AU621936B2 - Method for the isolation of dna - Google Patents

Description

63 DATE 23/03/90 6-2 1 3 P DATE 26/04/90

I:

APPLN. ID 42012 89 PCT NUMBER PCT/US89/03476 INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (51) International Patent Classification 5 (11) International Publication Number: WO 90/02179 C12N 15/10, C12P 19/34 Al C12N 11/68 (43) International Publication Date: 8 March 1990 (08.03.90) (21) International Application Number: PCT/US89/03476 (81) Designated States: AT (European patent), AU, BE (European patent), BF (OAPI patent), BJ (OAPI patent), CF (22) International Filing Date: 14 August 1989 (14.08.89) (OAPI patent), CG (OAPI patent), CH (European patent), CM (OAPI patent), DE (European patent), FR (European patent), GA (OAPI patent), GB (European Priority data: patent), IT (European patent), JP, LU (European pa- 238,938 31 August 1988 (31.08.88) US tent), ML (OAPI patent), MR (OAPI patent), NL (Euro- 373,721 29 June 1989 (29.06.89) US pean patent), SE (European patent), SN (OAPI patent), SU, TD (OAPI patent), TG (OAPI patent).

(71) Applicant: LIFECODES CORPORATION [US/US]; Old Saw Mill River Road, Valhalla, NY 10595 Published With international search report.

(72) Inventors: GRIMBERG, Jacob 113 Hobart Street, Ridge- Before the expiration of the time limit for amending the field Park, NJ 07660 NAWOSCHIK, Stanley 6 claims and to be republished in the event of the receipt of Secora Road, Apt. 19-G, Monsey, NY 10952 amendments.

(74) Agents: SALIWANCHIK, Roman et al.; 2421 N.W. 41st Street, Suite A-i, Gainesville, FL 32606 (US).

(54)Title: METHOD FOR THE ISOLATION OF DNA (57) Abstract The subject invention concerns a novel process for the isolation of substantially pure DNA from biological samples. 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.

WO 90/02179 PCT/US89/03476 1

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.

WO 90/02179 PCT/US89/03476 2 143:320-324), 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. 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% polyethylene glycol (PEG) in 10 mM Tris pH 8.0, 1 mM ethylenediaminetetraacetate (EDTA) (TE buffer).

The sample must then be desalted by microdialysis 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 -j I: i -iii WO 90/02179 PCr/US89/03476 formamide is then removed by dialysis and/or ethanol precipitation, both timeconsuming 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 the treatment of biological samples comprising the desired DNA and protein with lysing means (excluding enzyme inhibitors), and 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

I

i'1 jo :t r i

I$

."t i3 d t i i~ M I IT 1 MM 4 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 inventive process comprises treating a biological sample comprising DNA and protein with lysing means without enzyme inhibitors; and contacting the lysate obtained in with proteinase K at a temperature of about 60 0 C to about 70 0 C, for at least about hours to obtain a preparation of DNA substantially devoid 10 of protein and proteolytic activity.

Alternatively, step can be used and then step can be modified by contacting the lysed biological sample with any proteolytic enzyme at its working temperature, 37 0 C, and then deactivating the enzyme at a deactivating temperature, about According to a first embodiment of this invention, there is provided a process for isolating DNA from a biological sample which comprises lysing a biological sample comprising the desired DNA without enzymatic inhibitors; and 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.

According to a second embodiment of this invention, there is provided a process for isolating DNA from a biological sample which comprises lysing a biological sample comprising the desired DNA without enzymatic inhibitors; and contacting the lysed biological sample with an effective proteolysis amount of proteinase K at a temperature of about j to about 70°C, for at least about 1.5 hour.

According to a third embodiment of this invention, there is provided a process for isolating DNA from a biological sample which comprises lysing a biological sample comprising the desired DNA without enzymatic inhibitors; contacting the lysed biological sample with a proteolytic enzyme at its working temperature for a time sufficient to destroy protein; and deactivating said enzyme at a deactivating temperature.

S /642Z 1 4A According to a fourth embodiment of this invention, there is provided a forensic test procedure which comprises the use of DNA isolated from a biological sample which comprises lysing a biological sample selected from blood, blood stain, urine, semen, semen stain, tissue, hair or other animal cells; and contacting the lysed biological sample with an effective proteolysis amount of proteinase K at a temperature of about 0 C to about 70 0 C, for at least about 1.5 hour.

10 According to a fifth embodiment of this invention, there is provided a paternity test procedure which comprises the use of DNA isolated from a biological sample which comprises lysing a biological sample selected from blood, semen, or amniotic fluid; and contacting the lysed biological sample with an effective proteolysis amount of proteinase K at a temperature of about 60 0 C to about 70 0 C, for at least about 1.5 hour.

'According to a sixth emoodiment of this invention, there is provided a diagnostic test procedure which comprises the use of DNA isolated from a biological sample which comprises lysing a biological sample selected from blood, urine, semen, tissue, hair or other animal cells; and contacting the lysed biological sample with an effective proteolysis amount of proteinase K at a temperature of about 60 0 C to about 70°C, for at least about 1.5 hour.

According to a seventh embodiment of this invention, there is provided a kit when used for the forensic test procedure of claim 16, the paternity test procedure of claim 17, or the diagnostic procedure of 1 claim 18, said kit comprising a container with a lysing means for a biological sample which does not contain an enzyme inhibitor; and a container with an effective proteolysis amount of a proteolytic enzyme. r 4B 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: r DNA Isolation Reagent 1: 320 mM sucrose, 10 mM Tris-HCI pH 7.6, mM MgC12, 1% TRITONTMX-100 1

SI~

I' /4t i I 1 i i

-J;

WO 90/02!179

DN/

DNA

DTT

EDi

TAIS

Tris PCI/US89/03476 Sisolation Reagent 2: k Isolation Reagent 3:

FA:

4 buffer:

-HCI:

10 mM Tris-HCI pH 7.4, 10 mM EOTA, 10 mM NaCl 10 mg/ml proteinase K dithiothreitol ethylenediamine tetracetate 40 mM Tris-HCI, pH 7.9, 20 mM Sodium Acetate, 2 mM EDTA 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.

I WO 90/02179 PCT/US89/03476 6 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 r 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: digesting to completion a sample of DNA with a restriction endonuclease; 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 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, 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 cancer), which may be compared to determine somatic changes in the chromosomes. The detected changes may then be compared to a reference standard.

m WO 90/02179 PCT/US89/03476 7 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 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 anticoagulants such as heparin or ACD were also found to be stable if the vacutainers were unopened. However, EDTA was found to be the best anticoagulant for prolonged storage of whole blood at 40C, 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 rl oi blood were aliquoted 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 MgCl 2 1% TRITONTX-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 WO 90/02179 PCT/US89/03476 8 EDTA, 10 mM NaCI) and vortexed. The solution was centrifuged at 525 yg for min at 4 0 C. After pouring off the supernatant, the pellet was resuspended in 450 /I DNA Isolation Reagent 2, 50 pl 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 DNAs isolated from human blood were characterized quantitatively and qualitatively 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 A-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 ,l of whole blood. In some aquatic species DNA isolation buffer I should not have MgCL 2 but should include 100 mM NaCI, otherwise DNA degradation occurs.

Blood Stains 5-100 p1 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; 3i 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 :he: :e J WO 90/02179 PCT/US89/03476 9 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 ul/ml and incubated for 2 hr at 650C. 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.

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 was used.

Semen DNA was isolated form 100 ,l of human semen. Th. 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 The supernatant was poured off, the pellet was resuspended in DNA Isolation Reagent 1, and the same procedure as in was followed except that 10 mM DTT was added before the proteinase K in order to lyse the sperm cells.

Semen and semen blood stains differential lvsis.

The same procedure as in 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.

A

WO 90/02179 PCT/US89/03476 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 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 Hair human hair roots with shafts were suspended in 90 /l of DNA Isolation Reagent 2, 10 ul 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.

E. coli E. coli DNA has also been obtained by using 1.5 pl of overnight grown cells, pelleting the cells, washing with 1 ml Isolation Reagent 2, pelleting again, then resuspending in 270 p1, again adding 15 ul Isolation Reagent 3 and incubating 2 hr at 650C. Plasmid DNA can also be obtained without chromosomal DNA by first lysing the cells with lysozyme and 1% TRITONTMX- 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.

i' i I I II I.;:r*r-c;irn r WO 90102179 PC/US89/03476 11 Example 2 Analysis of Isolated Eukarvotic HMW DNA RFLP Analysis RFLP analysis was performed on the various purified DNAs after restriction with Pstl 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.

c 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 electrophoresis, lithium chloride is added to a final concentration of 3.75M, placed at 4 0 C and centrifuged for 20 minutes in a microfuge (at least 12000 g).

The supernatant is collected and the DNA is precipitated at 250 by adding ethanol to a final concentration of 70%, for 30 minutes; then centrifuged for 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 electrophoresis.

Claims (22)

1. A process for isolating DNA from a biological sample which comprises lysing a biological sample comprising the desired DNA without enzymatic inhibitors; and 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.

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 0 C to about 700C, for at least about 1.5 hours. A Si! I 'WO 90/02179 PCT/US89/03476 13 1

8. A process for isolating DNA from a biological sample which comprises t-\tC\-oiA- erv-z^^\O- \C~n\oC 2 lysing a biological sample comprising the desired DNA and 3 contacting the lysed biological sample with an effective 4 proteolysis amount of proteinase K at a temperature of about 60°C to about 70C, for at least about 1.5 hour. 1

9. The process, according to claim 7, wherein said biological sample is 2 selected from the group consisting of blood, blood stain, urine, semen, semen 3 stain, spleen tissue, HeLa cells, hair, and other animal cells. 1

10. The process, according to claim 7, wherein high molecular weight 2 DNA is isolated from said biological sample. 1

11. A process for isolating DNA from a biological sample which 2 comprises 3 lysing a biological sample comprising the desired DNA 4 without enzymatic inhibitors; contacting the lysed biological sample with a proteolytic 6 enzyme at its working temperature for a time sufficient to destroy protein; and 7 deactivating said enzyme at a deactivating temperature. 1

12. The process, according to claim 11, wherein said biological sample 2 is eukaryotic or prokaryotic. 1

13. The process, according to claim 12, wherein high molecular weight 2 DNA is isolated from said eukaryotic biological sample. IR T 14

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

The process, according to claim 13, wherein said eukaryotic biological sample is selected from blood, blood stain, urine, semen, semen stain, spleen tissue, HeLa cells, hair, or other animal cells.

16. A forensic test procedure which comprises the use of DNA isolated from a biological sample which comprises lysing a biological sample selected from blood, blood stain, :urine, semen, semen stain, tissue, hair or other animal cells; and 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 15 isolated from a biological sample which comprises lysing a biological sample selected from blood, semen, or amniotic fluid; and contacting the lysed biological sample with an effective proteolysis amount of proteinase K at a temperature of about S 20 60 0 C to about 70 0 C, for at least about 1.5 hour.

18. A diagnostic test procedure which comprises the use of DNA isolated from a biological sample which comprises lysing a biological sample selected from blood, urine, semen, tissue, hair or other animal cells; and contacting the lysed biological sample with an effective proteolysis amount of proteinase K at a temperature of about 0 °C to about 70 0 C, for at least about 1.5 hour.

19. A kit when used for the forensic test procedure of claim 16, the paternity test procedure of claim 17, or the diagnostic procedure of claim 18, said kit comprising i 1/642Z MM/642Z a container with a lysing means for a biological sample which does not contain an enzyme inhibitor; and a container with an effective proteolysis amount of a proteolytic enzyme.

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

21. A process for isolating DNA from a biological sample, which process is substantially as hereinbefore described with reference to Example 1. 10 22. The process of claim 21, wherein said biological sample is blood, blood stain, urine, semen, semen stain, spleen tissue, HeLa cells, hair or E. coli cells. Lifecodes Corporation Patent Attorneys for the Applicant SPRUSON FERGUSON kI LMM/642Z 'i LMM/642Z i U~ i 1 INTERNATIONAL SEARCH REPORT International Application No PCT/TUS 89/03476 I. CLASSIFICATION OF SUBJECT MATTER (it several classification symbols acoly, indicate all) 4 :111 According to International Patent Classification (IPC) or to both National Classification and IPC IPC 5 C 12 N 15/10, C 12 P 19/34, C 12 Q 1/68 II. FIELDS SEARCHED Minimum Documentation Searched 7 Classification System I Classification Symbols IPC 5 C 12 N, C 12 P, C 07 H Documentation Searched other than Minimum Documentation to the Extent that such Documents are Included In the Fields Searched Ill. DOCUMENTS CONSIDERED TO BE RELEVANT' Category Citation of Document, with Indication, where appropriate, of the relevant passagea 1 Relevant to Claim No. A Nucleic Acids Research, vol. 15, no.

22, 1,2,4-10 November 1987, IRL Press M. Jeanpierre: "A rapid method for the purification of DNA from blood", page 9611, see the whole article (cited in the application) A Eur. J. Biochem. vol. 36, 1973, 1-6 M. Gross-Bellard et al.: "Isolation of high-molecular-weight DNA from mammalian cells" pages 32-38, see the whole article SSpecial categorles of cited documents: io later document published after the International filing date "A document definng the general state the art which I not or priority date and not In conflict with the application but "A document dfining the gneral tat o the art which I not cited to understand the principle or theory underlying the considered to be of particular relevance invention earlier document but published on or after the International document of particular relevance; the claimed invention filing date cannot be considered novel or cannot be considered to document which may throw doubts on priority claim(s) or Involve an inventive step which is cited to establish the publication date of another document of particular relevance;' the claimed Invention citation or other special reason (as specified) cannot be considered to Involve an Inventive step when the document referring to an oral disclosure, use, exhibition or document is combined with one or more other such docu- other means ments, such combination being obvious to a person skilled document published prior to the International filing date but in the art. later than the priority date claimed document member of the same patent family IV. CERTIFICATION Date of the Actual Completion of the International Search Date of Mailing of this International Search Report 28th November 1989 19. Q0 CO International Searching Authority Sig~1nturn tia i EUROPEAN PATENT OFFICE T.K. L Form PCTIISA210 (second sheet) (January 1985) I ,~J IM Ill fT I MM