WO1990002179A1 - Procede d'isolement d'adn - Google Patents

Procede d'isolement d'adn Download PDF

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Publication number
WO1990002179A1
WO1990002179A1 PCT/US1989/003476 US8903476W WO9002179A1 WO 1990002179 A1 WO1990002179 A1 WO 1990002179A1 US 8903476 W US8903476 W US 8903476W WO 9002179 A1 WO9002179 A1 WO 9002179A1
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WO
WIPO (PCT)
Prior art keywords
biological sample
dna
blood
semen
isolated
Prior art date
Application number
PCT/US1989/003476
Other languages
English (en)
Inventor
Jacob Grimberg
Stanley Nawoschik
Original Assignee
Lifecodes Corporation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Lifecodes Corporation filed Critical Lifecodes Corporation
Publication of WO1990002179A1 publication Critical patent/WO1990002179A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay

Definitions

  • HMW DNA eukaryotic high molecular weight DNA
  • SDS proteinase K/sodium dodecyl sulfate
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • lysing means excluding enzyme inhibitors
  • a proteolytic enzyme at a temperature and for a time sufficient to destroy protein and autodigest or inactivate said proteolytic enzyme
  • 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.
  • 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.
  • DNA Isolation Reagent 1 320 mM sucrose, 10 mM Tris-HCI pH 7.6, 5 mM MgCI 2 , 1% TRITONTMX-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
  • 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
  • DNA Isolation Reagent 1 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 ⁇ DNA Isolation Reagent 2, 50 ⁇ 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 ⁇ -DNA standards. Alternatively, DNA concentration can be determined by using a fluorometer with
  • 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).
  • DNA isolation buffer I When isolating DNA from nucleated red blood cells, use only 10-50 ⁇ 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.
  • 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
  • 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.
  • 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).
  • PBS phosphate buffered saline
  • 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.
  • 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
  • PCR can also be performed with the DNA and amplification of various fragments has been obtained.
  • 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.

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  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Analytical Chemistry (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Plant Pathology (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Immunology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

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.
PCT/US1989/003476 1988-08-31 1989-08-14 Procede d'isolement d'adn WO1990002179A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US23893888A 1988-08-31 1988-08-31
US238,938 1988-08-31
US37372189A 1989-06-29 1989-06-29
US373,721 1989-06-29

Publications (1)

Publication Number Publication Date
WO1990002179A1 true WO1990002179A1 (fr) 1990-03-08

Family

ID=26932104

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1989/003476 WO1990002179A1 (fr) 1988-08-31 1989-08-14 Procede d'isolement d'adn

Country Status (4)

Country Link
EP (1) EP0431052A1 (fr)
JP (1) JPH04503901A (fr)
AU (1) AU621936B2 (fr)
WO (1) WO1990002179A1 (fr)

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0587541A1 (fr) * 1992-09-09 1994-03-16 Tecnogen S.C.P.A. Procédé de purification de protéine Big-endothéline
WO1996000228A1 (fr) * 1994-06-23 1996-01-04 Dade International Inc. Procede d'isolation rapide d'acide nucleique
US5595876A (en) * 1993-08-13 1997-01-21 E. I. Du Pont De Nemours And Company In situ extraction of microbial DNA
EP0938583A1 (fr) * 1996-07-23 1999-09-01 University of North Dakota Medical Education Research Foundation Procedure de purification d'adn
WO2001042456A2 (fr) * 1999-12-10 2001-06-14 Genespan Corporation Isolation et purification d'acides nucleiques
CN103243090A (zh) * 2013-05-31 2013-08-14 遵义医学院 一种提取昆虫dna的方法
CN108728435A (zh) * 2018-08-14 2018-11-02 苏州博睿义达生物科技有限公司 一种毛干样本裂解和dna提取纯化方法及系统

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2013523142A (ja) * 2010-04-08 2013-06-17 キアゲン ゲーエムベーハー 核酸の選択的単離および精製のための方法

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
Eur. J. Biochem. Vol. 36, 1973, M. GROSS-BELLARD et al.: "Isolation of High-Molecular-Weight DNA from Mammalian Cells" pages 32-38 *
Nucleic Acids Research, Vol. 15, No. 22, 25 November 1987, IRL Press M. JEANPIERRE of DNA from Blood", page 9611 *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0587541A1 (fr) * 1992-09-09 1994-03-16 Tecnogen S.C.P.A. Procédé de purification de protéine Big-endothéline
US5595876A (en) * 1993-08-13 1997-01-21 E. I. Du Pont De Nemours And Company In situ extraction of microbial DNA
WO1996000228A1 (fr) * 1994-06-23 1996-01-04 Dade International Inc. Procede d'isolation rapide d'acide nucleique
EP0938583A1 (fr) * 1996-07-23 1999-09-01 University of North Dakota Medical Education Research Foundation Procedure de purification d'adn
EP0938583A4 (fr) * 1996-07-23 1999-10-13
WO2001042456A2 (fr) * 1999-12-10 2001-06-14 Genespan Corporation Isolation et purification d'acides nucleiques
WO2001042456A3 (fr) * 1999-12-10 2002-01-03 Genespan Corp Isolation et purification d'acides nucleiques
CN103243090A (zh) * 2013-05-31 2013-08-14 遵义医学院 一种提取昆虫dna的方法
CN108728435A (zh) * 2018-08-14 2018-11-02 苏州博睿义达生物科技有限公司 一种毛干样本裂解和dna提取纯化方法及系统

Also Published As

Publication number Publication date
EP0431052A1 (fr) 1991-06-12
AU4201289A (en) 1990-03-23
AU621936B2 (en) 1992-03-26
JPH04503901A (ja) 1992-07-16

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