EP0431052A1

EP0431052A1 - Method for the isolation of dna

Info

Publication number: EP0431052A1
Application number: EP19890910010
Authority: EP
Inventors: Jacob Grimberg; Stanley Nawoschik
Original assignee: Lifecodes Corp
Current assignee: Lifecodes Corp
Priority date: 1988-08-31
Filing date: 1989-08-14
Publication date: 1991-06-12
Also published as: AU621936B2; JPH04503901A; WO1990002179A1; AU4201289A

Abstract

Un procédé nouveau d'isolement d'un ADN essentiellement pur à partir d'échantillons biologiques permet d'obtenir de manière avantageuse un ADN suffisamment pur pour être utilisé ultérieurement sans qu'il soit nécessaire de procéder à une purification séparée, comme c'est le cas dans des procédés de l'état antérieur de l'art.A novel process for the isolation of essentially pure DNA from biological samples makes it possible to advantageously obtain DNA which is sufficiently pure to be used later without the need for separate purification, as is the case. the case in methods of the prior art.

Description

DESCRIPTION

METHOD FOR THE ISOLATION OF DNA

Cross-Reference to a Related Application This application is a continuation-in-part of our co-pending application Serial No. 238,938, filed August 31, 1988.

Background of the Invention In order to study the biological, chemical, and physical properties of DNA or use recombinant DNA methodology, the DNA must be obtained from a given organism in its native, double-stranded form. A number of procedures are currently used to isolate DNA. Although the approaches vary considerably, the final step of each of the methods currently used involves the purification and/or concentration of the DNA.

One of the methods which is most commonly used to isolate eukaryotic high molecular weight (HMW) DNA involves lysis of the cell and nuclear membranes with a saline solution or tissue homogenization followed by the removal of proteins and other contaminants from the DNA by treatment with proteinase K/sodium dodecyl sulfate (SDS). These proteolytic digestion products, as well as proteinase K, are removed from the sample by phenol and/or phenol/chloroform extraction. The phenol/chloroform or phenol is subsequently removed by ethanol precipitation, dialysis, and/or ultracentrifugation in cesium chloride. This method has been used to extract DNA from eukaryotic cells grown in monolayer (Maniatis et al. [1982] in

Molecular Cloning. Cold Spring Harbor Press, Cold Spring Harbor, NY), organs (Graham [1978] Anal. Biochem. 85:609-613), tissue (Dubeau et al. [1986] Cancer Res. 46:2964, 2969), leukocytes (Gautreau et al. [1983] Anal. Biochem. 143:320-324), and plant tissue (Murray and Thompson [1980] Nucl. Acids Res. 8:4321-4325). This technique, however, is time consuming. In addition, phenol/chloroform extractions sometimes cause mechanical shearing of DNA. An alteration of the above technique has been devised for the isolation of high molecular weight DNA from cultured cells or chromosomes (Longmire et al. [1987] Nucl. Acids Res. 15:859). This procedure, as before, also requires that protein be removed by proteinase K/SDS treatment. The SDS concentration was 10-fold less than the SDS concentration used in the previous method: 0.1% vs. 1%. Detergent and proteolytic digestion products are removed in this method by dialyzing and concentrating nucleic acid-containing solutions against four 60 min changes of 20% (w/v) polyethylene glycol (PEG) in 10 mM Tris pH 8.0, 1 mM ethylenediaminetetraacetate (EDTA) (TE buffer). The sample must then be desalted by microdiatysis against two 15-30 minute changes of TE. Although the phenol/chloroform step is eliminated, the dialysis steps are time consuming. The preparation for the dialysis step is somewhat tedious since large volumes of buffer must be prepared, and a 20% PEG solution is somewhat viscous.

Another alternative to the phenol/chloroform extraction used to remove proteolytic digestion products known in the art involves the salting out of the cellular proteins by dehydration and precipitation with a saturated cesium chloride solution (Miller et al. [1988] Nucl. Acids Res. 16:1215). After centrifuging the solution, the supernatant containing the DNA was transferred to another tube. The DNA, however, had to be ethanol-precipitated and spooled out in order to remove excess salt as well as to concentrate the DNA. High molecular weight DNA has also been isolated from eukaryotic cells and solid tumors and organs using a method involving the denaturation of proteinase K digestion products with formamide (Reymond [1987] Nucl. Acids Res. 15:8118; and Kupiec et al. [1987] Anal. Biochem. 164:53-59). The formamide is then removed by dialysis and/or ethanol precipitation, both time- consuming procedures.

A final technique described in the art for the extraction of eukaryotic HMW DNA involves the use of the chaotropic agents guanidinium hydrochloride or guanidinium isothiocyanate to disrupt blood cells (JeanPierre [1987] Nucl.

Acids Res. 15:9611), cells grown in monolayer (Botwell [1987] Anal. Biochem. 162:463-465), tissue (Botwell, supra), and semen. Since guanidinium hydrochloride is a relatively weak chaotropic agent, it is sometimes necessary to further disrupt the nucleoprotein structure of a given organism by proteinase K treatment. Guanidinium isothiocyanate, though a very effective chaotropic agent, must be used under a chemical hood due to its noxious odor. Both guanidinium hydrochloride and guanidinium isothiocyanate may be removed by dialysis and/or ethanol precipitation.

Brief Summary of the Invention

The subject invention concerns a novel process for the isolation of substantially pure DNA from biological samples. The invention process is particularly useful for isolating eukaryotic HMW DNA of at least about 250 kilobase pairs. The process, advantageously, provides DNA of sufficient purity for subsequent use, as disclosed herein, without the necessity of a separate purification step as is used in prior art processes. The yield obtained is 100% since nothing is removed from the proteolysed lysate.

Specifically, the invention process comprises (1) the treatment of biological samples comprising the desired DNA and protein with lysing means (excluding enzyme inhibitors), and (2) contacting the lysed biological sample with a proteolytic enzyme at a temperature and for a time sufficient to destroy protein and autodigest or inactivate said proteolytic enzyme (in one step or two separate steps). Unexpectedly, and advantageously, the result of this procedure is that the DNA in the biological sample is provided in sufficient purity to be used for a variety of purposes, examples are disclosed herein, without the necessity for an added purification step.

In a preferred embodiment, the invention process comprises (a) treating a biological sample comprising DNA and protein with lysing means without enzyme inhibitors; and (b) contacting the lysate obtained in (a) with proteinase K at a temperature of about 60°C to about 70°C, for at least about 1.5 hours to obtain a preparation of DNA substantially devoid of protein and proteolytic activity.

Alternatively, step (a) can be used and then step (b) can be modified by contacting the lysed biological sample with any proteolytic enzyme at its working temperature, e.g., 37°C, and then deactivating the enzyme at a deactivating temperature, e.g., about 65°C.

Detailed Description of the Invention

Upon treating a biological sample comprising DNA with lysing means and then contacting the resulting lysate with a proteolytic enzyme which autodigests or is inactivated at a high temperature, there is obtained a sufficiently pure preparation of DNA which can be used for various purposes. The yield is 100% since there is no need for manipulation of the lysate.

As used herein, the following abbreviations will have the meanings indicated:

DNA Isolation Reagent 1: 320 mM sucrose, 10 mM Tris-HCI pH 7.6, 5 mM MgCI₂, 1% TRITON™X-100 DNA Isolation Reagent 2: 10 mM Tris-HCI pH 7.4, 10 mM EDTA, 10 mM NaCI

DNA Isolation Reagent 3: 10 mg/ml proteinase K

DTT: dithiothreitol

EDTA: ethylenediamine tetracetate

TAN buffer: 40 mM Tris-HCI, pH 7.9, 20 mM Sodium Acetate, 2 mM EDTA

Tris-HCI: Tris (Hydroxylmethyl) Aminomethane Hydrochloride

The process of the subject invention can be used to isolate DNA from any source. Examples of some of the sources are a blood cell sample, a urine sample, a tissue sample, a semen sample, cultured cells, a hair sample, amniotic fluid, bacteria (chromosomal and plasmid), yeast, other animal cells, and the like. Blood samples may be obtained from peripheral blood, cord, or organ blood. DNA from blood samples may be isolated from leukocytes. Cells in urine samples may include but are not limited to leukocytes, erythrocytes, epithelial cells, or fibroblasts. Cells in forensic semen samples may include but are not limited to spermatogonia and leukocytes or erythrocytes. Cultured cells may be epithelial cells or fibroblasts grown in suspension or on monolayers. Hair cells may be defined as those cells comprising the hair roots and the hair shafts, which may include epithelial cells. Animal cells may be from fish, reptiles, amphibians, birds or mammals. DNA isolated according to the invention method can be used for any enzymatic reaction, for example, restriction, DNA polymerization, phosphatased, RNA polymerization, etc. Any such use may require diluting or desalting the DNA in accord with procedures well known to those skilled in the art. A particular use of the DNA would be in the determination of an individual's identity using well-known nucleic acid hybridization technology. Such a method of genetic analysis of a DNA sample comprises: (a) digesting to completion a sample of DNA with a restriction endonuclease; (b) separating the digested DNA by gel electrophoresis according to the size of the digested DNAs; (c) transferring the separated DNA to a binding surface; and (d) hybridizing the separated DNA with an appropriate radioactively-labelled polymorphic probe using procedures known in the art. The pattern of signal so generated is preferably compared to a reference pattern or patterns.

The information generated by the comparison may be useful for situations requiring the determination of an individual's identity. Such information may be useful for forensic studies, i.e., the matching of physical evidence left at the scene of a crime with a particular suspect. Alternatively, such information may be used in paternity in which the child's DNA pattern is compared to that of the mother and "alleged" father. The information generated by the comparison may also be useful for diagnostic purposes. One example involves the identification of a gene or genotype related to a trait or medical condition. Another example involves analyzing DNA samples of a patient before and after the onset of a medical condition (e.g., cancer), which may be compared to determine somatic changes in the chromosomes. The detected changes may then be compared to a reference standard. Following are examples which illustrate procedures, including the best mode, for practicing the invention. These examples should not be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted.

Example 1 - Isolation of DNA from Blood, Urine. Semen. Tissue, HeLa Cells, and Hair

(a) Blood

Human blood specimens for DNA analysis were collected in vacutainer tubes containing the anti-coagulant ethylenediamine tetracetate (EDTA) and stored at 4°C immediately following collection. DNA collected in other anti¬ coagulants such as heparin or ACD were also found to be stable if the vacutainers were unopened. However, EDTA was found to be the best anti¬ coagulant for prolonged storage of whole blood at 4°C, or if repetitive sampling from a tube was required. During the isolation procedure, all tubes and solutions were kept at 4°C.

Samples of approximately 3-4 ml of blood were first spun for 10 min at 2000 xg to separate the buffy coat. 0.3 ml of the buffy coat was removed from each sample and placed into a 1.5 ml microcentrifuge tube (when large quantities of DNA are required. For smaller amounts, 300 μ\ of blood were aiiquoted and treated as follows: The blood cell membranes were ruptured by the addition of 1.0 ml of DNA Isolation Reagent 1 (320 mM sucrose, 10 mM Tris-HCI pH 7.6, 5 mM MgCI₂, 1% TRITON™X-100). After mixing the samples, the tubes were centrifuged at 900 xg for 5 minutes at 4°C. The supernatant was poured off, the pellet was resuspended in DNA Isolation Reagent 1 , and the above procedure was repeated.

After the second treatment with DNA Isolation Reagent 1 , the pellet was resuspended in 1.0 ml DNA Isolation Reagent 2 (10 mM Tris-HCI pH 7.4, 10 mM EDTA, 10 mM NaCI) and vortexed. The solution was centrifuged at 525 xg for 5 min at 4°C. After pouring off the supernatant, the pellet was resuspended in 450 μ\ DNA Isolation Reagent 2, 50 μ\ of DNA Isolation Reagent 3 (10 mg/ml proteinase K) were added, and incubated for 2 hr at 65°C. The DNA samples were stored at 4°C.

DNAs isolated from human blood were characterized quantitatively and qualitatively (0.6% agarose gel). The concentration of each sample was determined by comparing the fluorescent intensity of ethidium bromide bound to the DNA of the sample with those of bacteriophage λ-DNA standards. Alternatively, DNA concentration can be determined by using a fluorometer with

Houechst dye. DNA was also isolated from horse, antelope, gazelle, cow, dog, bear, dolphin, bird and chimpanzee blood using the above technique for white cells of blood, or nucleated red blood cells (fish, birds, tortoise).

When isolating DNA from nucleated red blood cells, use only 10-50 μ\ of whole blood. In some aquatic species DNA isolation buffer I should not have MgCI_₂, but should include 100 mM NaCI, otherwise DNA degradation occurs.

(b. Blood Stains

5-100 μ\ of a human blood sample was applied to a cotton cloth. The cloth containing the blood stain was cut into small pieces (up to about 2 cm x

2 cm), and transferred to a 15 ml conical centrifuge tube. The stains were dry; DNA has been isolated using the above method from stains in which the sample was applied up to 1 month prior to isolation. The pieces of cloth were suspended in 8 ml DNA Isolation Reagent 1 and incubated at 4°C for 2 hr with gentle rocking using a Nutator. After centrifuging the tubes for 5 min at 2,000 g, the supernatant was removed and the pellet was resuspended in 8 ml of DNA Isolation Reagent 1 and again incubated at 4°C for 30 min with gentle rocking. The tubes were centrifuged and the pellet containing nuclei was resuspended in 8 ml DNA Isolation Reagent 2. The lysate was centrifuged at 2,000 g for 5 min at 4°C. The pellet was resuspended in up to 700 I Isolation Reagent 2 and 10 mg/ml proteinase K (DNA Isolation Reagent 3) was added to a final concentration of 500 μ\/m\ and incubated for 2 hr at 65°C. DNAs isolated from blood stains were characterized on a 0.6% agarose yield gel. 2% of bloodstain DNA out of the total volume was loaded onto the gel. Approximately the same quantity and quality of DNA was obtained when isolated from a given volume of the same blood in liquid form or applied to a cloth. (c) Urine

DNA was isolated from 40 ml of human urine. The urine was centrifuged for 15 min at 2000 xg. The cell pellet was resuspended in 1.0 ml DNA Isolation

Reagent 1 , then centrifuged for 5 min at 4°C. The supernatant was poured off, the pellet was resuspended in DNA Isolation Reagent 1, and the same procedure as in blood (a) was used.

(d) Semen

DNA was isolated form 100 l of human semen. The cell membranes were ruptured by the addition of 1.0 ml of DNA Isolation Reagent 1. After mixing the samples, the tubes were centrifuged at 2,000 xg for 5 min at 4°C. The supernatant was poured off, the pellet was resuspended in DNA Isolation

Reagent 1, and the same procedure as in (a) was followed except that 10 mM DTT was added before the proteinase K in order to lyse the sperm cells.

(e. Semen and semen blood stains - differential Ivsis.

The same procedure as in (b) was used to obtain the non-semen DNA (usually contaminating a forensic sample) and discarded. The cloth was then treated again with proteinase K in Isolation Reagent 2 containing 10 mM DTT to lyse the sperm cells. ff. Spleen Tissue

Human spleen tissue was crushed with a mortar in the presence of liquid nitrogen. Samples were spun for 10 min at 1000 xg at 4°C. The cell membranes were ruptured by the addition of 1.0 ml of DNA Isolation Reagent 1 and then DNA was isolated as in (a).

(a) HeLa Cells

This procedure relates to HeLa cells grown in suspension. After the suspension was centrifuged, the pellet was washed with phosphate buffered saline (PBS). DNA was then isolated from these cells by treating cells with DNA Isolation Reagent 2. Treatment with DNA Isolation Reagent 1 was not necessary since blood was not present in these samples. The samples were subsequently treated with Isolation Reagent 3 for 2 hr at 65°C as in (a).

(h. Hair

5 human hair roots with shafts were suspended in 90 μ\ of DNA Isolation Reagent 2, 10 μ\ DNA Isolation Reagent 3 were added, and incubated for 2 hr at 65°C. Tubes were subsequently centrifuged for 60 seconds in a microfuge to separate the remaining undissolved hair and the supernatant containing DNA was collected.

(i. E. coli JE cgli DNA has also been obtained by using 1.5 μ\ of overnight grown cells, pelleting the cells, washing with 1 ml Isolation Reagent 2, pelleting again, then resuspending in 270 μ\, again adding 15 l Isolation Reagent 3 and incubating 2 hr at 65°C. Plasmid DNA can also be obtained without chromosomal DNA by first lysing the cells with lysozyme and 1% TRITON™X- 100, then centrifuging in a microfuge for 30 min, taking the plasmid-containing supernatant, and treating it with 500 g/ml proteinase K at 65°C for 2 hr. This method is good for plasmid screening and mapping. Example 2 - Analysis of Isolated Eukaryotic HMW DNA fa. RFLP Analysis

RFLP analysis was performed on the various purified DNAs after restriction with PstI or Hinfl followed by Southern blot transfer and hybridization to VNTR probes for the determination of identity or paternity.

DNA isolated was also tested with a battery of restriction enzymes

(BstN1, EcoR1. BamH1. Hindlll and others) and in all cases complete digestion can be obtained.

PCR can also be performed with the DNA and amplification of various fragments has been obtained.

When mobilities of DNA fragments have to be compared to standard fragments from more purified DNA, prior to sizing the restricted DNA's by gel eiectrophoresis, lithium chloride is added to a final concentration of 3.75M, placed at 4°C and centrifuged for 20 minutes in a microfuge (at least 12000 g). The supernatant is collected and the DNA is precipitated at 25° by adding ethanol to a final concentration of 70%, for 30 minutes; then centrifuged for 20 minutes in a microfuge. The resulting pellet is washed with 70% ethanol and reprecipitated; the DNA can now be resuspended in water and can be sized by gel eiectrophoresis.

Claims

1. A process for isolating DNA from a biological sample which comprises (a) lysing a biological sample comprising the desired DNA without enzymatic inhibitors; and (b) contacting the lysed biological sample with a proteolytic enzyme at a temperature and for a time sufficient to destroy protein and autodigest said proteolytic enzyme.

2. The process, according to claim 1 , wherein said biological sample is eukaryotic or prokaryotic.

3. The process, according to claim 2, wherein high molecular weight DNA is isolated from said eukaryotic biological sample.

4. The process, according to claim 2, wherein DNA is isolated from any cells from eukaryotic organisms.

5. The process, according to claim 3, wherein said eukaryotic biological sample is selected from the group consisting of blood, blood stain, urine, semen, semen stain, spleen tissue, HeLa cells, hair, and other animal cells.

6. The process, according to claim 1 , wherein said proteolytic enzyme is proteinase K.

7. The process, according to claim 1 , wherein said biological sample is contacted with proteinase K at a temperature of about 60°C to about 70°C, for at least about 1.5 hours.

8. A process for isolating DNA from a biological sample which comprises (a) lysing a biological sample comprising the desired DNA; and (b) contacting the lysed biological sample with an effective proteolysis amount of proteinase K at a temperature of about 60°C to about 70°C, for at least about 1.5 hour.

9. The process, according to claim 7, wherein said biological sample is selected from the group consisting of blood, blood stain, urine, semen, semen stain, spleen tissue, HeLa cells, hair, and other animal cells.

10. The process, according to claim 7, wherein high molecular weight DNA is isolated from said biological sample.

11. A process for isolating DNA from a biological sample which comprises (a) lysing a biological sample comprising the desired DNA without enzymatic inhibitors; (b) contacting the lysed biological sample with a proteolytic enzyme at its working temperature for a time sufficient to destroy protein; and (c) deactivating said enzyme at a deactivating temperature.

12. The process, according to claim 11, wherein said biological sample is eukaryotic or prokaryotic.

13. The process, according to claim 12, wherein high molecular weight DNA is isolated from said eukaryotic biological sample.

14. The process, according to claim 12, wherein DNA is isolated from any cells from eukaryotic organisms.

15. The process, according to claim 13, wherein said eukaryotic biological sample is selected from the group consisting of blood, blood stain, urine, semen, semen stain, spleen tissue, HeLa cells, hair, and other animal cells.

16. A forensic test procedure which comprises the use of DNA isolated from a biological sample which comprises (a) lysing a biological sample selected from the group consisting of blood, blood stain, urine, semen, semen stain, tissue, hair, and other animal cells; and (b) contacting the lysed biological sample with an effective proteolysis amount of proteinase K at a temperature of about 60°C to about 70°C, for at least about 1.5 hour.

17. A paternity test procedure which comprises the use of DNA isolated from a biological sample which comprises (a) lysing a biological sample selected from the group consisting of blood, semen, or amniotic fluid; and (b) contacting the lysed biological sample with an effective proteolysis amount of proteinase K at a temperature of about 60°C to about 70°C, for at least about 1.5 hour.

18. A kit for forensic or paternity testing use which comprises (a) a container with a lysing means for a biological sample which does not contain an enzyme inhibitor; and (b) a container with an effective proteolysis amount of a proteolytic enzyme.

19. The kit, according to claim 18, wherein said proteolytic enzyme is proteinase K.