+

WO1995006750A1 - Procede de quantification du nombre de cellules contenant une sequence d'acide nucleique selectionnee dans une population heterogene de cellules - Google Patents

Procede de quantification du nombre de cellules contenant une sequence d'acide nucleique selectionnee dans une population heterogene de cellules Download PDF

Info

Publication number
WO1995006750A1
WO1995006750A1 PCT/US1994/009719 US9409719W WO9506750A1 WO 1995006750 A1 WO1995006750 A1 WO 1995006750A1 US 9409719 W US9409719 W US 9409719W WO 9506750 A1 WO9506750 A1 WO 9506750A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
acid sequence
sequence
suspension
nucleic acid
Prior art date
Application number
PCT/US1994/009719
Other languages
English (en)
Inventor
Jeffrey M. Hall
David A. Molesh
Original Assignee
Cellpro, Incorporated
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 Cellpro, Incorporated filed Critical Cellpro, Incorporated
Priority to AU76404/94A priority Critical patent/AU7640494A/en
Publication of WO1995006750A1 publication Critical patent/WO1995006750A1/fr

Links

Classifications

    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification
    • 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
    • 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
    • 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
    • C12Q1/6809—Methods for determination or identification of nucleic acids involving differential detection
    • 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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer

Definitions

  • the present invention generally relates to methods for quantifying cells, and more specifically, to methods for amplifying the amount of a selected nucleotide sequence in a cell, and, quantifying the number of cells containing that sequence in a heterogenous population of cells.
  • PCR is a method of synthesizing specific segments of DNA or RNA in vitro. Briefly, two oligonucleotides complementary to sequences flanking the sequence of interest are utilized as primers for DNA synthesis. Multiple cycles of heat denaturation (to separate the DNA into single strands), annealing of the primers to their complementary sequences, and extension of the annealed primers with DNA polymerase result in amplification of the sequence flanked by the primers. Since the extension products themselves are capable of serving as templates for synthesis in subsequent cycles of amplification, there is an exponential increase in the number of copies of the sequence of interest, such that after n cycles of amplification, there will be approximately 2 n copies present. Since the amplification is somewhat less than 100% efficient, the actual number of copies will in fact be slightly less than 2 n .
  • RNA must be extensively purified prior to amplification.
  • PCR has proved to be a powerful tool for the detection and amplification of specific nucleic acid sequences, it has remained difficult to quantify the amount of a target sequence initially present in a sample. This is due to the fact that PCR is an exponential process of amplification, and hence small differences in reaction variables result in large differences of product yield.
  • RNA quantification using an internal standard.
  • competitive PCR Gilliland et al, ibid., pp. 60-69
  • a second template is added to the reaction which can be amplified using the same primers as for the target sequence, but which can be distinguished from the target sequence at completion of the PCR protocol.
  • the second template serves as an internal standard which, when added to the reaction mixture in a known amount, is amplified to the same extent as the target.
  • the competitive template is a sequence which differs by a single base pair from the target and which can be distinguished from the target by a restriction digest. This method has been reported to be applicable to quantification of mRNA from as few as 10 cells.
  • Wang and Mark (ibid., pp. 70-75) describe a plasmid which can serve as an internal standard for PCR of specific mRNAs.
  • the plasmid is designed such that its PCR product is easily separated on the basis of size from the PCR product of the target sequence.
  • Both Wang and Mark, and Gilliland et al. require highly purified mRNA as the starting material for PCR, and both require the synthesis of standard sequences which are custom to one or several related target sequences.
  • the present invention provides methods for quantifying the number of cells containing a selected nucleic acid sequence in a suspension of cells heterogenous with respect to the presence of said sequence, as well as methods for increasing the number of copies of a selected ribonucleic acid sequence in a sample.
  • methods of increasing the number of copies of a selected ribonucleic acid sequence in a sample comprising the steps of (a) disrupting cells to expose ribonucleic acids, and (b) amplifying a selected ribonucleic acid sequence from the exposed ribonucleic acids, such that the ribonucleic acid sequence is increased in number, and wherein, between the steps of disrupting and amplifying, no step of purification is performed.
  • such methods may be accomplished utilizing viruses or cell free extracts as samples.
  • methods for quantifying the frequency of cells containing a selected nucleic acid sequence in a suspension of cells heterogenous with respect to the presence of said sequence, comprising the steps of (a) determining the total number of cells in the suspension, (b) dispensing the suspension into a plurality of vessels in order to provide a series of multiply replicated aliquots at decreasing concentrations of cells, (c) determining the presence or absence of the selected sequence in each vessel, and (d) relating the number of vessels containing the selected sequence to the total number of cells assayed at each dilution, such that the frequency of cells containing the selected nucleic acid sequence in the suspension may be determined.
  • methods for quantifying the number of cells containing a selected ribonucleic acid sequence in a sample containing a heterogenous population of cells, comprising the steps of (a) determining the total number of cells in the suspension, (b) dispensing the suspension into a plurality of vessels in order to provide a series of multiply replicated aliquots at decreasing concentrations of cells, (b) disrupting cells within the suspension to expose ribonucleic acids, (c) amplifying a selected ribonucleic acid sequence from the exposed ribonucleic acids, such that the ribonucleic acid sequence is increased in number, and wherein, between the steps of disrupting and amplifying, no step of purification is performed, (d) determining the presence or absence of the selected sequence in each vessel, and (e) relating the number of vessels containing the selected sequence to the total number of cells assayed at each dilution, such that the frequency of cells containing the selected nucleic acid sequence in the
  • the selected nucleic acid sequence may be amplified by, for example, polymerase chain reaction, Q ⁇ -replicase amplification, or isothermal amplification.
  • the cell suspension prior to the step of determining, the cell suspension may be fractionated into subpopulations of cells.
  • a ribonuclease inhibitor is not added during the steps of disrupting or amplifying.
  • cells are disrupted by heating the cells.
  • preferred cells within the context of the present invention include fetal cells, CD34 + cells, and tumor cells.
  • the frequency of cells containing the selected nucleic acid sequence may be determined by the equation ⁇ jkj / (N- ⁇ ik j C j /2).
  • the present invention provides methods for quantifying the number of cells containing a selected nucleic acid sequence in a sample containing a heterogenous population of cells.
  • methods comprising the steps of (a) determining the total number of cells in the suspension, (b) dispensing the suspension into a plurality of vessels in order to provide a series of multiply replicated aliquots at decreasing concentrations of cells, (c) determining the presence or absence of the selected sequence in each vessel, and (d) relating the number of vessels containing the selected sequence to the total number of cells assayed at each dilution, such that the frequency of cells containing the selected nucleic acid sequence in said suspension may be determined.
  • the target sequence is an mRNA sequence which is obtained from a cell lysate and subjected to amplification by PCR without further extraction or purification of the mRNA.
  • samples which contain cells may be utilized within the context of the present invention.
  • Representative examples include samples taken from the environment (e.g., samples of water, air or earth), and biological samples.
  • Environmental samples may contain a variety of different cell types, including for example, plant cells, parasites, fungi, viruses and bacteria.
  • biological samples may contain a variety of cell types, and may be obtained from a variety of sources, including for example bodily fluids such as peripheral blood, cord blood urine, or cerebrospinal fluid, from bone-marrow, or from single-cell suspensions of body tissues (e.g., liver, spleen, brain, etc.).
  • suitable cells include human cells such as endothelial cells, tumor cells, pancreatic islet cells, macrophages, monocytes, NK cells, B lymphocytes, T lymphocytes, and hematopoietic stem cells.
  • Representative T lymphocytes include CD4 + cells, CD8 + cells, and specific subsets such as IL- 2R + , CD19 + , and transferrin receptor (TrR) + cells.
  • Hematopoietic stem cells include cells with differentiation markers such as CD34. Cell-free as well as cell-associated virus may also be utilized in the methods of this invention.
  • Such samples may be used as obtained, or may be subjected to one or more fractionation, concentration or culture procedures designed to enrich for the presence of a particular cell type or types from among a multiplicity of types initially present in the sample.
  • Representative fractionation methods include, for example, density gradient centrifugation, sedimentation field flow fractionation, fluorescence-activated cell sorting (FACS), and immunoselection using antibodies immobilized to a solid support, such as beads, fibers, filters, or plastic dishes (see Kumar and Lykke, Pathol. 7(5:53-62, 1984, and Basch et al., J. Immunol. Meth. 5(5:269-280, 1983, for reviews of these cell fractionation methods)).
  • the sample is fractionated by a combination of density gradient centrifugation (to produce a mononuclear cell suspension) and positive immunoselection, using a directly or indirectly biotinylated antibody to an antigen expressed by the cells of interest and immobilized avidin (U.S. Patent No. 5,215,927; U.S. Patent No. 5,225,353; and Berenson et al., WO 87/04628, published August 13, 1987; all of which are herein incorporated by reference).
  • density gradient centrifugation to produce a mononuclear cell suspension
  • positive immunoselection using a directly or indirectly biotinylated antibody to an antigen expressed by the cells of interest and immobilized avidin (U.S. Patent No. 5,215,927; U.S. Patent No. 5,225,353; and Berenson et al., WO 87/04628, published August 13, 1987; all of which are herein incorporated by reference).
  • a wide variety of antibodies specific for different cell types
  • antibodies to human leukocytes are extensively described in Knapp et al. (ed.), Leucocyte Typing IV, Oxford: Oxford University Press, 1989; such antibodies can be used advantageously to separate peripheral blood lymphocytes from other leukocytes, for example, or to separate one subpopulation of lymphocytes from another, such as B cells from T cells.
  • the mononuclear cell fraction is then incubated with a biotinylated antibody to the CD34 antigen and passed over a column of immobilized avidin (hereinafter referred to as the immunoadsorbent).
  • the immunoadsorbent retains those cells to which the anti-CD34 antibody has bound, while unbound cells flow through the column. After washing, the immunoadsorbent is agitated gently to elute the CD34-positive cells.
  • Fractionation of a heterogenous population of cells into pure subpopulations is rarely either possible or necessary. In general, it is sufficient to enrich the proportion of cells of interest in the population by ten to one-thousand fold, such that the cells of interest are present after enrichment in a ratio of between 1:100 to 1:100,000 relative to cells not of interest.
  • fetal cells are typically present in the maternal circulation at a ratio of about 1 : 1,000,000.
  • the proportion of fetal cells in the sample is approximately 1 :1,000 to 1 :10,000, which represents a 100- 1000-fold enrichment relative to the starting material.
  • the number of cells in the sample or subpopulation thereof is determined. This may be accomplished by manual counting, for example, utilizing a hemocytometer and a light microscope, or automatically or semi-automatically, utilizing an electronic impedance counter, such as a Coulter counter (Coulter Electronics, Hialeah, FL).
  • a Coulter counter Coulter Electronics, Hialeah, FL.
  • the sample is then dispensed to provide a series of multiply replicated aliquots containing decreasing concentrations of cells.
  • This may be accomplished by dispensing the cells into a plurality of vessels, such as test tubes or the wells of a microtiter tray, in numbers ranging from less than one cell per vessel, to several hundred thousand per vessel.
  • the cells are usually diluted in logs, such that the highest number of cells per vessel is about 1-5 X 10 ⁇ and the lowest number per vessel is about 1-5 X 10 ⁇ .
  • doubling dilutions may be employed.
  • the cells are aliquoted into between ten and fifty vessels, more often between about ten and twenty vessels. The more replicates of each concentration are prepared, the greater certainty as to the quantity of cells containing the sequence of interest in the original sample when statistical methods are utilized (as described below).
  • the cells are diluted such that the range of concentrations assayed encompasses at least one concentration at which some of the vessels are likely to contain cells which in turn contain the selected sequence, while other vessels at that dilution are likely to lack cells which contain the target sequence.
  • An experimental situation in which every vessel contains cells containing the selected sequence, or conversely, no vessel contains cells containing the selected sequence, is relatively uninformative, providing only an upper or a lower limit for the number of target cells in the population.
  • Cells are then disrupted in order to expose nucleic acids.
  • Cell disruption may be effected by any of a variety of means, including detergent lysis, enzymatic digestion, freezing and thawing, heating to temperatures between about 56°C and 95°C, etc. (see Kawasaki, op. cit., for a review of sample preparation methods). In the present invention, it is generally preferred to effect disruption of the cells by heating the cells to 95°C for approximately 5 minutes.
  • the selected sequence to be amplified is DNA
  • proteinase K may be included in the lysis buffer to lyse nuclei, but no other manipulation of the sample is generally necessary prior to PCR.
  • the target sequence is RNA
  • it is art-accepted practice to perform modified or additional extraction and purification steps prior to PCR (Villarmal et al., Blood 75:1216-1222, 1991 ; Schnerer et al., Blood 77:287-789, 1991).
  • the cells are lysed by guanidinium isothiocyanate, followed by centrifugation over a cesium chloride gradient, two or more phenol/chloroform extractions, and an ethanol precipitation step. Purity of the starting RNA is generally believed to have a direct bearing on the sensitivity of PCR, higher purity correlating with higher sensitivity. However, every manipulation of the sample results in some loss of starting material, which can seriously compromise sensitivity when the amount of starting material is small.
  • RNA can be utilized in PCR amplification without extensive purification. Briefly, within a preferred embodiment cells are lysed by heating to about 95°C for about 5 minutes and amplified directly. No further purification is undertaken, and no ribonuclease inhibitors are added. In another embodiment of the invention, 1.5 to 2 volumes of diethylpyrocarbonate (DEPC) -treated water is added to each sample after heating.
  • DEPC diethylpyrocarbonate
  • DEPC-treated water is prepared by adding DEPC to distilled water to a final concentration of 0.01% and autoclaving the water, which inactivates the DEPC and sterilizes the solution.)
  • DEPC-treated water is not believed to act as an RNase inhibitor because of its inactivation by autoclaving; it serves solely as a diluent for the cells.
  • RNA is minimized relative to conventional techniques in which the sample is manipulated five or more times.
  • each aliquot is then assayed in order to determine the presence or absence of a selected nucleic acid sequence.
  • sequence selected should be specific for the cell of interest. For example, Y-chromosome sequences can be amplified for the detection of male fetal cells. The sequence encoding the CD34 gene or its message can be amplified for the detection of hematopoietic stem cells.
  • each aliquot is subjected to nucleic acid amplification, for example, by PCR, using primers specific for the sequence which it is desired to detect. Briefly, for the amplification of a selected DNA sequence, a buffered solution containing MgCl2, reverse transcriptase, the four deoxynucleoside triphosphates, and the two primers (hereinafter referred to as DNA master mix) is added to the vessel containing each aliquot.
  • DNA master mix a buffered solution containing MgCl2, reverse transcriptase, the four deoxynucleoside triphosphates, and the two primers
  • the master mix with the restriction endonuclease Eco RI for approximately one hour at 37°C, prior to adding it to the vessel containing the cell lysate. This is done to eliminate carry-over products, amplification of which may result in false positive results. If the primers contain an Eco RI site, the Eco RI activity must be eliminated from the master mix by incubating it for about 5 minutes at 95 °C. A number of cycles of amplification may then be performed, preferably using a Perkin Elmer 9600 thermocycler (Perkin-Elmer Corp., Norwalk, CT).
  • the number of cycles, the temperature and length of time allowed for primer annealing, the time and temperature for primer extension, and the time and temperature for denaturation of product and template will vary, depending on the concentration of the target sequence, the concentration of the primers, the sequence of the primers, the magnesium concentration, etc. and are determined empirically for each application. In general, about thirty cycles of amplification are performed, each cycle consisting of approximately 30 seconds at 95°C, 30 seconds at 55°C, and 30 seconds at 72°C.
  • Sensitivity may be improved if PCR amplification is conducted in two stages (Orrego and King, in PCR Protocols: A Guide to Methods and Applications, M.A. Innis et al. (eds.), New York: Academic Press, pp. 416-426), using two different pairs of primers, the second pair chosen to be internal to the sequence amplified by the first pair (hereinafter referred to as nested primers).
  • nested primers the second pair chosen to be internal to the sequence amplified by the first pair
  • one microliter of the product of the first round of amplification is added to a master mix containing the second set of (nested) primers.
  • a second round of amplification is then performed, using empirically- determined conditions.
  • the second round consists of 15 cycles of amplification, each cycle consisting of 30 seconds at 95°C, 30 seconds at 50°C, and 30 seconds at 72°C. Again, the optimal number of cycles, the cycling temperatures and times may be determined empirically.
  • RNA master mix For amplification of a selected RNA, such as mRNA, within one embodiment a suitable primer is added to the cell lysate in each vessel, and allowed to anneal to the RNA at 70°C for about 10 minutes. The exact time and temperature will vary, depending on the sequence of the primer and the concentrations of the primer and the template. The mixture is then chilled on ice for one to five minutes before a buffered solution containing MgCl2, dithiothreitol (DTT), the four deoxynucleoside triphosphates, and reverse transcriptase (hereinafter referred to as RNA master mix) is added.
  • MgCl2, dithiothreitol (DTT) the four deoxynucleoside triphosphates
  • RNA master mix reverse transcriptase
  • a complementary DNA (cDNA) is synthesized from mRNA by reverse transcriptase during incubation at about 42 °C for approximately one hour. Following denaturation for about 5 minutes at 90°C, PCR amplification of the resultant cDNA is performed essentially as described above with respect to nested primer amplification of a DNA target.
  • nucleic acid amplification has been concerned with PCR, it will be evident to one skilled in the art given the disclosure provided herein that a variety of other amplification methods may also be utilized, including, for example, ligation amplification (LCR, Wu and Wallace, Genomics 4:560, 1989), Q ⁇ -replicase amplification (Kramer and Lizardi, Nature 55P.401, 1989), and transcription-based amplification (Kwoh et al., Proc. Natl. Acad. Sci. (USA) £7:1874, 1989). Isothermal amplification methods may also be employed. In addition, a variety of other methods may be utilized in order to determine the presence or absence of a selected nucleic acid sequence, including for example, cycling probe reactions (U.S. Patent Nos. 4,876,187 and 5,011,769).
  • cycling probe reactions U.S. Patent Nos. 4,876,187 and 5,011,769
  • the presence or absence of the selected nucleic acid sequence is detected, for example, by agarose electrophoresis or by Southern blotting using a labeled probe for the product (Ausubel et al. (ed.), Current Protocols in Molecular Biology, New York: Wiley Interscience, 1987). Knowing the number of cells input to each vessel and the number of vessels at each cell concentration which contain the desired product, one may then readily determine the number of cells in the original sample which contain the selected nucleic acid sequence, for example, by Poisson analysis as described below.
  • a suspension of cells at a known concentration is aliquoted into a plurality of vessels at a number of dilutions.
  • the number of cells per well is given by the expression c .
  • n vessels of which are observed to contain the selected nucleic acid sequence.
  • C(n,k) is the binomial coefficient for n and k.
  • Equation (5) The approximation given by equation (5) is used only with those dilutions which yield both positive and negative vessels, as well as the first neighboring dilutions (if any) which yield exclusively positive or exclusively negative vessels. If one has no information regarding the target cell frequency initially, it is preferred to test a large number of dilutions first in order to identify the range which will be most informative.
  • An alternative procedure may be utilized to calculate /when each dilution tested yields both positive and negative vessels, i.e. when some vessels at every dilution contain the selected nucleic acid sequence while other vessels do not.
  • the value of / may be estimated for each dilution along with its standard error, and a single estimate may be developed by weighting the individual estimates with weights proportional to the inverse of the standard error squared.
  • the methods of the instant invention may be utilized to quantify the presence of rare cells present in a heterogenous population, which cells exhibit sequence differences determinative of their type.
  • Applications include the quantification of virus-infected cells in tissue (such as HIV-infected cells in the peripheral blood), of cancer cells in peripheral blood (for early detection of metastasis), and of cancer cells with a high metastatic or growth potential in a tumor (such as estrogen receptor expression for determining prognosis in breast cancer).
  • the instant invention also enables quantification of the number of cells containing a given mRNA. This method is useful for all of the aforementioned applications, as well as for quantifying cell-free virus and levels of expression of exogenously-introduced genes in cells.
  • a single male (fetal) cell present among one million female (maternal) cells can be detected and quantified utilizing methods of the present invention to amplify Y-chromosome-specific DNA sequences.
  • the number of CD34+ cells in peripheral blood is also quantified utilizing the methods described above.
  • the number of tumor cells in a peripheral blood specimen is quantified utilizing the methods of the present invention to amplify the estrogen receptor mRNA from breast carcinoma cells.
  • the cells were lysed by heating the microtiter tray to 95°C for 5 minutes in a Perkin Elmer Model 9600 Thermocycler.
  • a DNA master mix (10 mM Tris HC1, pH 8.3 / 1.5 mM MgC_2 / 50 mM KCl / 1 mM each of dATP, dTTP, dGTP, dCTP (Boehringer Mannheim)/ 25 units per ml Taq polymerase/ 1 umol of each of two primers, designated Yl and Y2, the sequences of which are shown in Table 1) was prepared and incubated at 37°C for one hour with 800 U/ml of Eco RI (Boehringer Mannheim). Residual Eco RI activity was destroyed by heating to 95 °C for 5 minutes in the thermocycler. Fifty microliters of Eco RI -treated DNA master mix was then added to each well of the microtiter tray.
  • the tray was then subjected to 30 cycles of amplification. Each cycle consisted of 30 seconds at 95°C, 30 seconds at 55°C, and 30 seconds at 72°C. At the conclusion of this first round of amplification, a 154 base pair product was expected.
  • PCR product from each well was transferred to a fresh well in a second microtiter plate.
  • 50 ul of Eco RI- treated DNA master mix identical to that used above, except for the substitution of primers Y3 and Y4 for Yl and Y2.
  • Y3 and Y4 the sequences of which are shown in Table 1, are located internal to Yl and Y2.
  • the tray was then subjected to an additional 15 cycles of amplification, each cycle consisting of 30 seconds at 95°C, 30 seconds at 50°C, and 30 seconds at 72°C.
  • reaction products in each well were analyzed by subjecting 25 ul to electrophoresis in a 4%> agarose gel.
  • the expected product of this reaction a 108 base pair DNA fragment, was detected by agarose gel electrophoresis in the presence of appropriate DNA standards.
  • the number of wells at each cell concentration containing the expected 108 base pair fragment is shown in Table 2. Each positive well is assumed to contain a single cell containing the sequence of interest. Accordingly, the number of male (and necessarily, fetal) cells present in the maternal blood specimen assayed in this example is approximately 1 in one million. The same result is obtained by applying equation (5) to the data in Table 2, and solving for "fq":
  • a mononuclear cell fraction was prepared from ten milliliters of peripheral blood, obtained by venipuncture of the antecubital vein, by density gradient centrifugation on Ficoll-Hypaque.
  • To 1 ml of the resultant MNC fraction was added 3 ml of hemolysis buffer (150 mM NH4CI / 12 mM NaHC ⁇ 3 / 0.1 mM EDTA, pH 7.3). Hemolysis was allowed to take place for approximately 2 minutes, after which the cells were washed twice in PBS/1% BSA. The remaining cells were counted using a hemocytometer.
  • a sufficient volume of the cell suspension to yield 100, 500, 1000, 5000, or 10,000 cells per tube was dispensed and the volume in each tube adjusted to 5 ul with PBS, as necessary.
  • Ten tubes were set up at each cell number for a total of 50 tubes in all. The cells were heated to 95 °C for 5 minutes in the thermocycler, after which 8 ul of DEPC-treated water was added to each tube. To each tube was then added 0.1 nmoles of CD34-1 primer (see Table 1 for sequence). The primer was allowed to anneal with the target mRNA for 10 minutes at 70°C.
  • RNA master mix (2 ul 10X synthesis buffer (200 mM TrisHCl, pH 8.4, 500 mM KCl, 25 mM MgCl, 1 mg/ml BSA), 2 ul 0.1 M DTT, 1 ul 10 mM dNTP mix (10 mM each in dATP, dTTP, dCTP, and dGTP), 1 ul reverse transcriptase (at 200 units/ ml)) was added to each tube and allowed to incubate for 5 minutes at room temperature. The tubes were then incubated at 42°C for 50 minutes, followed by a 5 minute incubation at 90°C. This completed cDNA synthesis.
  • 10X synthesis buffer 200 mM TrisHCl, pH 8.4, 500 mM KCl, 25 mM MgCl, 1 mg/ml BSA
  • 2 ul 0.1 M DTT 2 ul 0.1 M DTT
  • 1 ul 10 mM dNTP mix 10 mM each in d
  • PCR amplification of the resultant cDNA, using nested primers was performed as follows. To each tube was added 1 ul of Taq polymerase (at 5 units/ul), 8 ul of 10X synthesis buffer, 68 ul of water, and 1 ul (1 nmole each) of CD34-1 and CD34-2 primers. Thirty cycles of amplification were conducted, each cycle consisting of 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 60 seconds.
  • the methods of the instant invention may be utilized to detect and quantify the presence of rare cells in a heterogenous population, provided that the rare cells exhibit sequence differences determinative of their type.
  • these methods are applied to the detection and quantification of tumor cells (breast carcinoma) in peripheral blood, using the mRNA for the estrogen receptor (ER) as the target sequence for nested primer amplification.
  • a mononuclear cell (“MNC") fraction was prepared from ten milliliters of peripheral blood, obtained by venipuncture of the antecubital vein of a healthy volunteer, by density gradient centrifugation on Ficoll-Hypaque, as described in Examples 1 and 2 above. An aliquot of the MNC suspension was counted on a hemocytometer.
  • a single-cell suspension of BT-20 cells (ATCC No. HTB19; a breast cancer line available from the American Type Culture Collection, Rockville, MD) was prepared and spiked into the MNC suspension at a ratio of 1 BT-20 cell to 1000 mononuclear cells to simulate a peripheral blood specimen derived from a tumor-bearing host. Sufficient volumes of this mock tumor cell suspension to yield 100,000 cells, 10,000 cells, and 1,000 cells per well were dispensed into the wells of a microtiter tray. The volume in each well was adjusted, as necessary, with PBS to 10 ul.
  • Nested primer amplification was performed essentially as described in Examples 1 and 2 above.
  • the sequences of the primers used are given in Table 1, where ER-1 and ER-2 were used in the first 30 cycles of amplification and ER-N1 and ER-N2, in the next 15 cycles.
  • the reaction products in each well were analyzed by subjecting an aliquot to agarose gel electrophoresis, as described in the above Examples.
  • the expected product of this reaction a 129 base pair DNA fragment, was detected in the well containing 100,000 cells (100 of which would be expected to be tumor cells). The expected product was not seen in the wells containing only 10,000 cells (10 tumor cells) or 1000 cells (1 tumor cell).
  • the methods of this invention can be utilized to detect the presence of metastasizing tumor cells in a tumor-bearing host.
  • the fraction of cells which are tumor cells was then enriched by subjecting the mock tumor cell suspension to immunoselection using a biotinylated anti-breast antibody and immobilized avidin.
  • the anti-breast antibody is designated NRLu-10 and is available from NeoRx (Seattle, WA).
  • the immunoselection method and apparatus are described in U.S. Patents 5,215,927, issued June 1, 1993 to Berenson et al.; 5,225,353, issued July 6, 1993 to Berenson et al.; and 5,240,856, issued August 31, 1993 to Goffe et al.; and in PCT/US91/07646, published April 30, 1992, all of which patents or patent applications are herein incorporated by reference.
  • the suspension of eluted cells was then dispensed into the wells of a microtiter tray in volumes sufficient to yield 15,000 cells, 10,000 cells, 1,000 cells, 100 cells, 10 cells or 1 cell per well and the volume in each well adjusted as necessary to 10 uL.
  • RT-PCR was performed as described above using nested primers specific for ER-mRNA (see Table 1 for primer sequences). The resultant reaction products were analyzed by agarose gel electrophoresis.
  • CD34-1 5 CACCCTGTGTCTCAACATGG
  • CD34-2 6 GGAGATGTTGCAAGGCTAG CD34-3 7 AGGCCAGAACAAACATCACA

Landscapes

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

Abstract

Un procédé de quantification de la fréquence de cellules contenant une séquence d'acide nucléique sélectionnée dans une suspension de cellules hétérogènes par rapport à la présence de la séquence consiste à (a) déterminer le nombre total de cellules dans la suspension, (b) distribuer la suspension dans plusieurs récipients afin d'obtenir une série d'aliquotes multiples répliquées à des concentrations décroissantes de cellules, (c) déterminer la présence ou l'absence de la séquence sélectionnée dans chaque récipient, et (d) mettre en relation le nombre de récipients contenant la séquence sélectionnée avec le nombre total de cellules analysées à chaque dilution, de sorte que la fréquence de cellules contenant la séquence sélectionnée d'acides nucléiques dans la suspension puisse être déterminée.
PCT/US1994/009719 1993-09-03 1994-08-26 Procede de quantification du nombre de cellules contenant une sequence d'acide nucleique selectionnee dans une population heterogene de cellules WO1995006750A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU76404/94A AU7640494A (en) 1993-09-03 1994-08-26 Methods for quantifying the number of cells containing a selected nucleic acid sequence in a heterogenous population of cells

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US11737193A 1993-09-03 1993-09-03
US08/117,371 1993-09-03

Publications (1)

Publication Number Publication Date
WO1995006750A1 true WO1995006750A1 (fr) 1995-03-09

Family

ID=22372545

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1994/009719 WO1995006750A1 (fr) 1993-09-03 1994-08-26 Procede de quantification du nombre de cellules contenant une sequence d'acide nucleique selectionnee dans une population heterogene de cellules

Country Status (2)

Country Link
AU (1) AU7640494A (fr)
WO (1) WO1995006750A1 (fr)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5858665A (en) * 1996-07-25 1999-01-12 Navix, Inc. Homogeneous diagnostic assay method utilizing simultaneous target and signal amplification
FR2810677A1 (fr) * 2000-06-27 2001-12-28 Urogene Procede de diagnostic in vitro du cancer de la prostate et kit de mise en oeuvre
WO2005028677A3 (fr) * 2003-05-07 2005-07-14 Agilent Technologies Inc Systemes et procedes permettant de determiner la composition d'un type de cellule d'une population cellulaire melangee a l'aide de signatures d'expression genique
DE102005059227A1 (de) * 2005-12-12 2007-06-14 Advalytix Ag Verfahren zur Bestimmung des Genotyps aus einer biologischen Probe enthaltend Nukleinsäuren unterschiedlicher Individuen
US9970058B2 (en) 1999-08-02 2018-05-15 The Johns Hopkins University Digital amplification
JP2018515074A (ja) * 2015-05-04 2018-06-14 エピバックス インコーポレーテッド インフルエンザａ／上海／２／２０１３ ｈ７配列の改変ｈ７赤血球凝集素糖タンパク質

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0330191A2 (fr) * 1988-02-25 1989-08-30 The General Hospital Corporation ADN encodant CD40
EP0390323A2 (fr) * 1989-03-29 1990-10-03 The Johns Hopkins University Détection de l'écoulement du type sauvage du p53 gène
WO1991002817A1 (fr) * 1989-08-21 1991-03-07 Cetus Corporation Quantification d'acides nucleiques a l'aide de la reaction en chaine de la polymerase
EP0438115A2 (fr) * 1990-01-19 1991-07-24 F. Hoffmann-La Roche Ag Procédé pour la détection des pathogènes microbiens aqueuses et l'indicateur de la contamination fécale humaine dans des échantillons aqueuses et kits pour cela
EP0512334A2 (fr) * 1991-05-02 1992-11-11 F. Hoffmann-La Roche Ag Procédé pour la détection d'ADN dans un échantillon
JPH05100A (ja) * 1990-11-09 1993-01-08 Nippon Flour Mills Co Ltd 細菌の薬剤感受性試験法
EP0524808A2 (fr) * 1991-07-23 1993-01-27 F. Hoffmann-La Roche Ag Préparations PCR in situ, leur utilisation et équipement à cycles thermiques correspondant
EP0534640A1 (fr) * 1991-09-23 1993-03-31 Pfizer Inc. Procédé pour détecter ARNm et ADN spécifiques dans les cellules

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0330191A2 (fr) * 1988-02-25 1989-08-30 The General Hospital Corporation ADN encodant CD40
EP0390323A2 (fr) * 1989-03-29 1990-10-03 The Johns Hopkins University Détection de l'écoulement du type sauvage du p53 gène
WO1991002817A1 (fr) * 1989-08-21 1991-03-07 Cetus Corporation Quantification d'acides nucleiques a l'aide de la reaction en chaine de la polymerase
EP0438115A2 (fr) * 1990-01-19 1991-07-24 F. Hoffmann-La Roche Ag Procédé pour la détection des pathogènes microbiens aqueuses et l'indicateur de la contamination fécale humaine dans des échantillons aqueuses et kits pour cela
JPH05100A (ja) * 1990-11-09 1993-01-08 Nippon Flour Mills Co Ltd 細菌の薬剤感受性試験法
EP0512334A2 (fr) * 1991-05-02 1992-11-11 F. Hoffmann-La Roche Ag Procédé pour la détection d'ADN dans un échantillon
EP0524808A2 (fr) * 1991-07-23 1993-01-27 F. Hoffmann-La Roche Ag Préparations PCR in situ, leur utilisation et équipement à cycles thermiques correspondant
EP0534640A1 (fr) * 1991-09-23 1993-03-31 Pfizer Inc. Procédé pour détecter ARNm et ADN spécifiques dans les cellules

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
AOKI-SEI S ET AL: "PLASMA HIV-1 VIREMIA IN HIV-1 INFECTED INDIVIDUALS ASSESSED BY POLYMERASE CHAIN REACTION", AIDS RESEARCH AND HUMAN RETROVIRUSES, vol. 8, no. 7, 1992, NEW YORK, pages 1270 - 1263 *
DATABASE WPI Section Ch Week 9306, Derwent World Patents Index; Class B04, AN 93-049157 *
MOLESH D.A. ET AL: "Quantitative analysis of CD34+ stem cells using RT-PCR on whole cells", PCR METHODS AND APPLICATIONS, vol. 3, no. 5, April 1994 (1994-04-01), USA, pages 278 - 284 *
TASWELL C: "LIMITING DILUTION ASSAYS FOR THE DETERMINATION OF IMMUNOCOMPETENT CELL FREQUENCIES 3. VALIDITY TESTS FOR THE SINGLE-HIT POISSON MODEL", JOURNAL OF IMMUNOLOGICAL METHODS, vol. 72, no. 1, 1984, AMSTERDAM, pages 29 - 40 *
VILLARREAL XC ET AL: "Demonstration of osteonectin mRNA in megakaryocytes: the use of the polymerase chain reaction.", BLOOD, vol. 78, no. 5, 1991, NEW YORK, pages 1216 - 1222 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5858665A (en) * 1996-07-25 1999-01-12 Navix, Inc. Homogeneous diagnostic assay method utilizing simultaneous target and signal amplification
US6114117A (en) * 1996-07-25 2000-09-05 Medical Analysis Systems, Inc. Homogeneous diagnostic assay method utilizing simultaneous target and signal amplification
US9970058B2 (en) 1999-08-02 2018-05-15 The Johns Hopkins University Digital amplification
FR2810677A1 (fr) * 2000-06-27 2001-12-28 Urogene Procede de diagnostic in vitro du cancer de la prostate et kit de mise en oeuvre
WO2005028677A3 (fr) * 2003-05-07 2005-07-14 Agilent Technologies Inc Systemes et procedes permettant de determiner la composition d'un type de cellule d'une population cellulaire melangee a l'aide de signatures d'expression genique
DE102005059227A1 (de) * 2005-12-12 2007-06-14 Advalytix Ag Verfahren zur Bestimmung des Genotyps aus einer biologischen Probe enthaltend Nukleinsäuren unterschiedlicher Individuen
JP2018515074A (ja) * 2015-05-04 2018-06-14 エピバックス インコーポレーテッド インフルエンザａ／上海／２／２０１３ ｈ７配列の改変ｈ７赤血球凝集素糖タンパク質

Also Published As

Publication number Publication date
AU7640494A (en) 1995-03-22

Similar Documents

Publication Publication Date Title
JP7203128B2 (ja) 血液試料中の循環腫瘍細胞のデジタル分析
KR101781147B1 (ko) 대립유전자, 게놈 및 전사체 검출을 위한 방법 및 유전자형 분석 패널
US7258976B2 (en) Method of preparing cell lysate
EP1862545B1 (fr) Procédé pour l'analyse quantitative d'ADNC dans une cellule unique
EP1997912B1 (fr) Dispositif et procédé pour quantification à haut rendement de MRNA à partir de sang total
EP2652155B1 (fr) Procédés pour l'analyse parallèle massive des acides nucléiques contenus dans des cellules individuelles
JP5133238B2 (ja) 遠隔サンプル由来のｄｎａ断片を提供する方法
EP3485043A1 (fr) Séquençage de produits de transcription unicellulaires
CA2437737A1 (fr) Procedes et compositions d'amplification d'arn
US20210301329A1 (en) Single Cell Genetic Analysis
JP2002505080A (ja) オリゴヌクレオチド固定化ｐｃｒマイクロプレート上での直接ｒｔ−ｐｃｒ
JPH05269000A (ja) 核酸増幅による悪性腫瘍転移の検出方法
HUE034937T2 (en) Enrichment and detection of fetal cells from maternal blood and ligands for this purpose
Rasmussen et al. Circulating clonal cells in multiple myeloma do not express CD34 mRNA, as measured by single‐cell and real‐time RT‐PCR assays
WO1995006750A1 (fr) Procede de quantification du nombre de cellules contenant une sequence d'acide nucleique selectionnee dans une population heterogene de cellules
Zhou et al. High throughput analysis of TCR-β rearrangement and gene expression in single T cells
Tubo et al. The nuclear matrix continues DNA synthesis at in vivo replicational forks
EP1329506A1 (fr) Procédé de quantification du niveau in vivo d'ARN
JP2018525034A (ja) 腫瘍特異的ｔ細胞を提供するための方法
Ali et al. Application of differential display to immunological research
LARA‐MARQUEZ et al. Vasoactive intestinal peptide (VIP) receptor type 2 (VPAC2) is the predominant receptor expressed in human thymocytes
Che¹ et al. RNA amplification methodologies
Bertrand et al. Use of serial analysis of gene expression (SAGE) technology reveals new granulocytic markers
AU756357B2 (en) Methods for identifying therapeutic targets
Rasmussen et al. Identification and characterisation of malignant cells using PT-PCR on single flow-sorted cells

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU CA FI JP NO NZ

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IE IT LU MC NL PT SE

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: CA

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载