Process for rapid isolation of high molecular weight DNA

A procedure for isolating high molecular weight nucleic acids utilizing a mixture of lytic enzymes and a chaotropic agent to complete protein denaturation and dissociation from nucleic acids is provided. The nucleic acids so obtained are useful for restriction enzyme analysis and DNA probe hybridization.

TECHNICAL FIELD 
This invention relates to the field of molecular biology and more 
particularly to the isolation of high molecular weight DNA suitable for 
restriction enzyme analysis. 
BACKGROUND ART 
There have been great advances in molecular biology in recent years in 
terms of new restriction enzymes, improved DNA cloning methods, improved 
hybridization probes, and improved hybridization supports: however, 
little, if any, attention has been paid to preparation of the samples for 
use in these procedures. That is, little has been done to improve the 
speed and efficiency of isolation and preparation of nucleic acids, 
specifically DNA, for use in these procedures. 
The classic reference for isolation of high molecular weight DNA is Marmur 
[Journal of Molecular Biology, Volume 3, 208-218 (1961)]. The process 
taught by Marmur includes 14 steps, some of which must be repeated to 
insure completeness. Many of these steps are time consuming and most 
require a highly skilled technician to assure successful isolation of the 
DNA. The individual steps are discussed below showing all details provided 
by the author: 
1. Cell lysis 
A cellular suspension is first treated with a detergent, sodium dodecyl 
sulfate, at 60.degree. C. in an attempt to lyse the cells. If lysis is 
achieved, one proceeds to the next step; if not, an enzyme, lysozyme, is 
added and allowed to digest the cells for 30-60 minutes. These steps can 
be reversed but, if they are, the detergent must be added for the 
subsequent processes to work correctly. This method of cell lysis, while 
broadly effective, is not universally so. There are organisms with cell 
walls which are resistant to both lysozyme and detergent treatment (e.g. 
Streptococcus pyogenes). 
2. Deproteinization 
The viscous lysed suspension is made 1M in perchlorate and extracted with a 
chloroform-isoamyl alcohol mixture. This mixture is then centrifuged to 
form 3 layers, the middle layer containing the proteins. The upper aqueous 
layer contains the nucleic acids and is removed for further processing. 
3. Nucleic acid precipitation 
The nucleic acids are precipitated by layering ethanol on top of the 
aqueous layer and collecting them on a glass rod. The precipitate is 
drained of excess ethanol by pressing the precipitate against the side of 
the flask. This step requires a highly skilled technician because, as is 
the case with all DNA precipitation steps to follow, significant losses of 
the desired DNA can occur. These losses occur due to incomplete 
precipitation, redissolution of precipitate during washing, and binding of 
precipitating DNA to the walls of the vessel. 
4. Dissolution of nucleic acids 
The precipitate is transferred to dilute saline-citrate buffer and the 
nucleic acid is redissolved by gentle stirring. Excessive stirring causes 
shearing of the DNA resulting in low molecular weight DNA being formed 
and, therefore, great care must be exercised. 
5. Deproteinization 
The soluble nucleic acids are re-extracted as described in Step 2 to remove 
any remaining proteins. This step may be repeated several times to assure 
complete removal of the proteins. Complete removal is assumed when very 
little protein is seen at the interface of the solvent layers. 
6. Nucleic acid precipitation 
Step 3 is repeated to purify further the nucleic acids. 
7. Dissolution of nucleic acids 
Step 4 is repeated to obtain a nucleic acid solution for further 
processing. 
8. Ribonuclease treatment 
The mixture of nucleic acids present in the solution is treated with 
ribonuclease for 30 minutes at 37.degree. C. to digest any RNA present in 
the sample. Following digestion, it is possible to remove proteins which 
were resistant to earlier extractions (steps 2 and 5). 
9. Deproteinization 
Step 2 is repeated to obtain DNA free of RNA, any proteins released by the 
ribonuclease treatment, and ribonuclease itself. 
10. DNA precipitation 
DNA is precipitated as described in step 3. 
11. Dissolution of DNA 
DNA is redissolved as described in step 4. 
12. Isopropyl alcohol precipitation 
To the DNA solution is added an acetate-EDTA buffer and the solution is 
mixed rapidly. While the solution is being mixed, 0.54 volume of isopropyl 
alcohol is added dropwise into the vortex. Then, according to the author, 
"DNA usually precipitates in a fibrous form after first going through a 
gel phase at about 0.5 vol(umes) isopropyl alcohol"--another difficult 
step. 
13. Isopropyl alcohol precipitation 
Steps 4 and 12 can be repeated "if the yield is good". 
14. Final washing 
The final precipitate is washed free of acetate and salt by gently stirring 
the adhered precipitate in aqueous ethanol containing progressively 
increasing (70-95%) portions of ethanol. The DNA is then available for 
dissolution in the buffer of choice for use in further analysis. 
The process described by Marmur and followed to date by molecular 
biologists is complex and prone to loss of the desired DNA. According to 
Marmur, recovery of up to 50% of the DNA from the cells can be achieved by 
carefully following this process, not a very high yield. This process also 
generally requires 1 to 2 days, an undesirably lengthy processing period. 
Furthermore, according to Marmur, it would be very difficult to devise a 
technique for the efficient isolation of DNA from a wide variety of 
microorganisms. Although Marmur's method is effective against various 
organisms including almost all Gram negative organisms and many Gram 
positive organisms, one organism of great interest, Streptococcus pyogenes 
is not lysed by this method. This organism is the causative agent for the 
common illness, strep throat. 
An alternative procedure for isolating DNA also disclosed by Marmur 
utilizes cesium chloride centrifugation. This process, while having many 
fewer steps, requires centrifugation for 3 days and, therefore, is also 
not a rapid method for isolating DNA. Additionally, cesium ions have a 
detrimental effect on the biological activity of the recovered DNA. 
Carter et al. [Biotechniques, Volume 1(3), 142-147 (1983)] disclose an 
improved isopycnic centrifugation medium which uses cesium 
trifluoroacetate instead of cesium chloride. The cesium trifluoroacetate 
is used in a fashion similar to cesium chloride. The trifluoroacetate 
anion, however, imparts properties to cesium trifluoroacetate that result 
in higher quality nucleic acid preparations when compared to traditional 
cesium density gradient media. The use of cesium trifluoroacetate has 
extended the application of isopycnic centrifugation in nucleic acid 
separations and purifications but the procedure is still inherently time 
consuming and labor intensive. 
Gross-Bellard et al. [European Journal of Biochemistry, Volume 36, 32-38 
(1973)] disclose a similar method useful for isolating high molecular 
weight DNA from mammalian cells. In this method, Proteinase K and a 
detergent, sodium dodecyl sulfate (SDS), are used to lyse the cells. This 
method is also applicable to microorganisms susceptible to these lysis 
conditions. Deproteinization is accomplished using phenol saturated 
buffers rather than chloroform:isoamyl alcohol. The use of phenol, 
however, requires a 4-hour dialysis to remove it before proceeding 
further. Any RNA present is digested using ribonuclease and then the 
ribonuclease is digested using proteinase K and SDS. The DNA is then 
deproteinized twice more and dialyzed again. Finally, the DNA is 
precipitated with ethanol. This procedure offers little advantage over 
that of Marmur. There are fewer DNA precipitations, but the procedure 
introduces two long dialysis steps. 
Chassy et al. [Applied and Environmental Microbiology, Volume 39(1), 
153-158 (1980)] disclose a procedure for extending the usefulness of 
lysozyme in lysing microorganisms. This procedure depends upon growing the 
organism in a modified medium, particularly one containing L-threonine. 
This leads to organisms with weakened cell wall crosslinks which are 
susceptible to lysozyme treatment. This procedure is useful only when the 
organism to be lysed is known to grow in the modified medium and precludes 
any possibility of using DNA collected directly from a clinical specimen 
without culturing the organism. Therefore, this procedure is not of 
general utility. 
Potter et al. [Cancer Letters, Volume 26, 335-341 (1985)] disclose a method 
for rapid extraction and purification of DNA from human leukocytes. This 
method includes detergent lysis of the cells, potassium acetate 
precipitation of cellular material, ribonuclease digestion, adsorption 
chromatography using DEAE-cellulose to purify the DNA, and ethanol 
precipitation of the DNA. This method is applicable only to those 
microorganisms susceptible to detergent lysis, generally the Gram negative 
organisms. 
Potter et al.'s method is different from Marmur's in the use of potassium 
acetate to precipitate the cellular contents and the use of adsorption 
chromatography. These authors acknowledge that DNA can be lost in the 
potassium acetate precipitation step. Also, as with many precipitation 
methods, the sample must be centrifuged in order to assure complete 
recovery of the precipitate and centrifugation requires expensive 
equipment and valuable time. Avoidance of precipitation and centrifugation 
steps would be advantageous. 
The use of adsorption chromatography to purify DNA can also have certain 
disadvantages since DNA with the highest molecular weight tends to bind 
most strongly to the support and, therefore, is not easily eluted from the 
column. This can result in selective loss of the highest molecular weight 
DNA. 
De Klowet [Journal of Microbiological Methods, Volume 2, 189-196 (1984)] 
discloses a method for rapid isolation of high molecular weight RNA and 
DNA from yeast through the use of a single glucanase enzyme, lyticase, 
isolated from Oerskovia xanthineolytica, in the presence or absence of a 
detergent to lyse the cells. After lysing, the sample is deproteinized by 
extraction with an equal volume of a phenol:chloroform (4:1) solution. The 
mixture is centrifuged to separate the layers and the aqueous nucleic 
acid-containing phase is collected. This phase is made 0.3M in sodium 
acetate (pH 5.0) and the nucleic acids precipitated with two volumes of 
ethanol. Alternatively, high molecular weight RNA can be isolated by 
selective precipitation with lithium chloride. The high molecular weight 
DNA can be isolated from the supernatant of that precipitation or directly 
from the previously precipitated nucleic acids. In the former case, DNA 
isolation proceeds with ribonuclease treatment to destroy the RNA present. 
DNA is then deproteinized again by phenol:chloroform extraction and 
precipitated with ethanol. This overall procedure is very similar to that 
of Marmur in that it entails repeated deproteinization with organic 
solvents and repeated precipitation with ethanol, both of which are 
undesirable. These types of treatments are undesirable in that they 
require skilled technicians, extensive equipment and facilities and take 
substantial amounts of time. 
Monsen et al. [FEMS Microbiology Letters, volume 16, 19-24 (1983)] disclose 
a general method for cell lysis and preparation of DNA from streptococci. 
This method uses the lytic enzyme mutanolysin (endo-N-acetylmuraminidase) 
isolated from Streptomyces globisporus 1829 to lyse the organism. As noted 
above, these organisms are resistant to lysozyme and detergent-induced 
lysis. Monsen et al. also showed that streptococci are resistant to a 
general protease, Proteinase K. High molecular weight DNA was isolated by 
Monsen et al. using cesium chloride centrifugation. As noted above, this 
is a very time consuming procedure not appropriate for routine preparation 
of clinical samples. 
None of the methods discussed offers a completely general method for lysis 
of microorganisms of interest in clinical diagnostic applications and 
isolation of their DNA. In general, these methods utilize a single enzyme 
and/or a detergent to lyse a limited group of organisms. 
Gillespie et al. (U.S. Pat. No. 4,483,920, issued Nov. 20, 1984) disclose 
the immobilization onto filters of messenger RNA in the presence of a high 
concentration (80%) of a chaotropic salt, sodium iodide. Here the 
chaotropic salt is used to denature proteins, to dissociate them from mRNA 
and to solubilize substantially all cellular components to allow them to 
pass through the hybridization filter. 
Von Hippel et al., Science Volume 145, 577-580 (1964), studied the 
denaturation of proteins and nucleic acids with chaotropic salts and found 
that proteins are more susceptible to such denaturation than nucleic 
acids. One can conclude from such findings that it might be possible to 
select concentrations of chaotropic salts which will denature proteins, 
thus aiding their dissociation from nucleic acids, without denaturing 
double stranded high molecular weight DNA. 
There remains a need for a rapid, efficient and simple process for 
isolating high molecular weight nucleic acids from a wide variety of 
sources. 
DISCLOSURE OF THE INVENTION 
A method of isolating high molecular weight nucleic acids from their source 
organism comprising the steps of: 
(A) forming a suspension of said organism containing or suspected of 
containing the desired nucleic acid; 
(B) treating said organism with at least one lytic enzyme; 
(C) treating said organism with surfactant prior to, simultaneously with or 
subsequent to step (B); 
(D) degrading unwanted classes of nucleic acids by treatment with nucleases 
specific for the unwanted nucleic acids; 
(E) degrading proteins by digestion with at least one broadly active 
protease; 
(F) denaturing remaining proteins and dissociating them from the nucleic 
acid by adding at least one chaotropic agent; and 
(G) dialyzing and concentrating the nucleic acid. 
DESCRIPTION OF THE INVENTION 
As discussed above, there is a need for an improved method of isolating 
nucleic acids, particularly DNA, from their source organism. By source 
organism is meant any organism which contains a nucleic acid, including 
cells, particularly microbial cells, viruses, and mycoplasma. The cells 
subjected to this process include cells of mammalian or bacterial origin 
or a mixture of mammalian and bacterial cells. This improved method allows 
rapid, high yield recovery of high molecular weight nucleic acids from a 
wide variety of source organisms especially from most clinically relevant 
cell types. Surprisingly, it has been found that the method of this 
invention allows isolation of high molecular weight nucleic acids from a 
very wide variety of organisms in approximately 90 minutes. This method is 
expected to find the greatest utility in the isolation of DNA, but is also 
useful in isolating RNA. 
For sake of convenience, the process will be described as being carried out 
with a suspension of cells but it should be understood that other nucleic 
acid-containing source organisms can be treated similarly. As a practical 
matter, it is convenient to begin this process of isolation with a cell 
suspension containing 1.times.10.sup.7 to 1.times.10.sup.8 cells in 500 
.mu.L buffer. A variety of buffers can be used, including sodium borate 
and sodium phosphate, but tris(hydroxymethyl)aminomethane hydrochloride 
(Tris) is preferred. The preferred buffer concentration is 10 mM, although 
a concentration from about 1 mM to about 500 mM is acceptable. The 
preferred pH is about 8 although a pH ranging from about 4 to about 9 is 
acceptable. The buffer can also contain 1-500 mM sodium chloride with 10 
mM being preferred and, similarly, about 1-10 mM 
ethylenediaminetetraacetic acid (EDTA) with about 1 mM being preferred. 
The preferred buffer composition is 10 mM Tris, 10 mM sodium chloride, 1 
mM EDTA, pH 8.0 (Tris buffer, pH 8.0). The process can be readily adapted 
to accommodate larger volumes and/or different numbers of cells by 
adjusting dilutions and process timing. 
The method of this invention can be carried out using a single lytic enzyme 
but it is preferred that a mixture of such enzymes can be used. The 
mixture of lytic enzymes to be added to the organism suspension is 
generally a bacteriolytic enzyme "cocktail" and can include lysozyme, 
endo-N-acetylmuraminidase and achromopeptidase, among others. The final 
concentrations of the enzymes in this lytic mixture can be in the range of 
50-500 .mu.g/mL, 10-500 .mu.g/mL and 50-500 .mu.g/mL, respectively. The 
preferred concentrations are 300 .mu.g/mL, 30 .mu.g/mL and 300 .mu.g/mL, 
respectively. 
This lytic enzyme cocktail can be prepared in the Tris buffer, pH 8.0 
described above, or any of the generally acceptable buffers can be 
utilized. This cocktail can be added to the suspension described above at 
a temperature range of about 20.degree. C.-70.degree. C. and incubated for 
a time period of about 1-60 minutes. It is known that, in general, 
enzymatic processes proceed more rapidly at higher temperatures provided 
the enzyme is not denatured by the higher temperature. It is thus 
preferred to operate at the highest possible (non-denaturing) temperature 
for a given enzyme cocktail in order to minimize the time required to 
complete digestion. This same consideration will apply to all subsequent 
enzymatic degradation steps. The preferred treatment conditions for the 
above enzyme mixture are a digestion temperature of about 37.degree. C. 
for about 10 minutes. Optionally, a reducing agent can be added to the 
lytic mixture to further improve cell lysis. A variety of reducing agents 
can be used including dithiothreitol, dithioerythritol, cysteine and 
ascorbic acid, 2-mercaptoethanol being preferred. The final concentrations 
of any of the reducing agents can be in the range of 0.1-30 mM. The 
preferred concentration is 5 mM. To further improve both cell lysis and 
solubilization, aprotic solvent can be added to the lytic mixture. Among 
such solvents are N,N-dimethylformamide (DMF) and sulfolane with 
dimethylsulfoxide (DMSO) being a preferred one. The final concentration of 
the aprotic solvent can be in the range of 1-20% (v/v). The preferred 
concentration is 10% (v/v). 
The lytic effect of this particular enzyme mixture is broad and includes 
microorganisms that can be found in the following genera: 
______________________________________ 
Aerobacter sp. Microccocus sp. 
Acholeplasma sp. Mycoplasma sp. 
Achromobacter sp. Pediococcus sp. 
Arthrobacter sp. Proteus sp. 
Azotobacter sp. Protaminobacter sp. 
Bacillus sp. Pseudomonas sp. 
Blevibacterium sp. Salmonella sp. 
Clostridium sp. Sarcina sp. 
Enterobacter sp. Serratia sp. 
Escherichia sp. Shigella sp. 
Flavobacterium sp. Staphylococcus sp. 
Klebsiella sp. Streptococcus sp. 
Kurthia sp. Streptomyces sp. 
Lactobacillus sp. 
Leuconostoc sp. 
______________________________________ 
Other enzymes can also be added to the above enzyme cocktail to further 
broaden its bacteriolytic action. Suitable enzymes include: lipase, 
lysopeptase, endo-N-acetylglucosaminidase D or H, dextranase, cellulase, 
glucoamylase, hyaluronidase, N-acetylmuramyl-L-alanine amidase, 
streptomyces KM endopeptidase, streptomyces SA endopeptidase and 
streptomyces ML endopeptidase. Such enzymes will generally be useful in 
concentration ranges from about 10-500 .mu.g/mL of the mixture. Selection 
of one or more additional enzymes for inclusion in the lytic cocktail will 
depend upon the cell wall and cell membrane structure of the cell to be 
lysed. For example, the streptomyces ML endopeptidase can be added to 
improve lysis of cells containing type III peptidoglycans. The enzymatic 
specificity of each of these enzymes is generally known and enzyme 
selection is expected to be based on the type of organism present. 
Complete lysis of the cells can be assured by addition of surfactant to the 
lytic mixture, increasing the temperature of lysis and/or by using a 
longer reaction time. The surfactant addition can be prior to, 
simultaneous with or subsequent to the lytic enzyme treatment. It is 
generally preferred to add sodium dodecylsulfate (SDS) to a final 
concentration of about 0.1% (w/v) and to increase the temperature to about 
60.degree. C. for 10 minutes. The concentration of SDS can be from about 
0.05% to about 1% (w/v); the temperature can range from about 20.degree. 
C. to about 70.degree. C.; and the time from about 1 minute to about 60 
minutes. Other surfactants can be utilized under similar conditions. These 
include cationic, anionic and nonionic surfactants such as Triton X-100, 
octyl-.beta.-D-glucopyranoside, 
3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS) and 
3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propane sulfonate 
(CHAPSO). 
At this stage of the procedure it is necessary to decide whether one wishes 
to isolate DNA or RNA. Until this stage, the processing does not degrade 
either DNA or RNA. It is now advantageous to degrade one or the other so 
that desired nucleic acid can be isolated. Since this invention is 
expected to have its greatest utility in the isolation of DNA, the 
remainder of the process will be discussed in terms of DNA isolation and 
reference will only be made to RNA isolation when specific modifications 
to the procedure are required. 
The RNA in the lysate can be degraded using a ribonuclease (RNase). RNase I 
A is the preferred enzyme although other RNases such as RNase T, RNase U, 
RNase H or RNase B can be substituted. The RNase is added to a final 
concentration of about 50-500 .mu.g/mL of the lytic mixture at a 
temperature of about 20.degree. C. to about 70.degree. C. for about 1 to 
about 60 minutes. The preferred treatment conditions are approximately 300 
.mu.g/mL RNase IA for approximately 10 minutes at approximately 60.degree. 
C. These conditions serve to degrade rapidly the RNA in the lysate without 
degrading the DNA. If RNA isolation is desired, digestion with a 
deoxyribonuclease (DNase) is substituted for RNase digestion. DNase I is a 
suitable enzyme for use in this manner. In order to avoid degradation of 
the desired class of nucleic acid, however, it is important to utilize 
RNase or DNase of appropriate purity. 
It is optional, but frequently desirable, at this point to add some Tris 
buffer, pH 8.0, or water to the lytic mixture to reduce its viscosity. 
High viscosity can cause reduced recovery of isolated DNA due to 
inefficient operation of the remaining steps in the process. This optional 
dilution can be incorporated as part of the RNase treatment step. 
The cellular proteins present in the lytic mixture and the RNase can then 
be degraded with a non-specific protease or a mixture of such broadly 
active proteases. The preferred protease is Proteinase K, but others such 
as an alkaline protease isolated from Streptomyces griseus can be 
substituted. The final concentration is preferably 50-500 .mu.g/mL for 
about 5-120 minutes at about 20-70.degree. C. A preferred specific 
treatment condition is approximately 300 .mu.g/mL of Proteinase K at 
approximately 60.degree. C. for approximately 30 minutes. This protein 
degradation step generally replaces the organic solvent extractions of the 
prior art. By eliminating these extractions, the yield of recoverable DNA 
can be greatly improved because the extractions expose the DNA to a great 
deal of shear force and frequently cause the high molecular weight DNA to 
break into smaller pieces which would be lost during processing. 
Optionally, a reducing agent can be added to the lytic mixture to further 
improve the protein degradation. A variety of reducing agents can be used 
including dithiothreitol, dithioerythritol, cysteine and ascorbic acid, 
2-mercaptoethanol being preferred. The final concentrations of any of the 
reducing agents can be in the range of 1-100 mM. The preferred 
concentration is 5 mM. To further improve both cell lysis and 
solubilization, aprotic solvent can be added to the lytic mixture. Among 
such solvents are N,N-dimethylformamide (DMF) and sulfolane with 
dimethylsulfoxide (DMSO) being a preferred one. The final concentration of 
the aprotic solvent can be in the range of 1-50% (v/v). The preferred 
concentration is 10% (v/v). 
Any proteins still associated with nucleic acids or present in the mixture 
up to this point can be denatured and dissociated from the nucleic acids 
by the addition of one or more chaotropic agents. By chaotropic agent is 
meant any substance capable of altering the secondary and tertiary 
structure of proteins and nucleic acids. The chaotropic agent should be 
added to the mixture under conditions to avoid denaturation of 
double-stranded DNA. This means that the temperature of chaotrope 
treatment should be below approximately 40.degree. C., preferably about 
30.degree. C. These lower temperatures are important because 
double-stranded DNA is more susceptible to heat denaturation when the 
binding proteins which stabilize it have been degrade, denatured or 
dissociated from it and because the chaotropic agent can lower the thermal 
melting temperature of double-stranded DNA. Temperatures as low as about 
4.degree. C. can be used. The preferred chaotropic agents are salts such 
as sodium trifluoroacetate, sodium perchlorate and sodium iodide. The 
preferred salt is sodium trifluoroacetate at a final concentration of 
approximately 0.5M, but can range from about 0.1 to about 1.0M. The 
duration of the chaotrope treatment is about 1-30 minutes with 5 minutes 
being preferred. In some instances, the contents released from cells 
during the process of this invention can be of such quantity that the 
cellular components can precipitate upon the addition of the chaotropic 
agent reducing the purity and the amount of recovered DNA. Such 
precipitation can be avoided by adding a buffer, for example, Tris buffer, 
pH 8.0, or water to the mixture just prior to the addition of the 
chaotropic agent and/or by using less chaotropic agent. 
At this stage, DNA is ready for final purification and concentration. This 
can be accomplished by collodion membrane dialysis-concentration. Before 
beginning the dialysis-concentration process, the mixture can be diluted 
with a low salt buffer such as 10 mM Tris, 1 mM EDTA, pH 8.0 to prevent 
the concentration process from proceeding too rapidly and causing the 
cellular components to precipitate. It may be desirable to add more buffer 
during the concentration process to assure complete removal of 
contaminants (degradation products of the organisms and the reagents 
utilized in the process of this invention) before achieving the desired 
concentration. The concentration process can be stopped when a convenient 
volume of DNA containing solution remains. 
The DNA thus prepared is pure as can be shown by UV spectral analysis. The 
ratio of the absorbance at 260 nm to that at 280 nm for DNA is between 1.5 
and 2. The process of this invention affords products of such a ratio, 
indicating a high level of purity. Lower ratios are indicative of 
contamination by proteins and higher ratios indicative of RNA 
contamination of a DNA preparation. 
The purified DNA so obtained is ready for use or can be stored in this form 
for later use. It is immediately available for restriction endonuclease 
digestion, molecular weight determination and hybridization analysis, 
among other uses. Further processing may be necessary for DNA cloning or 
in recombinant work. 
The following examples illustrate the invention. In a method substantially 
identical to the process described in Examples 1 and 2, genomic DNA was 
successfully isolated and analyzed from Klebsiella pneumoniae, 
Staphylococcus aureus, Enterobacter aerogenes, Proteus mirabilis, E. coli, 
and Staphylococcus epidermis.

EXAMPLE 1 
Isolation and Analysis of Bacterial Genomic DNA from Pseudomonas Aeruginosa 
A. DNA Isolation 
A cell suspension of Pseudomonas aeruginosa DP 295, (ATCC accession number 
10145) was prepared by first removing several single colonies from a 
37.degree. C. overnight growth on a Petri dish of blood agar base medium 
with a Pasteur pipet. The cells were then suspended and mixed by vortexing 
them in 1 mL of 10 mM Tris buffer, pH 8.0, containing 10 mM NaCl and 1 mM 
EDTA in a 12.times.75 mm borosilicate glass test tube. A concentration of 
approximately 3.times.10.sup.8 cells/mL was achieved by diluting the cell 
suspension with the above-mentioned buffer. The cell concentration was 
determined by a visual comparison of the cell suspension with a McFarland 
nephelometric turbidity standard of 1 in a 12.times.75 mm borosilicate 
glass test tube. 500 .mu.L of this cell suspension was then transferred to 
another 12.times.75 mm borosilicate glass test tube for further 
processing. 
A lytic enzyme cocktail was prepared by combining 150 .mu.L of 10 mg/mL 
lysozyme in water, 150 .mu.L of 10 mg/mL achromopeptidase in water and 150 
.mu.L of 1 mg/mL endo-N-acetylmuraminidase in water. This cocktail was 
stored at -20.degree. C. 45 .mu.L of this cocktail was added to 500 .mu.L 
of the cell suspension. The mixture was vortexed and incubated at 
37.degree. C. for 10 minutes. 
5.6 .mu.L of 10% SDS and 15 .mu.L of 10 mg/mL ribonuclease I A in water 
were added to the tube, vortexed and incubated at 60.degree. C. for 10 
minutes. 
100 .mu.L of 10 mM Tris buffer, pH 8.0, containing 10 mM NaCl and 1 mM EDTA 
was added to the tube and vortexed to dilute the sample. 
20 .mu.L of 10 mg/mL Proteinase K in water was added to the tube, vortexed 
and incubated at 60.degree. C. for 30 minutes. 
The tube was cooled to 20.degree. C. and to it was slowly added 686 .mu.L 
if 1M sodium trifluoroacetate that was first filtered with a 0.22 .mu.m 
Nalgene filter unit. The tube was vortexed and incubated at 20.degree. C. 
for 5 minutes. 
750 .mu.L of TE buffer (10 mM Tris buffer, pH 8.0, containing 1 mM EDTA) 
was added to the tube and vortexed to dilute the sample. 
The sample was then transferred from the test tube into a 2-mL capacity 
collodion membrane (available from Schleicher & Schuell, Inc. Keene, NH) 
for dialysis and concentration. The average retention of the collodion 
membrane was 25,000 Daltons and above. TE buffer, pH 8.0, was used for the 
dialysis. A vacuum of 19 inches of mercury was used to facilitate the 
concentration of the sample in a Schleicher & Schuell dialysis and 
concentration apparatus. 
After the sample volume was reduced to approximately 200 .mu.L, the vacuum 
was released and 500 .mu.L of TE buffer, pH 8.0, was added to the sample 
and mixed by drawing the ample into a 2 mL glass pipet and expelling it 
back into the collodion membrane. The dialysate was also removed and 
replaced with TE buffer, pH 8.0. The dialysis and concentration of the 
sample was resumed until a final volume of approximately 50 .mu.L was 
achieved. The purified DNA sample was then removed from the collodion 
membrane with a Rainin P-200 pipet tip and placed into a 1.5-mL 
polypropylene Eppendorf tube for storage at 4.degree. C. 
B. Restriction Endonuclease Digestion 
23 .mu.L of the approximately 50 .mu.L purified DNA sample from above was 
transferred to another 1.5-mL Eppendorf tube and to it were added: 6 .mu.L 
of a 5X EcoRI restriction enzyme buffer (500 mM Tris buffer, pH 7.5, 
containing 50 mM MgCl.sub.2, 250 mM NaCl and 500 .mu.g/mL bovine serum 
albumin from Bethesda Research Laboratories) and 1 .mu.L of 100 
units/.mu.L EcoRI restriction endonuclease from Boehringer Mannheim. The 
tube was gently vortexed and incubated at 37.degree. C. for 4 hours. The 
reaction was stopped with the addition of 7.5 .mu.L of a 5X Ficoll dye 
solution (12.5% Ficoll type 400-DL from Sigma, 50 mM EDTA, 0.13% 
bromphenol blue and 0.13% xylene cyanol). 
C. Agarose Gel Electrophoresis 
A 0.8% gel was prepared by melting 1.6 g of Seakem LE agarose at 
100.degree. C. in 200 mL of a Tris-acetate buffer (20 mM Tris-acetate 
buffer containing 2 mM EDTA), pH 8.0. The molten agarose was cooled to 
50.degree. C. and a gel was cast in a 15.times.20 cm BioRad 
electrophoresis tray with a 20 well comb. After the gel cooled to room 
temperature, the gel was allowed to stand at 4.degree. C. for 1 hour. The 
comb was removed and the gel was placed into a BioRad submarine gel 
electrophoresis unit with 1500 mL of Tris-acetate buffer, pH 8.0, at room 
temperature. 30 .mu.L of the DNA-ficoll dye containing solution was added 
to a well in the agarose gel. The electrophoretic separation of the DNA 
fragments was then allowed to proceed for 15 hours at 1.5 volts/cm with 
the buffer circulating in the unit at 250 mL/hour. 
D. Transfer of Size-Separated DNA Fragments onto a Membrane Support 
An electroblotting method was used. The DNA fragments in the gel were first 
denatured in 700 mL of 0.4N sodium hydroxide at room temperature for 30 
minutes with a gently rocking motion. The gel was then treated for 5 
minutes in 700 mL of electroblot buffer (12 mM Tris buffer, pH 7.5, 
containing 6 mM sodium acetate and 0.3 mM EDTA). The electrophoretic 
transfer of the denatured DNA fragments from the gel onto a membrane 
support (electroblotting) was carried out using a Hoefer TE 42 Transphor 
Electrophoresis Cell. To prepare the gel for electroblotting, one side of 
the Hoefer Transphor Cassette was submerged in an 8-L polypropylene tray 
containing 3 L of electroblot buffer, pH 7.5, at room temperature. A 
15.5.times.22 cm Dacron.RTM. polyester fiber sponge (a registered 
trademark of E .I. du Pont de Nemours and Company) was placed on top of 
the inside surface of the Transphor Cassette and layered with the 
following materials: 1 sheet of 15.times.21.5 cm blotter paper, a piece of 
charged modified nylon membrane (GeneScreen.TM. Plus, available from E. I. 
du Pont de Nemours and Company) cut to the same size as the gel, the 
agarose gel with the denatured DNA fragments and 2 sheets of 15.times.21.5 
cm blotter paper. To finish the assembly of the Transphor Cassette, the 
Dacron.RTM. polyester sponge/blotter paper/agarose gel/blotter paper 
sandwich was locked between the tow sides of the Transphor Cassette and 
placed into a Transphor Cell containing 4.5 L of electroblot buffer, pH 
7.5, chilled to 5.degree. C. The operation of the Transphor Cell was 
carried out at 2 different voltage settings as follows: first at 10 volts 
for 60 minutes and then at 40 volts for 60 minutes. A temperature of 
5.degree. C. was maintained throughout the entire operation of the 
Transphor Cell. The membrane was then removed from the Transphor Cassette, 
rinsed in electroblot buffer, pH 7.5, and air dried for 30 minutes at room 
temperature. 
E. DNA Probe Preparation 
Plasmid DNA [pKK3535, Brosius et al., Plasmid, Volume 6, 112-118 (1981)], 
known to contain DNA sequences which are capable of hybridizing with those 
portions of the genomic DNA which code for rRNA, was labeled with .sup.32 
P for use as a DNA probe using a Nick-translation Kit (available from E. 
I. du Pont de Nemours and Company). The following were added to a 1.5-mL 
Eppendorf tube: 9 .mu.L of 550 .mu.g/mL plasmid DNA; 61 .mu.L of water, 50 
.mu.L of nick-translation buffer; 40 .mu.L of a solution containing dATP, 
dGTP, and dTTP: 1 millicurie of .sup.32 P-dCTP with a specific activity of 
3000 Curies/mmole in a volume of 100 .mu.L; 20 .mu.L of DNA polymerase I; 
and 20 .mu.L of DNase I. The contents were mixed by drawing them into a 
Rainin P-200 pipet tip and expelling them back into the tube. The reaction 
was then allowed to proceed at 15.degree. C. for 60 minutes. The reaction 
was stopped with the addition of 6 .mu.L of 500 mM EDTA. The .sup.32 
P-labeled probe DNA was then separated from the unincorporated 
deoxynucleotide triphosphates by size separation on a 0.7.times.30 cm 
Sephadex G-50 column with TE buffer, pH 8.0. The specific activity of the 
labeled doublestranded DNA probe was approximately 3.times.10.sup.8 
dpm/.mu.g DNA. 
F. DNA Probe Hybridization 
The hybridization membrane containing the transferred DNA fragments 
(prepared in step D above) was pre-hybridized with 100 .mu.g/mL sonicated, 
denatured, salmon sperm DNA for 30 minutes in 200 mL of 3X SSC buffer (3X 
SSC buffer contains 0.45M NaCl and 0.045M sodium citrate), containing 0.5% 
SDS, 10X Denhardt's solution (10X Denhardt's solution contains 0.2% 
ficoll, 0.2% polyvinylpyrolidone and 0.2% bovine serum albumin, fraction 
5), at 60.degree. C. in a sealed plastic box on a rocker platform. The 
purified .sup.32 P-labeled DNA probe was then denatured by heating it to 
100.degree. C. in a boiling water bath for 5 minutes, quickly cooled to 
4.degree. C. on ice and added immediately to the pre-hybridization mix on 
the membrane to achieve approximately 4.times.10.sup.6 dpm/mL. 
Hybridization was allowed to proceed for 20 hours at 60.degree. C. with 
continuous rocking. After 20 hours, any unreacted .sup.32 P-labeled DNA 
probe was removed by four successive 15-minute washes with 3X SSC buffer 
containing 0.5% SDS at 60.degree. C. with continuous rocking. The membrane 
was then removed from the wash solution and air dried at room temperature. 
G. Analysis of the Restriction Fragment Length Polymorphisms 
The ribosomal RNA operon(s) contained between the EcoRl restriction sites 
of the genomic DNA were detected by visualization of the labeled hybrids 
by autoradiography of the membrane on x-ray film. The same fragment sizes 
and number of fragments were observed from the Pseudonomas aeruginosa 
cells as have previously been obtained by using the DNA isolation method 
of De Klowet with phenol/chloroform extractions and alcohol 
precipitations. 
EXAMPLE 2 
Isolation and Analysis of Bacterial Genomic DNA From Streptococcus Faecalis 
A. DNA Isolation 
A cell suspension of Streptococcus Faecalis DP 283 (ATCC accession number 
19433) was prepared by first removing several single colonies from a 
37.degree. C. overnight growth on a Petri dish of blood agar base medium 
with a Pasteur pipet. The cells were then suspended and mixed by vortexing 
them in 1 mL of 10 mM Tris buffer, pH 8.0, containing 10 mM NaCl and 1 mM 
EDTA in a 12.times.75 mm borosilicate glass test tube. A concentration of 
approximately 6.times.10.sup.8 cells/mL was achieved by diluting the cell 
suspension with the above-mentioned buffer. The cell concentration was 
determined by a visual comparison of the cell suspension with a McFarland 
nephelometric turbidity standard of 2 in a 12.times.75 mm borosilicate 
glass test tube. 500 .mu.L of this cell suspension was then transferred to 
another 12.times.75 mm borosilicate glass test tube for further 
processing. 
45 .mu.L of the lytic enzyme cocktail prepared in Example 1(A) was added to 
500 .mu.L of the cell suspension. The mixture was vortexed and incubated 
at 37.degree. C. for 10 minutes. 5.6 .mu.L of 10% SDS was added to the 
tube, vortexed and incubated at 60.degree. C. for 10 minutes. 
15 .mu.L of 10 mg/mL ribonuclease I A in water was added to the tube, 
vortexed and incubated at 60.degree. C. for 10 minutes. 
100 .mu.L of 10 mM Tris buffer, pH 8.0, containing 10 mM NaCl and 1 mM EDTA 
was added to the tube and vortexed to dilute the sample. 
20 .mu.L of 10 mg/mL Proteinase K in water was added to the tube, vortexed 
and incubated at 60.degree. C. for 30 minutes. 
The tube was cooled to 20.degree. C. and to it was slowly added 686 .mu.L 
of 1M sodium trifluoroacetate that was first filtered with a 0.22 .mu.m 
Nalgene filter unit. The tube was vortexed and incubated at 20.degree. C. 
for 5 minutes. 
750 .mu.L of TE buffer was added to the tube and vortexed to dilute the 
sample. 
The sample was then transferred from the test tube into an 8-mL capacity 
Schleicher & Schuell collodion membrane, average retention of above 75,000 
Daltons, for dialysis and concentration. TE buffer, pH 8.0, was used for 
the dialysis. A vacuum of 19 inches of mercury was used to facilitate the 
concentration of the sample in a Schleicher & Schuell flatbottom 
apparatus. A magnetic stirring bar was used inside of the apparatus to 
facilitate mixing of the dialysis buffer during the process. 
After the sample volume was reduced to approximately 200 .mu.L, the vacuum 
was released and 500 .mu.L of TE buffer, pH 8.0, was added to the sample 
and mixed by drawing the sample into a 2-mL glass pipet and expelling it 
back into the collodion membrane. The dialysate was also removed and 
replaced with TE buffer, pH 8.0. The dialysis and concentration of the 
sample was resumed until a final volume of approximately 50 .mu.L was 
achieved. The purified DNA sample was then removed from the collodion 
membrane with a Rainin P-200 pipet tip and placed into a 1.5-mL 
polypropylene Eppendorf tube for storage at 4.degree. C. 
Steps (B) through (F) were carried out as described in Example 1(B) through 
(F). 
G. Analysis of the Restriction Fragment Length Polymorphisms 
The ribosomal RNA operon(S) contained between the EcoRI restriction sites 
of the genomic DNA were detected by visualization of the labeled hybrids 
by autoradiography of the membrane on x-ray film. The same fragment sizes 
and number of fragments were observed from the Streptococcus faecalis 
cells as have previously been obtained by using the DNA isolation method 
of De Klowet with phenol/chloroform extractions and alcohol 
precipitations. 
EXAMPLE 3 
Isolation and Analysis of Bacterial DNA 
A. DNA Isolation 
An unamplified cell suspension of Escherichia coli K12 strain LM1035 
containing the plasmid pKK3535 [Brosius et al. Plasmid, Volume 6, 112-118 
(1981)] was prepared by first removing several single colonies from a 
37.degree. C. overnight growth on a Petri dish of trypticase soy agar 
containing 100 .mu.g/mL ampicillin with pasteur pipet. The cells were then 
suspended and mixed by vortexing them in 1 mL of 10 mM Tris buffer, pH 
8.0, containing 10 mM NaCl and 1 mM EDTA in a 12.times.75 mm borosilicate 
glass test tube. A concentration of approximately 3.times.10.sup.8 
cells/mL was achieved by diluting the cell suspension with the 
above-mentioned buffer. The cell concentration was determined by a visual 
comparison of the cell suspension with a McFarland nephelometric turbidity 
standard of 1 in a 12.times.75 mm borosilicate glass test tube. 500 .mu.L 
of this cell suspension was then transferred to another 12.times.75 mm 
borosilicate glass test tube for further processing. 
45 .mu.L of the lytic enzyme cocktail prepared in Example 1(A) was added to 
500 .mu.L of the cell suspension in a test tube. The mixture was vortexed 
and incubated at 37.degree. C. for 10 minutes. 
5.6 .mu.L of 10% SDS and 15 .mu.L of 10 mg/mL ribonuclease I A in water 
were added to the tube, vortexed and incubated at 60.degree. C. for 10 
minutes. 
100 .mu.L of 10 mM Tris buffer, pH 8.0, containing 10 mM NaCl and 1 mM EDTA 
was added to the tube and vortexed to dilute the sample. 
20 .mu.L of 10 mg/mL Proteinase K in water was added to the tube, vortexed 
and incubated at 60.degree. C. for 30 minutes. 
The tube was cooled to 20.degree. C. and to it was slowly added 686 .mu.L 
of 1M sodium trifluoroacetate that was first filtered with a 0.22 .mu.m 
Nalgene filter unit. The tube was vortexed and incubated at 20.degree. C. 
for 5 minutes. 
750 .mu.L of TE buffer was added to the tube and vortexed to dilute the 
sample. 
The sample was then transferred from the test tube into an 8-mL capacity 
Schleicher & Schuell collodion membrane for dialysis and concentration. 
The average retention of the collodion membrane was 75,000 Dalons and 
above. TE buffer, pH 8.0, was used for the dialysis. A vacuum of 19 inches 
of mercury was used to facilitate the concentration of the sample in the 
Schleicher & Schuell flat-bottom apparatus. 
After the sample volume was reduced to approximately 200 .mu.L, the vacuum 
was released and 500 .mu.L of TE buffer, pH 8.0, was added to the sample 
and mixed by drawing the sample into a 2-mL glass pipet and expelling it 
back into the collodion membrane. The dialysate was also removed and 
replaced with TE buffer, pH 8.0. The dialysis and concentration of the 
sample was resumed until a final volume of approximately 50 .mu.L was 
achieved. The purified DNA sample so obtained was then removed from the 
collodion membrane with a Rainin P-200 pipe tip and placed into a 1.5-mL 
polypropylene Eppendorf tube for storage at 4.degree. C. 
B. Agarose Gel Electrophoresis 
Electrophoresis was carried out as described in Example 1(B) with the 
following modifications: 
6 .mu.L of a 5X Ficoll dye solution (12.5% Ficoll type 400-DL from Sigma, 
50 mM EDTA, 0.13% bromphenol blue and 0.13% xylene cyanol) was added to 25 
.mu.L of the purified DNA sample in an Eppendorf tube and gently vortexed. 
30 .mu.L of the DNA-ficoll dye containing solution was then added to a 
well in the agarose gel. The electrophoretic separation of the DNA was 
then allowed to proceed for 2 hours at 3 volts/cm with the buffer 
circulating in the unit at 250 mL/hour. 
C. Plasmid DNA Detection 
The presence of plasmid DNA in E. coli was ascertained by exposing the gel 
to ultraviolet light, 302 nm, and by observing the fluorescence of the 
nucleic acids. Proof of the presence of the plasmid pKK3535 was obtained 
by photographing the fluorescent nucleic acids and observing a band 
corresponding to the molecular weight of the plasmid. The results of this 
Example 3 demonstrate that the process of this invention is useful for 
isolation of intact plasmid DNA also.