WO1995006750A1

WO1995006750A1 - Methods for quantifying the number of cells containing a selected nucleic acid sequence in a heterogenous population of cells

Info

Publication number: WO1995006750A1
Application number: PCT/US1994/009719
Authority: WO
Inventors: Jeffrey M. Hall; David A. Molesh
Original assignee: Cellpro, Incorporated
Priority date: 1993-09-03
Filing date: 1994-08-26
Publication date: 1995-03-09
Also published as: AU7640494A

Abstract

A method of quantifying the frequency of cells containing a selected nucleic acid sequence in a suspension of cells heterogenous with respect to the presence of the sequence, comprising (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.

Description

METHODS FOR QUANTIFYING THE NUMBER OF CELLS CONTAINING A SELECTED NUCLEIC ACID SEQUENCE IN A HETEROGENOUS POPULATION OF CELLS

Technical Field

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.

Background of the Invention

The first technique for amplifying nucleic acids (ex vivo) was described by Saiki et al. in Science 250:1350-1354, 1985. This technique, later designated "polymerase chain reaction" or "PCR," was applied to detect mutations in the beta-globin gene. A more general description of the methodology was subsequently provided by Mullis (Mullis et al., Cold Spring Harbor Symp. Quant. Biol. 57:263-273, 1986; Mullis and Faloona, Methods Enzymol. 755:335-350, 1987; U.S. Patent No. 4,683,195, issued to Mullis; U.S. Patent No. 4,683,202, issued to Mullis). Since that time, more than 18,000 publications have appeared in the scientific literature describing various applications of and variations on PCR, as well as numerous patents on various aspects of the technology (see, for example, U.S. Patent No. 4,965,188; U.S. Patent No. 5,008,182; U.S. Patent No. 5,057,410; U.S. Patent No. 5,091,310; U.S. Patent No. 5,079,352; U.S. Patent No. 5,066,584; and U.S. Patent No. 5,075,216).

Generally, 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 2ⁿ copies present. Since the amplification is somewhat less than 100% efficient, the actual number of copies will in fact be slightly less than 2ⁿ.

Shortly after the first description of PCR, Seeburg et al. described PCR amplification of mRNA from cDNA (1986, UCLA Symposium, unpublished). Subsequently, Veres et al. (Science 257:415-417, 1987) provided the earliest published description of mRNA amplification by PCR. These and other references rely on purified, or partially purified RNA as the starting material. Methods of RNA extraction and purification have been described in numerous publications, including, for example, Davis et al., Basic Methods in Molecular Biology, New York: Elsevier, 1986; Ausubel et al. (ed.), Current Protocols in Molecular Biology, New York: Wiley Interscience, 1987; and Berger and Kimmel, Methods Enzymol. 752:215-304, 1987. When high sensitivity is not required and the target sequence is relatively abundant, it may be feasible to utilize crude RNA, without purification by cesium chloride centrifugation or phenol extraction provided a ribonuclease inhibitor is employed. (Kawasaki, ES., in PCR Protocols: A Guide to Methods and Applications, MA. Innis et al. (eds.), New York: Academic Press, pp. 146-152, 1990). However, many tissues, such as blood and pancreas, contain too much ribonuclease activity to be amenable to the use of such shortened purification protocols and it is presently axiomatic in molecular biology that RNA must be extensively purified prior to amplification.

Although 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.

Various methods of quantification, however, have been proposed, such as competitive PCR using an internal standard. Briefly, in 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. Thus, 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. Typically, 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. Despite advances in the ability to quantify specific, amplified nucleic acid sequences, it is still not possible to routinely quantify the number of cells in a biological sample containing a specific target sequence. This limitation is inherent in the PCR methodology, which produces an imprecise degree of amplification from an imprecise amount of starting material contributed by a multiplicity of cells which are not necessarily equal with regard to their contribution of target sequence.

Thus, there is a need in the art for a means of quantifying the number of cells in a biological sample which contain a specific nucleic acid sequence. The present invention fulfills this need, and further, provides other related advantages.

Summary of the Invention

Briefly stated, 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. More specifically, within one aspect of the present invention methods of increasing the number of copies of a selected ribonucleic acid sequence in a sample are provided, 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. Within related aspects of the present invention, such methods may be accomplished utilizing viruses or cell free extracts as samples. Within another aspect of the present invention, methods are provided 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.

Within yet another aspect of the present invention, methods are provided 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 suspension may be determined.

Within various embodiments of the above-described inventions, the selected nucleic acid sequence may be amplified by, for example, polymerase chain reaction, Qβ-replicase amplification, or isothermal amplification. Within another embodiment, prior to the step of determining, the cell suspension may be fractionated into subpopulations of cells. Within yet another embodiment, a ribonuclease inhibitor is not added during the steps of disrupting or amplifying. Within other embodiments, cells are disrupted by heating the cells. Representative examples of preferred cells within the context of the present invention include fetal cells, CD34⁺ cells, and tumor cells. Within yet other embodiments of the invention, the frequency of cells containing the selected nucleic acid sequence may be determined by the equation ∑jkj / (N- ∑ik_jC_j/2).

These and other aspects of the present invention will become evident upon reference to the following detailed description. In addition, various references are set forth below which describe in more detail certain procedures or compositions, and are therefore incorporated by reference in their entirety.

Description of the Invention

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. Within one aspect of the invention, methods are provided 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. In one embodiment of the invention, 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.

A variety of 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. Likewise, 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.). Representative examples of 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)). Many of the above fractionation methods, as well as more common methods such as centrifugation and filtration, may likewise be utilized to concentrate cells. In a preferred embodiment of the instant invention, 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). A wide variety of antibodies specific for different cell types have been described in the literature. For example, 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.

In one embodiment of this invention, it is desired to separate fetal cells from maternal cells present in the peripheral blood of a pregnant female. Methods by which this may be accomplished are described in commonly owned, co-pending patent applications U.S.S.N. 07/946,803, filed September 16, 1992 and entitled "Method for Enriching Fetal Cells from Maternal Blood," which is a continuation of U.S.S.N. 07/513,057, filed April 23, 1990 and entitled "Method for Enriching Fetal Cells from Maternal Blood," and U.S.S.N. 07/782,148, filed October 23, 1991 and entitled "Method for Enriching Fetal Progenitor Cells from Maternal Blood," which is a continuation-in-part of U.S.S.N. 513,057, cited above (all of which are herein incorporated by reference). Briefly, maternal peripheral blood is collected in anticoagulant by venipuncture of the antecubital vein. The blood is centrifuged to produce a buffy coat from which a mononuclear cell fraction is prepared by density gradient centrifugation using Ficoll-Hypaque. 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. For example, fetal cells are typically present in the maternal circulation at a ratio of about 1 : 1,000,000. After positive immunoselection using an anti-CD34 antibody, 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. Once the sample has been fractionated into two or more subpopulations of cells (if desired), 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).

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. For convenience, 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^. Alternatively, doubling dilutions may be employed. Typically, at each of about 4 or 5 concentrations 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).

Ideally, 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.

If 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. However, if 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). Typically, 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.

In one embodiment of the present invention, it has been found that 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 is a ribonuclease inhibitor which is inactivated by heating. 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.) As utilized in the instant invention, 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. These methods represent a considerable time savings, typically as much as four hours, over the art-accepted methods of RNA purification and eliminate the need for sophisticated equipment (an ultracentrifuge is required for cesium chloride gradients) arid hazardous chemicals (phenol and chloroform), yet there is no loss of sensitivity. Furthermore, the fact that the sample needs to be manipulated only once (to aliquot it into vessels for amplification) means that loss of RNA is minimized relative to conventional techniques in which the sample is manipulated five or more times. After the cells have been disrupted to expose nucleic acids, each aliquot is then assayed in order to determine the presence or absence of a selected nucleic acid sequence. Within the context of the present invention it should be understood that numerous sequences may be selected for amplification. Generally, the 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. Similarily, the estrogen receptor message can be amplified for the detection of estrogen receptor positive tumor cells (e.g. breast carcinomas) (see Example 3 below.). Within one embodiment of the invention, 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. It is generally preferred to incubate 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). Briefly, to employ nested primers, 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. Typically, 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.

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. 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. Although the foregoing description of 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), Qβ-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).

After amplification (whether of DNA or RNA), 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.

In particular, within a preferred embodiment of the invention, a suspension of cells at a known concentration is aliquoted into a plurality of vessels at a number of dilutions. At each dilution i (where = 1, 2, ... D), the number of cells per well is given by the expression c . For the f^m dilution, there are n vessels, of which are observed to contain the selected nucleic acid sequence. (Note that in the method of this invention, there are only two possible outcomes on a per-vessel basis, namely, that a given vessel contains the selected nucleic acid sequence or that it does not.) If Xj equals the number of cells in a single vessel at dilution i, X\ will be distributed according to the Poisson distribution with a mean equal to fcf. The probability that a vessel will contain the selected nucleic acid sequence is given by equation (1).

(1) P(Xi > 0) = 1 - e-fci The probability of observing the experimental outcome of k out of nj vessels containing the selected nucleic acid sequence at dilution i is given by equation (2),

(2) C(n,k) • (l-e-f j)^ki • e-fc/nf ,

where C(n,k) is the binomial coefficient for n and k. The logarithm of this expression can be maximized across all of the different dilutions i = 1, 2, ... D, according to the equation given in (3).

(3) log L = ∑i log C(n kj) + ∑k_t log(l-ef^Cj) + ∑_z-/c_zf/__z-

Taking the first derivative of logL with respect to / and setting it equal to 0 will determine the value of/ the concentration of target cells, that maximizes the probability of the observed outcome. This equation is given by (4), where N equals the total number of cells in all vessels at all dilutions.

(4) ∑ikiCi/ (l-e-f i) = N

An approximate solution for / can be found by using a quadratic approximation to the function on the left-hand side of equation (4). Using this, the approximate solution, fq, is given by equation (5).

(5) f_q = ∑ikj/ ^• (N- ∑ik_iCi/2)

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. In this case, 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.

In summary, 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.

In the following examples, which are offered by way of illustration, and not by way of limitation, it is shown that 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. In another example, the number of CD34+ cells in peripheral blood is also quantified utilizing the methods described above. In yet another example, 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.

EXAMPLES

EXAMPLE 1 QUANTIFICATION OF MALE FETAL CELLS IN MATERNAL BLOOD BY NESTED PRIMER AMPLIFICATION OF Y-CHROMOSOME-SPECIFIC DNA

Ten milliliters of blood in ACD were collected from a pregnant female and separated on a discontinuous Ficoll gradient (1.077 g/ml; Bio- Whitaker, Walkersville, MD). The mononuclear cell fraction sedimenting at 1.077 g/1 was harvested, resuspended in phosphate buffered saline (PBS 0.15 M NaCl/ 0.15 M phosphate, pH 7.4) and counted on a hemocytometer. A sufficient volume of the resultant cell suspension to yield 20,000 cells, 50,000 cells, 100,000 cells, or 200,000 cells per well was dispensed into the wells of a microtiter tray. The volume of cell suspension in each well was adjusted to 10 ul with PBS, as necessary. Twenty wells were seeded at each cell concentration, for a total of 80 wells (20 x 20,000 cells/well, 20 x 50,000 cells/well, etc.).

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.

One microliter of PCR product from each well was transferred to a fresh well in a second microtiter plate. To each well was added 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.

At the conclusion of the second round of amplification, the 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":

f_q = 7/(7.4 x lθ6 - 525,000) = 0.000001

EXAMPLE 2 QUANTIFICATION OF CD34+ CELLS IN PERIPHERAL BLOOD

BY AMPLIFICATION OF CD34 MRNA

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 NaHCθ3 / 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. The reaction was then chilled on ice for 3 minutes, at which time 6 ul of 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. 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. At the conclusion of the first round of amplification, 1 ul of product was removed from each tube and added to a fresh tube containing 1 nmole each of primers CD34-3 and CD34-4, in addition to Taq polymerase, 10 X synthesis buffer, and water in the proportions described above. A second round of PCR was performed, consisting of 20 cycles, each cycle consisting of 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 30 seconds. Finally, 25 ul of the product of each tube was analyzed by agarose gel electrophoresis for the presence of the expected amplification product, a 140 base pair DNA fragment.

The results of this experiment are presented in Table 3. Briefly, when the number of cells per well is 1000 or less, assay response is linear, giving an estimate of 1000 CD34+ cells per million mononuclear cells in peripheral blood. However, when the number of cells per well is increased above 1000 cells, the assay response plateaus, leading to an underestimate of the number of CD34 cells and indicating that there is more than one CD34+ cell per well at densities greater than 1000 cells per well.

EXAMPLE 3 DETECTION AND QUANTIFICATION OF TUMOR CELLS IN PERIPHERAL BLOOD

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. In the present example, 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. At the conclusion of the second round of amplification, 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). These results indicate that as few as 100 tumor cells can be detected among 100,000 non-tumor cells using RT-PCR on unextracted mRNA. Hence, 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.

Briefly, 1 ml of the mock tumor cell suspension was incubated with 1.25 ul of NRLu-10 (10 mg/mL) for 30 minutes twice, followed by a second incubation with biotinylated rat anti-mouse IgG (4ug/mL on ice for 30 minutes). The mixture was then passed through a pliable column containing 4 mL of avidin-conjugated Biόgel P60 equilibrated in PBS/BSA. After washing with PBS to remove unbound (non-tumor) cells, the specifically bound (tumor) cells were eluted by gently squeezing the column.

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.

A signal was detected in five of five wells containing 15,000 cells, enriched as described above, and in the wells containing 10,000, 1,000, and 100 cells (one well at each cell number). No signal was detected in wells containing only 10 cells or 1 cell. These results demonstrate that the limit of detection of unextracted mRNA by RT-PCR can be increased 1000-fold by a prior immunoenrichment step.

TABLE 1 PRIMER DNA SEQUENCES

Primer SEQ. ID NO: Sequence

Yl 1 TCCACTTTATTCCAGGCCTGTCC

Y2 2 TTGAATGGAATGGGAACGAATGG

Y3 3 ATTACACTACATTCCCTTCCA

Y4 4 AGTGAAATTGTATGCAGTAGA

CD34-1 5 CACCCTGTGTCTCAACATGG

CD34-2 6 GGAGATGTTGCAAGGCTAG CD34-3 7 AGGCCAGAACAAACATCACA

CD34-4 8 GAATAGCTCTGGTGGCTTGC

ER-1 9 CATAACGACTATATGTGTCCAGCC

ER-2 10 AACCGAGATGATGTAGCCAGCAGC

ER-3 11 AAAAACAGGAGGAAGAGCTGC

ER-4 12 ATCATCTCTCTGGCGCTTGT

TABLE 2 NUMBER OF FETAL (MALE) CELLS IN A MATERNAL BLOOD SPECIMEN

Cells/well Total Cells in 20 Number of Fetal Cells / Wells Positive Wells Maternal Cells

20,000 400,000 0 0

50,000 1,000,000 1 1/106

100,000 2,000,000 2 l/lθ6

200,000 4,000,000 4 l/lθ6

TABLE 3 NUMBER OF CD34 CELLS IN PERIPHERAL BLOOD

Cells/well Total cells in 10 Number of CD34+ Cells / Wells Positive Wells MNC

100 1000 1 1000/106

500 5000 5 1000/106

1000 10000 10 1000/106

5000 50000 10 200/106

10000 100000 10 100/106

From the foregoing, it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims. SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANTS: Hall, Jeffrey M. Molesh, David A.

(ii) TITLE OF INVENTION: METHODS FOR QUANTIFYING THE NUMBER OF CELLS CONTAINING A SELECTED NUCLEIC ACID SEQUENCE IN A HETEROGENOUS POPULATION OF CELLS

(iii) NUMBER OF SEQUENCES: 12

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Seed and Berry

(B) STREET: 6300 Columbia Center, 701 Fifth Avenue

(C) CITY: Seattle

(D) STATE: Washington

(E) COUNTRY: U.S.A.

(F) ZIP: 98104

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentln Release #1.0, Version #1.25

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: McMasters, David D.

(B) REGISTRATION NUMBER: 33,963 (C) REFERENCE/DOCKET NUMBER: 200072.415

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (206) 622-4900

(B) TELEFAX: (206) 682-6031

(C) TELEX: 3723836 SEEDANBERRY

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 23 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

TCCACTTTAT TCCAGGCCTG TCC 23

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 23 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

TTGAATGGAA TGGGAACGAA TGG 23 (2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

ATTAC ACTAC ATTCCCTTCC A 21

(2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

AGTGAAATTG TATGC AGTAG A 21

(2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:

CACCCTGTGT CTCAACATGG 20

(2) INFORMATION FOR SEQ ID NO:6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:

GGAGATGTTG CAAGGCTAG 19

(2) INFORMATION FOR SEQ ID NO:7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:

AGGCCAGAAC AAACATCACA 20 (2) INFORMATION FOR SEQ ID NO:8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:

GAATAGCTCT GGTGGCTTGC 20

(2) INFORMATION FOR SEQ ID NO:9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:

CATAACGACT ATATGTGTCC AGCC 24

(2) INFORMATION FOR SEQ ID NO: 10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:

AACCGAGATG ATGTAGCCAG CAGC 24

(2) INFORMATION FOR SEQ ID NO:l 1 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l 1 :

AAAAACAGGA GGAAGAGCTG C 21

(2) INFORMATION FOR SEQ ID NO: 12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:

ATCATCTCTC TGGCGCTTGT 20

Claims

1. A method of increasing the number of copies of a selected ribonucleic acid sequence in a sample, comprising:

(a) disrupting cells in the sample to expose ribonucleic acids; and

(b) amplifying a selected ribonucleic acid sequence from said exposed ribonucleic acids, such that said ribonucleic acid sequence is increased in number, and wherein, between the steps of disrupting and amplifying, no step of purification is performed.

2. A method of increasing the number of copies of a selected ribonucleic acid sequence in a sample, comprising:

(a) disrupting viruses in the sample to expose ribonucleic acids; and

3. The method of claim 1 or 2 wherein said selected ribonucleic acid sequence is amplified by polymerase chain reaction.

4. The method of claim 1 or 2 wherein said selected ribonucleic acid sequence is amplified by isothermal amplification.

5. The method of claim 1 or 2 wherein said selected ribonucleic acid sequence is amplified by Qβ-replicase amplification.

6. The method of claim 1, further comprising, prior to the step of disrupting, fractionating said sample into subpopulations of cells.

7. The method of claim 1 wherein said cells are disrupted by heating the cells.

8. The method of claim 1 wherein a ribonuclease inhibitor is not added during the steps of disrupting or amplifying.

9. The method of claim 1 wherein said cells are a CD 34⁺ cells.

10. The method of claim 1 wherein said cells are tumor cells.

11. The method of claim 1 wherein said cells are virally-infected cells.

12. The method of claim 1 wherein said cells are fetal cells.

13. A method of 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:

(a) determining the total number of cells in said 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.

14. The method of claim 13 wherein the frequency of cells containing the selected nucleic acid sequence is determined by the equation ∑jkj / (N- ∑ikjCj/2).

15. The method of claim 13 wherein said selected nucleic acid sequence is amplified by polymerase chain reaction.

16. The method of claim 13, further comprising, prior to the step of determining, fractionating said suspension into subpopulations of cells.

17. The method of claim 13 wherein said cells are disrupted by heating the cells.

18. The method of claim 13 wherein said cells are CD 34+ cells.

19. The method of claim 13 wherein said cells are fetal cells.

20. A method of quantifying the number of cells containing a selected ribonucleic acid sequence in a suspension of cells heterogenous with respect to the presence of said sequence, comprising:

(a) determining the total number of cells in said suspension;

(b) disrupting cells within the suspension to expose ribonucleic acids;

(c) amplifying a selected ribonucleic acid sequence from said exposed ribonucleic acids, such that said 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 said suspension may be determined.

21. The method of claim 20 wherein said selected ribonucleic acid sequence is amplified by polymerase chain reaction.

22. The method of claim 20 wherein said selected ribonucleic acid sequence is amplified by Qβ-replicase amplification.

23. The method of claim 20, further comprising, prior to the step of determining, fractionating said suspension into subpopulations of cells.

24. The method of claim 20 wherein said cells are disrupted by heating the cells.

25. The method of claim 20 wherein a ribonuclease inhibitor is not added during the steps of disrupting or amplifying.

26. The method of claim 20 wherein said cells are CD 34⁺ cells.

27. The method of claim 20 wherein said cells are fetal cells.