WO2001012847A2 - Isolation de cellules dans des echantillons judiciaires pour empreintes genetiques - Google Patents

Isolation de cellules dans des echantillons judiciaires pour empreintes genetiques Download PDF

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Publication number
WO2001012847A2
WO2001012847A2 PCT/EP2000/007771 EP0007771W WO0112847A2 WO 2001012847 A2 WO2001012847 A2 WO 2001012847A2 EP 0007771 W EP0007771 W EP 0007771W WO 0112847 A2 WO0112847 A2 WO 0112847A2
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specific
cells
species
molecule
cell
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WO2001012847A3 (fr
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Elfride Van Den Eeckhout
Dieter Deforce
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Universiteit Gent
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    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2833—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against MHC-molecules, e.g. HLA-molecules
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays

Definitions

  • the present invention relates to the field of DNA typing, more particularly the field of cell isolation for DNA typing, more particularly DNA typing on forensic samples.
  • the DNA present in the forensic sample can be from human, animal or vegetal origin.
  • PCR based DNA-typing is known to be a very sensitive technique, difficulties are often encountered when examining samples from dirty matrices. These problems can be attributed to the presence of PCR-inhibitors in these samples.
  • forensic samples are often exposed to different environmental conditions and substances which may inhibit the amplification process (Wallin et al. 1998), a specific isolation of human cells from forensic samples for DNA-typing can be of critical importance to obtain a DNA-profile of a sample.
  • Some biological samples which maybe of extreme importance to forensic casework such as feces, urine and skeletal muscle contain high amounts of PCR-inhibitors such as bile salts, polysaccharides, urea, myoglobin (Lantz et al.
  • the present invention relates to the use of a species -specific, cell-type-specific or individual-specific molecule in a method for isolating or capturing cells from a forensic sample with said species-specific, cell-type-specific or individual-specific molecule being preferably bound to a solid phase.
  • solid phase refers to any type of solid substrate known in the art which allows the binding of molecules or antibodies for the use according to the present invention, and which allows subsequently and easy recovery of the human cells.
  • solid phases include: beads, plates, dipsticks, membranes, tubes or others made of for example polystyrene, polyvinyl, latex, sepharose or other polymers.
  • the solid phase is composed of paramagnetic beads. Methods to prepare such paramagnetic or immunomagnetic beads are known to those skilled in the art. Suitable magnetic beads are also commercially available from for instance Dynal AS (Oslo, Norway). These beads may be coated directly with a molecule, for instance an antibody.
  • these beads may have been previously coated with a monoclonal antibody specific for Fc on all mouse IgG. Coating of the species-specific, cell-type specific or individual-specific antibody or molecule of the invention to the beads can be done directly (covalently) or indirectly (via another antibody).
  • a preferred type of immunomagnetic beads to be used in the methods of the present invention are the commercially available Paramagnetic Dynabeads® Pan Mouse IgG (cat. No. 110.22), coated with a monoclonal antibody specific for Fc on all mouse IgG as commercialised by Dynal AS, Oslo, Norway. Preferably an excess of said immuno- or paramagnetic beads are used to capture as many cells as possible in a sample.
  • kits-specific, cell-type-specific or individual-specific molecules refers to molecules which specifically bind to cells, essentially without binding to any compound of non-biological origin.
  • kits-specific molecule refers to molecules which bind to cells from a specific species.
  • the molecules may specifically bind to human cells, or may specifically bind to cells of an animal species, or may specifically bind to cells of a plant species.
  • human cell-specific molecules specifically bind to human cells essentially without binding any cell of non-human origin.
  • species refers to a group of related organisms which have certain characteristics in common and which, if they reproduce sexually, are able to interbreed freely in nature and produce fully fertile offspring. A species ranks next below a genus and may be divided into several varieties, races or breeds.
  • cell-type-specific molecule refers to molecules which bind to specific cell types for instance, molecules that specifically bind to epithelial cells, to mucus-secreting cells, to blood cells, to sperm cells, to muscle cells or to bone cells.
  • individual- specific molecule refers to molecules which specifically bind to an individual organism. This organism can be a specific human individual, a specific individual animal, a specific individual plant. For instance a human individual-specific molecule specifically binds to cells of said individual and not to cells from other human beings.
  • the present invention relates to the use of a species- specific, cell-type-specific or individual-specific molecule as defined above in a method for isolating mammalian cells from a forensic sample. In a more preferred embodiment, the present invention relates to the use of a species- specific, cell-type-specific or individual-specific molecule as defined above in a method for isolating human cells from a forensic sample.
  • the present invention relates to the use of a species-specific, cell-type-specific or individual-specific molecule as defined above in a method for isolating animal or vegetal cells from a forensic sample.
  • the present invention further relates to the use of a species-specific, cell-type-specific or individual-specific antibodies in a method for isolating or capturing cells from a forensic sample wherein said species-specific, cell-type-specific or individual-specific molecule is an antibody.
  • the present invention relates to the use of monoclonal as well as polyclonal antibodies or their derivatives.
  • the species-specific, cell-type-specific-, or individual specific antibody is specific for or specifically reacts with an HLA or an HLB antigen.
  • a preferred human (cell) specific antibody is for instance an HLB antibody.
  • HLB antibodies are well known in the art (see for instance Shipp, M.A. et al., 1983, 1989).
  • a preferred human (cell) specific molecule according the present invention is an HLA antibody.
  • HLA antibodies of class I and II are well known in the art (see for instance Brodsky etal., 1979 and Shipp et al., 1986).
  • Antibodies that are directed for instance against specific HLA class I types, such as but not limited to HLA A2, HLA.B7, HLA C7, or against specific HLA class II types, such as but not limited to HLA DR1 and HLADQ, are commercially available.
  • a preferred HLA antibody to be used according to the present invention is the AbHLAI antibody which is commercially available from Sigma (see Example 1 and 6).
  • Anti-HLA class I and class II can be used to distinguish between cells from two individuals if the HLA-type of the cells of at least one individual is known.
  • the specie-specific or cell-type-specific antibody is specific for or specifically reacts with an epithelial cell antigen, particularly an epithelial cell surface antigen.
  • a preferred human cell-type specific molecule according to the present invention is an epithelial specific antigen antibody.
  • Epithelial specific antigen antibodies are well known in the art (see for instance Tsubura et al., 1992).
  • a preferred commercially available epithelial specific antigen antibody to be used according to the present invention is AbESA from Sigma
  • the species-specific, cell-type-specific or individual-specific antibody is specific for blood cells or for specific types of blood cells.
  • Antibodies which are directed against specific blood cells are for instance antibodies against CD4 and CD8 (present on T cells in blood) or against CD19 (present on B cells in blood).
  • the species-specific, cell-type-specific or individual- specific antibody is specific for sperm or sperm cells.
  • Monoclonal antibodies can be prepared to techniques well known in the art. Derivatives or fragments of monoclonal antibodies can also be used as species-specific, cell type-specific or individual-specific molecules according to the present invention. The preparation of these derivatives or fragments is well known in the art.
  • immunomagnetic particles consist of supermagnetic microspheres which are coated with a monoclonal antibody specific for Fc on all mouse IgG (Dynabeads Pan Mouse IgG, product description). These supermagnetic micro spheres can be for instance coated with mouse monoclonal anti-human HLA class I antigen in order to selectively purify any human cell type.
  • This monoclonal antibody recognizes the human HLA class I antigen expressed on most human nucleated cells (Lawlor et al., 1990).
  • the supermagnetic microspheres can preferably be coated with for instance the mouse monoclonal anti-human epithelial specific antigen.
  • This monoclonal antibody binds to a cell surface glycoprotein called epithelial specific antigen (ESA) which is present on normal epithelial cells and carcinomas, such as skin appendages and their tumors (Tsubura et al., 1992).
  • ESA epithelial specific antigen
  • forensic sample broadly refers to a matrix or mixture which contains human biological materials such as blood, blood stains, saliva, saliva stains, skin debris, faeces, faeces stains, urine, sperm cells, vaginal epithelial cells, sperm epithelial cells, all other epithelial cells, muscle, bone, bone and muscle remains or mummified remains.
  • matrix refers to any non-biological material.
  • mixture refers to mixtures of different types of cells or cells from different species or individuals present in one forensic sample.
  • the invention relates to a method for isolating or capturing cells from a forensic sample, said method comprising the use of a species-specific, cell-type-specific or individual- specific molecule as defined above.
  • the invention relates to a method for isolating or capturing cells from a forensic sample comprising the steps of (a) capturing the cells from said forensic sample using a molecule as defined above, wherein said molecule is bound or can be bound to a solid phase and (b) possibly recovering said human cells from said solid phase.
  • Isolated cells can be further treated with Chelex-resin to extract their DNA for subsequent multiplex PCR analysis of Short Tandem Repeat (STR) fragments.
  • Short Tandem Repeats are well known in the art and represent multivariable loci present in the genomic DNA consisting of short sequences (2 to 4 bases) which are repeated a number of times, giving fragments of different lengths. Therefore, the present invention also relates to a method for preparing DNA from a forensic sample, said method comprising the use of a species-specific, cell-type-specific or individual - specific molecule as defined above.
  • the present invention thus further relates to a method for preparing DNA from a forensic sample, comprising the steps of (a) isolating or capturing cells from a forensic sample using a species-specific, cell-type-specific or individual-specific molecule as defined above, wherein said molecule is bound to a solid phase, (b) possibly recovering said cells isolated in step a) from said solid phase, (c) possibly resuspending said cells in a (5% (w/v)) Chelex suspension, and (d) extracting said DNA from said cells.
  • the present invention also relates to a method for analysing DNA from a forensic sample, said method comprising the use of a species-specific, cell-type-specific or individual-specific molecule as defined earlier.
  • the present invention thus also relates to a method for DNA analysis of cells in a forensic sample comprising the steps of (a) isolating or capturing cells from a forensic sample using a species-specific, cell-type-specific or individual-specific molecule as defined above, bound to a solid phase, (b) possibly recovering or isolating said cells from said solid phase, (c) possibly resuspending said cells in a (5% (w/v)) Chelex suspension, (d) possibly extracting DNA from said cells, (e) performing an amplification on said DNA of said cells, and (f) analyzing the amplification product(s).
  • the binding of the cells with the species-specific, cell-type-specific or individual-specific molecule can be accomplished first, whereafter a solid phase (e.g. immunomagnetic particles whereon an antibody against the first antibody is bound) is added.
  • a solid phase e.g. immunomagnetic particles whereon an antibody against the first antibody is bound
  • the amplification step is preferably done by the polymerase chain reaction (PCR; Saiki et al., 1988) but can also be done by any other type of nucleic acid amplification, such as ligase chain reaction (LCR; Landgren et al., 1988; Wu and Wallace, 1989; Barany, 1991 ), nucleic acid sequence based amplification (NASBA; Guatelli et al., 1990; Compton, 1991), transcription-based amplification systm (TAS; Kwoh et al., 1989), strand displacement amplificatin (SDA; Duck, 1990; Walker et al., 1992) or amplification by means of Qss replicase (Lizardi ef al., 1988; Lomeli ef al., 1989) or any other suitable method to amplify nucleic acid molecules.
  • the amplification reaction is preferably repeated between 20 and 70 times, advantageously between 25 and 45 times.
  • the amplification reaction is preferably a
  • the amplified products of the present invention can be analyzed by any method known in the art such as for instance any type of gel-electrophoresis possibly including hybridisation reactions to unique probes, restriction length polymorphism (RFLP) or mass spectrometry.
  • the amplified products can also be analyzed by direct sequencing techniques.
  • the analysis technique demonstrated in the present examples is a form of gel-electrophoresis using laser fluorescence detection.
  • the present invention also relates to a method for separating different cells present in a mixture using a species-specific, cell-type-specific or individual-specific molecule as described earlier.
  • the cells to be seperated belong to the same cell-type and can be isolated using a cell-type-specific molecule.
  • the present invention also relates to a method for DNA analysis of cells in a forensic sample as defined above comprising separating mixtures of cells of the same cell type from distict individuals or persons using individual-specific molecules as described above.
  • the cells to be seperated are from distinct individuals of the same species, for instance different races (human species), different breeds (animal species) or different varieties (plant species). Therefore, the invention also relates to the use of race-specific, breed-specific or variety-specific molecules for separating different cells from a forensic sample.
  • the present invention further relates to a method for DNA analysis of cells in a forensic sample as defined above comprising separating species-specific cells from a mixture of cells of the same cell-type wherein said cells are from distict species using a species-specific molecule as defined above.
  • the present invention relates to a method for extracting mitochondrial DNA from a forensic sample comprising the steps of a) capturing the cells from said forensic sample using a molecule as defined in any of claims 1 to 10 wherein said molecule is bound to a solid phase, and, b) possibly recovering said cells from said solid phase.
  • the invention further relates to the use of a species-specific, cell-type-specific or individual-specific molecule as defined above in a method for preparing and/or isolating cells from a forensic sample, and/or in a method for preparing and/or analysing DNA from cells in a forensic sample, and/or in a method for preparing mitochondrial DNA from cells in a forensic sample.
  • the methods of the invention are used to isolate and to analyse human cells and human DNA.
  • the species-specific, cell-type-specific or individual-specific molecules used in any of the methods of the examples are bound to (immuno)magnetic beads.
  • the present examples describe the use of immunomagnetic cell-separation to isolate cells from all kinds of matrices and mixtures with non-biological materials for subsequent DNA-typing using multiplex PCR.
  • this method can be extremely useful to isolate DNA for subsequent PCR-amplification from samples containing PCR-inhibitors.
  • a slight modification of the technique can also be used to purify or remove specific cell types from mixtures with other cell types, eg. the specific isolation of epithelial cells of the suspect from a knife covered with blood from the victim, for subsequent DNA-typing.
  • the methodology described in the present invention was performed successfully on a variety of samples, such as blood, blood stains, saliva, saliva stains (on pop cans) and on skin debris.
  • Figure 1 Photograph taken at a magnification power of 100 of an aliquot of a sample in which 10 ⁇ l of saliva was purified using the procedure described in the present examples. Note the presence of dark spheres, which are the Dynabeads coated with the HLAI antibody, on the isolated cell material.
  • Figure 2 Photograph taken at a magnification power of 400 of an aliquot of a sample in which a saliva stain taken from the drink opening of a pop can with a compress was purified using the procedure described in the examples section. Again note the presence of dark speres, which are the Dynabeads coated with the HLAI antibody, on the isolated cell material.
  • FIG. 3A Multiplex Short Tandem Repeat (STR) profiles obtained of a sample in which 10 ⁇ l of saliva was added prior to cell-isolation as described in the examples section.
  • the saliva sample which was completely prepared as described in Example 3, thus in which 200 ⁇ l of Chelex was added.
  • FIG. 3B Multiplex Short Tandem Repeat (STR) profiles obtained of a sample in which 10 ⁇ l of saliva was added prior to cell-isolation as described in the examples section.
  • the saliva sample was prepared as described for Figure 3A except that instead of 200 ⁇ l
  • Example 1 Materials, Instrumentation and methods
  • Dynal sample mixer® which is a mixing device which allows both gentle tilting and rotation was purchased from Dynal AS, Oslo, Norway.
  • a MagneSphere® Technology magnetic separation stand (MSS) was purchased from Promega, Madison, USA.
  • Capillary Electrophoresis was performed on an ABI prism 310 Genetic Analyzer from Applied Biosystems (Perkin Elmer, Foster City, USA). Collection was performed using the 310 Genetic Analyzer Data Collection software version 1.0.2 and the data were analyzed using the Genescan Analysis software version 2.0.2 (Applied Biosystems, Perkin Elmer, Foster City, USA).
  • Paramagnetic Dynabeads® Pan Mouse IgG (cat. no. 110.22), coated with a monoclonal antibody specific for Fc on all mouse IgG were purchased from Dynal AS, Oslo, Norway.
  • Sterile distilled water was prepared by autoclaving (121 °C during 21 min.), distilled water obtained from a Milli-Q RG®-apparatus (Millipore SA, Molsheim, France).
  • Example 2 Coating of the Dynabeads The coating procedure was carried out in a laminar air flow (LAF) hood, this to prevent contamination with microorganisms.
  • Phosphate buffered saline (PBS) pH 7.4 NaH 2 P0 4 .H 2 O 0.16 g; Na 2 HPO 4 .2H 2 O 0.98 g; NaCI 8.10 g in 1 L water
  • PBS/BSA buffer was prepared by addition of 0.1 % (w/v) of BSA and 0.02% of sodium azide (final concentrations) to PBS buffer.
  • the Dynabeads coated with AbHLAI were washed three times by subsequently placing the tube in the MSS, removing the fluid, removing the tube from the MSS and resuspending the coated beads with 1 ml PBS/BSA buffer. After the last washing the beads were resuspended in 400 ⁇ l PBS/BSA buffer. When stored for longer than two weeks the coated beads should be washed.
  • Example 3 Cell isolation and DNA isolation
  • the optimal procedure to isolate DNA from blood, blood stains, saliva, saliva stains or skin debris was as follows. 10 ⁇ l blood or saliva was suspended in 1 ml PBS/BSA buffer. Blood stains on sterile cotton compresses, saliva stains, which were rubbed of from pop cans with a piece (1 square cm) of sterile cotton compress moistened with PBS/BSA buffer or skin debris which was also rubbed of from its matrix with a moistened compress were put in 1 ml PBS/BSA buffer. All samples were then gently vortexed during 30 seconds and incubated at 4°C during 30 minutes. From this point on all manipulations were carried out in a cooling chamber at a temperature of 4°C.
  • DNA quantitation was performed using Quantiblot® Human DNA quantitation kit, following the procedure described by the manufacturer in the protocol present in the kit. This procedure is based on the hybridization of a biotinylated oligonucleotide probe to DNA samples immobilized on a nylon membrane (Walsh et al., 1992). The probe used is complementary to a specific alpha satellite DNA sequence at the locus D17Z1 (Waye et al., 1986). After hybridization and washing procedures, chemiluminescent detection was performed to quantitate the amount of DNA present in the samples using the ECLTM kit as indicated by the manufacturer.
  • Example 5 Multiplex PCR. separation and detection
  • the washing procedure was as follows: after the samples (being 10 or 100 ⁇ l saliva or blood, compresses with skin debris or saliva stains) were incubated during 15 min. in 1 ml PBS/BSA buffer, the compresses were removed, and the samples were centrifuged at 20000 g during 5 min to pellet the cells. After centrifugation the supernatant was removed and the pellets were resuspended in 1 ml PBS/BSA buffer by gentle vortexing. This washing removes contaminants like soluble antigens from the samples. However there was no detectable advantage linked to the washing of the cells. Signal strength of the STR-profiles was not enhanced by this additional procedure.
  • PCR polymerase chain reaction

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Abstract

L'invention concerne une molécule spécifique à l'espèce, à la cellule ou à l'individu, à utiliser dans un procédé destiné à isoler des cellules dans un échantillon judiciaire, ladite molécule étant, de préférence, liée à une phase solide. Plus précisément, ladite molécule est un anticorps monoclonal ou polyclonal. L'invention traite également d'un procédé permettant d'isoler des cellules dans un échantillon judiciaire, lesdites cellules étant capturées à l'aide de ladite molécule. Elle traite en outre de procédés de préparation et d'analyse d'ADN à partir d'un tel échantillon. Elle concerne enfin des procédés pour isoler des cellules spécifiques dans des mélanges de cellules présentes dans un échantillon judiciaire.
PCT/EP2000/007771 1999-08-11 2000-08-10 Isolation de cellules dans des echantillons judiciaires pour empreintes genetiques WO2001012847A2 (fr)

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Cited By (6)

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Publication number Priority date Publication date Assignee Title
US7320891B2 (en) 2004-09-10 2008-01-22 Promega Corporation Methods and kits for isolating sperm cells
US8993292B2 (en) 2007-01-16 2015-03-31 Applied Biosystems Llc Methods and systems for differential extraction
EP2144993A1 (fr) * 2007-05-09 2010-01-20 Applied Biosystems, LLC Procédés et systèmes pour extraction différentielle
EP2144993A4 (fr) * 2007-05-09 2010-07-28 Life Technologies Corp Procédés et systèmes pour extraction différentielle
CN103592432A (zh) * 2013-10-15 2014-02-19 重庆市公安局 一种免疫磁珠分离精子与上皮细胞混合斑中精子的方法
CN103592432B (zh) * 2013-10-15 2015-06-03 重庆市公安局 一种免疫磁珠分离精子与上皮细胞混合斑中精子的方法

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