min in a microfuge) after an overnight growth in Circlegrow.TM. (Bio 
101) containing the antibiotic ampicillin (100 .mu.g per ml). The 
supernatant was discarded and the pellet retained. To the pellet, 100 
.mu.l of Solution I (50 mM glucose, 10 mM EDTA and 25 mM Tris, pH 8.0), 
200 .mu.l of Solution II (0.2N sodium hydroxide, 1% APO-10), and 150 .mu.l 
of Solution III (3M potassium acetate, pH 4.8) were added sequentially at 
five-minute intervals to produce a sample. The APO-10 was obtained from 
Pierce Chemical Co., Rockford, Ill. Solution I is a buffer that resuspends 
the pellet, Solution II lysis the cell and Solution III neutralizes the 
base, i.e., the sodium hydroxide, in preparation for the next step. The 
sample was centrifuged for 15 minutes at 4.degree. C. in a microfuge, the 
pellet discarded and the supernatant precipitated with 1 ml of 100% 
ethanol for 5-10 minutes. The precipitated supernatant was then 
centrifuged for 15 minutes at 4.degree. C. in a microfuge, the supernatant 
discarded, the pellet washed with 500 .mu.l of 70% ethanol and then dried 
to produce isolated, purified plasmid DNA. The plasmid DNA was then 
brought up in 25 .mu.l of water or TE (10 mM Tris, 1 mM EDTA, pH 8) with 1 
.mu.l of 10 mg/ml RNase A (Sigma). The isolated plasmid DNA is ready for 
either restriction digestion or double stranded DNA sequencing. 
EXAMPLE 2: COMISON OF PROCEDURES AND YIELDS 
The plasmid digestion and/or double strand sequencing of DNA isolated using 
APO-10 (Procedure A--described above in EXAMPLE 1) and of DNA isolated 
using two other techniques (Procedures B and C) were compared. Procedure B 
differed from Procedure A only in that 1% SDS was substituted for the 1% 
APO-10 in Solution II. Procedure C was similar to Procedure B except that 
the supernatant was extracted sequentially with phenol and chloroform 
instead of 70% ethanol. For the sequential extraction, an equal volume of 
phenol was added to the supernatant, the tube vortexed and centrifuged for 
five minutes in a microfuge at room temperature. To extract the top phase, 
an equal amount of chloroform was added with the resultant mixture being 
vortexed, centrifuged and the DNA of the aqueous phase precipitated with 1 
ml of ethanol. The precipitated DNA was centrifuged for 15 minutes in a 
microfuge at 4.degree. C., washed and respun. The pellet was brought up in 
25 .mu.l of TE and 1 .mu.l of RNase A (10 mg/ml). 
The yields of DNA isolated by Procedures A, B and C were found to be nearly 
identical (within about 3% ) based on absorbance readings at 260 nm. The 
relative yield of isolated DNA was determined by a florescence technique 
using the preferred binding of Hoechst Dye 33258 (bis Benzimide) to DNA. 
The yield of DNA isolated by Procedures A, B and C were very similar 
(within about 7% ). 
EXAMPLE 3: DETERMINATION OF THE EFFECT OF APO DETERGENT ON DNA DIGESTION 
The digestion of DNA isolated using Procedure A was compared with the 
digestion of DNA isolated using Procedure B. Restriction was accomplished 
using one unit of the restriction enzyme Pst 1 per 0.5 .mu.g of isolated 
DNA for various lengths of time. Referring to FIG. 3, lanes 1, 2, 3 and 4 
are for DNA isolated using Procedure A and lanes 5, 6, 7 and 8 are for DNA 
isolated using Procedure B. The lengths of time are as follows: zero 
minutes for lanes 1 and 5; one minute for lanes 2 and 6; five minutes for 
lanes 3 and 7; and 30 minutes for lanes 4 and 8. After one minute, the DNA 
isolated by Procedure A was almost entirely convened into a linear form 
(see FIG. 3, lane 2). In contrast, the plasmid isolated by Procedure B is 
not linear at a time period of up to five minutes later (see FIG. 3, lane 
7). These results indicate that either the APO isolation yields a cleaner 
preparation or that residual APO detergent causes less inhibition of the 
restriction digest than residual SDS. In FIG. 3, the line marks the linear 
form. 
EXAMPLE 4: DETERMINATION OF THE EFFECT OF APO DETERGENT ON DNA SEQUENCING 
To test the relative ability of DNA isolated by each of the three 
procedures to be sequenced, a plasmid containing a plant lipoxygenase 
gene, which has significant problems with premature termination 
(Altschuler, unpublished), was used. For this comparison, an equal volume 
(2.5 .mu.l) of DNA solution from individually isolated plasmid using 
Procedures A, B and C was subjected to denaturation and annealed with a T7 
primer (see, for example, Barker, A More Robust, Rapid Alkaline 
Denaturation Sequencing Method, Biotechniques 14: 168-70 (1993). Samples 
(2.5 .mu.l) were heated to 65.degree. C. for 10 minutes prior to adding 7 
.mu.l of NSA buffer, i.e., 400 mM Tris-HCl, pH 7.5, 200 mM MgCl.sub.2, 
followed by returning the samples to a temperature of 65.degree. C. for 10 
minutes. The samples were permitted to cool to room temperature and then 
sequenced using the sequenase enzyme kit suggested by the manufacturer 
(USB, Cleveland, Ohio) with the following differences: a mixture was made 
for eight sets of reactions that included 16 .mu.l of diluted labeling 
mix, 8 .mu.l (0.1M) DTT, 4 .mu.l P.sup.32 dATP, 13 .mu.l Enzyme dilution 
buffer, 1 .mu.l Pyrophosphatase and 2 .mu.l Sequenase. To each sample, 5.4 
.mu.l of this mixture was introduced. After an incubation of five minutes 
at room temperature, 3.5 .mu.l of the labeling reactant was introduced 
into four tubes, each containing 2.5 .mu.l of the appropriate termination 
mixture containing the appropriate dideoxyribonucleotides. The samples 
were centrifuged, resuspended by shaking, centrifuged again and incubated 
at 37.degree. C. for five minutes. Then, 3.5 .mu.l of dye was added to 
each tube following by heating to 65.degree. for 15 minutes before 3 .mu.l 
of each reaction product was loaded and analyzed on a 7% polyacrylamide 
gel. As can be seen in FIG. 4, the DNA isolated by Procedures A, B and C 
are suitable templates for DNA sequencing. However, due to the enhanced 
efficiency of the polymerization step in the sequencing of the sample 
isolated by Procedure A, the sequencing band intensity was greatest for 
DNA isolated by Procedure A in a relative short period of time. When the 
gel is exposed to reveal sequential information for Procedures B and C, 
sequence information using Procedure A looks overexposed which again shows 
the superiority of Procedure A. In FIG. 4, termination regions are 
designated by A, C, G and T. Regions of premature sequencing termination 
are marked with a line. 
Of equal interest is when domains of high secondary sequence are analyzed. 
In this case, all treatments that utilize SDS (Procedures B and C) result 
in a significant amount of premature termination during the extension 
reaction. In contrast, the isolation Procedure A (which includes APO-10) 
did not result in significant premature termination. 
The use of the APO detergent produces isolated plasmid DNA that is more 
amenable to cutting with restriction enzymes. The use of APO detergent in 
DNA sequencing greatly reduces any problems in reading sequences that have 
secondary structures. The use of the APO detergent improves the speed of 
obtaining the DNA, lowers the cost of the method and advantageously 
permits sequence of DNA sequences with strong secondary structures which 
increase the cutting ability of the restriction enzymes and generates DNA 
templates that have less secondary structure problems with respect to 
sequencing. The use of the APO detergent to DNA isolation decreases the 
cost, time and effort needed to isolate clean DNA template for DNA 
manipulation, especially sequencing. These advantages are of great 
importance to projects that analyze large numbers of DNA sequences such as 
the human genome project. 
This invention has been described in terms of specific embodiments set 
forth in detail. It should be understood, however, that these embodiments 
are presented by way of illustration only, and that the invention is not 
necessarily limited thereto. Modifications and variations within the 
spirit and scope of the claims that follow will be readily apparent from 
this disclosure, as those skilled in the an will appreciate.