WO1997020951A1 - Titrage rapide concernant l'estimation de transferts geniques dans des cellules - Google Patents

Titrage rapide concernant l'estimation de transferts geniques dans des cellules Download PDF

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WO1997020951A1
WO1997020951A1 PCT/US1996/019495 US9619495W WO9720951A1 WO 1997020951 A1 WO1997020951 A1 WO 1997020951A1 US 9619495 W US9619495 W US 9619495W WO 9720951 A1 WO9720951 A1 WO 9720951A1
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seq
cell
sequence
pcr
primers
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PCT/US1996/019495
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William G. Kerr
Christopher J. Gerard
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Systemix, Inc.
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Priority to AU14613/97A priority Critical patent/AU1461397A/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • 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
    • C12Q1/686—Polymerase chain reaction [PCR]
    • 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
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage

Definitions

  • This invention relates to assays for detecting gene transfer into cells, for example, determining gene transfer into cells transfected with a known vector such as a Moloney-Murine leukemia virus (MMLV) -based vector.
  • MMLV Moloney-Murine leukemia virus
  • Retrovirus vectors are used to achieve stable, long-term expression of a foreign gene in a host cell.
  • Commonly utilized retroviral vectors include Moloney murine leukemia virus (MMLV) , Harvey murine sarcoma virus, murine mammary tumor virus, and Rous Sarcoma virus. These vectors can transfer or include a sequence for a selectable marker so that transduced cells may be identified and generated.
  • MMLV Moloney murine leukemia virus
  • Harvey murine sarcoma virus Harvey murine sarcoma virus
  • murine mammary tumor virus and Rous Sarcoma virus.
  • Rous Sarcoma virus Rous Sarcoma virus.
  • the gene of interest is cloned into a retrovirus vector that lacks most viral genes. The gene is usually expressed under the control of the strong viral promoter in the LTR.
  • the recombinant plasmid is transfected into a special packaging cell line that harbors an integrated provirus.
  • Packaging cell lines include, for example, ⁇ 2, PA317, and PA12.
  • the provirus is designed such that, although it produces all the proteins required to assemble infectious viruses, its own RNA cannot be packaged into a virus.
  • the packaging cell ⁇ produce empty virions, since no genome is packaged.
  • a retroviral vector containing a ⁇ packaging sequence is introduced into the packaging cells, RNA produced from the recombinant virus is packaged into infectious viral particles.
  • the virus stock released from the packaging cells thus contain only the recombinant virus.
  • the virus can be used to infect a host cell, resulting in the integration of the viral genome and the stable production of the foreign gene product (Armentano et al. (1987) J. Virol. 61:1647-1650; Bender et al.
  • PCR polymerase chain reaction
  • the basic principle of PCR is the exponential replication of a DNA sequence by successive cycles of primer extension.
  • the extension product of one primer when hybridized to another primer, becomes a template for the synthesis of another nucleic acid molecule.
  • the primer template complexes act as substrate for DNA polymerase which, in performing its replication function, extends the primers.
  • thermostable DNA polymerases e.g., such as that isolated from Thermus aquaticus or Taq DNA polymerase.
  • Other amplification methods currently under development include LCR (ligase chain reaction, BioTechnica International) which utilizes DNA ligase, and a probe consisting of two halves of a DNA segment that is complementary to the sequence of the DNA to be amplified; enzyme QB replicase (Gene-Trak Systems) and a ribonucleic acid (RNA) sequence template attached to a probe complementary to the DNA to be copied which is used to make a DNA template for exponential production of complementary RNA; and NASBA (nucleic acid sequence-based amplification, Cangene Corporation) which can be performed on RNA or DNA as the nucleic acid sequence to be amplified (Gemen et al. (1993) AIDS 7:5107-5110) .
  • LCR ligase chain reaction, BioTechnica International
  • QB replicase Gene-Trak Systems
  • RNA ribon
  • PCR is an extremely sensitive assay which has many uses in retroviral-mediated gene transfer protocols (Morgan et al. (1990) Human Gene Therapy 1:135) . PCR is routinely used to estimate gene transfer following retroviral transduction of cells. Typically, the PCR target is specific to the introduced gene, i.e., the presence of an introduced marker or therapeutic gene is used to detect transduction.
  • the present invention provides rapid and sensitive gene transfer assays applicable to gene transfers accomplished without regard to the specific vector used.
  • One non-limiting example of the invention provides an assay for the detection of gene transfer by Moloney- Murine leukemia virus (MMLV) -based vectors.
  • MMLV Moloney- Murine leukemia virus
  • the invention is directed to a method for detecting gene transfer by a vector which includes a gene and a sequence unique to that vector, such as a MMLV-based vector, by (1) obtaining a population of cells, (2) lysing the cells to obtain a cell lysate, (3) contacting the cell lysate with a pair of oligonucleotide primers corresponding to a segment of unique sequence, e.g. unique MMLV DNA, under conditions in which a DNA segment complementary to the primers is amplified, and (4) detecting the amplified DNA sequence, which is indicative of gene transfer.
  • a vector which includes a gene and a sequence unique to that vector, such as a MMLV-based vector
  • the method of the invention is sufficiently sensitive to detect gene transfer in a cell population having a single transduced cell in a population of cells.
  • the method is used to detect gene transfer in a single cell colony.
  • the method may also be used to detect gene transfer in a bulk cell population including transduced and non-transduced cells.
  • a colony of hematopoietic stem/progenitor cells is analyzed for gene transfer.
  • the oligonucleotide primer pair used for detection amplifies a sequence unique to a retroviral vector packaging gene and not present in the host cells, preferably the PCR amplifies a packaging sequence conserved in MMLV-based vectors.
  • the packaging sequence is the ⁇ packaging sequence.
  • the oligonucleotide primers amplifying the ⁇ packaging sequence are selected from the primers having the sequences of SEQ ID NO: 1-8. More preferably, the primers used are those having the sequence SEQ ID NO:l/SEQ ID NO:8 and SEQ ID NO:2/SEQ ID NO:8. Most preferably, the primers used have the sequence of SEQ ID NO:l and SEQ ID NO:8.
  • the method of the invention is used with more than one pair of oligonucleotide primers.
  • a second pair of primers may be used which correspond to a second marker of interest.
  • the second pair of primers correspond to 3-globin DNA as an internal control for the PCR reaction. The use of multiple primer pairs to other markers of interest are included in the method of the invention.
  • the amplified PCR product may be detected in a number of ways, including by gel electrophoresis and Southern blots.
  • the amplified PCR product may also be detected by incorporation of a label into the PCR product, including radioactive, fluorescent, chemiluminescent, etc.
  • the amplified PCR product is measured by the amount of fluorescence generated in the PCR reaction mixture.
  • a fluorescent oligonucleotide probe complementary to a segment of the amplified PCR product is included with the first primer pair, and the level of fluorescence in the PCR reaction mixture is proportional to the concentration of amplified PCR product.
  • the invention features oligonucleotide primers useful in the method of the invention for detection of a MMLV-based retroviral vector transduced cell.
  • the primers of the invention include those shown in Table 1 (SEQ ID NO:l-8) .
  • Preferred primers are those having the sequence of SEQ ID NO:l, SEQ ID NO:2, and SEQ ID NO:8.
  • the most preferred primer pair sequence are those having the sequence of SEQ ID NO:l and SEQ ID NO:8.
  • the invention features a kit for determining gene transfer in a cell, providing buffers and primers suitable for performing the assay of the invention.
  • a kit for the analysis of transduction frequency in single methylcellulose colonies includes a 96-well plate containing PCR lysing buffer, PCR reagents (sterile water, 10 x buffer, dNTPs, a pair of PCR primers, polymerase, and optionally, control primers such as 3-globin primers) , detection reagents (probe, either fluorescently labeled or 32 P-ATP labeled) , and instructions.
  • a kit for the estimation of transduction frequency in a bulk assay contains bulk standards (premade cell lysates) , PCR reagents as above, and instructions for use.
  • the assay of the present invention is convenient and simple, and it is universal in that it can be used without regard to the specific gene being transferred, thus lending it to formulation in kits without the need to develop unique primers and to optimize conditions for use of those primers.
  • Fig. 1 is a sequence comparison of the extended packaging signal ( ⁇ ) of several Moloney-based retroviruses.
  • a "•” indicates that the nucleotide is unchanged from the Moloney sequence, and a "-" indicates that a nucleotide has been deleted. Nucleotide substitutions are as indicated.
  • Both the upstream primer (Fl) and the downstream primer (B5) are underlined.
  • Fig. 2A is an ethidium bromide stained agarose gel showing a representative XP outcome (excessive non ⁇ specific bands) obtained with primers F2/B1.
  • Lanes 1, 2, 3, and 4 are the result of amplifying 250 cell equivalents of ⁇ l ( ⁇ positive) , KG-la ( ⁇ negative) , SV29 ( ⁇ positive) , and Sp6- ⁇ ( ⁇ positive) cells; "-" is a negative PCR buffer control.
  • An arrow indicates the size of the expected PCR product based on the sequence of the MMLV sequence (see Fig.l).
  • Fig. 2B is an ethidium bromide stained agarose gel showing a representative IP outcome (insufficient PCR product formation) obtained with primers F2/B4. Lanes 1, 2, 3, and 4 are as described in the legend to Fig. 2A.
  • Fig. 2C is an ethidium bromide stained agarose gel showing a representative NP outcome (no PCR product formation) with LXSN/LNL6-based vectors F3/B1. Lanes 1, 2, 3, and 4 are as described in the legend to Fig. 2A.
  • Fig. 2D is an ethidium bromide stained agarose gel showing a representative OK outcome (PCR product of the expected size with little or no non-specific bands) obtained with vectors F1/B5. Lanes 1, 2, 3, and 4 are as described in the legend to Fig. 2A.
  • Fig. 3A is an ethidium bromide stained agarose gel showing the absolute sensitivity of the primers F1/B5 in a 40 cycle PCR.
  • the primer pair was tested against a 2- fold dilution series of ⁇ l ( ⁇ positive) cell lysates at 200, 100, 50, 25, 12.5, 6.3, 3.1, 1.6, and 0.8 cell equivalents.
  • "-" is a negative PCR buffer control;
  • "L” is a 100 bp ladder (500, 400, 300, 200, 100, 50 bp; an arrow indicates the position of the 134 bp ⁇ PCR product.
  • Fig. 3B is an ethidium bromide stained agarose gel showing the detection capability of the F1/B5 ⁇ primers in a 30 cycle bulk PCR assay.
  • Bulk PCR controls were made by adding KG-la cells ( ⁇ negative) to a two-fold dilution series of SV29 ( ⁇ positive) cells.
  • Lysates "A” through “F” contain 10, 5, 2.5, 1.3, 0.6, and 0.3% SV29 cells, respectively. Lysate " ⁇ " contains 100% KG-la cells.
  • the total number of cell equivalents per PCR is 2,500.
  • "L” is a 100 bp ladder (500, 400, 300, 200, 100, 50 bp) ; an arrow indicates the position of the 134 bp f PCR product.
  • Fig. 4A is an ethidium bromide stained agarose gel of ⁇ l ( ⁇ positive) and KG-la ( ⁇ negative) colonies pooled and processed for the PCR having a ratio of 4:1, 3:2, and 2:3 KG-la x ⁇ l colonies.
  • Fig. 4B is an ethidium bromide stained agarose gel showing the effect of residual methylcellulose medium on PCR sensitivity in a lysate consisting of ⁇ l cells in a background of KG-la cells.
  • Samples in lanes 1-6 all contain 375 KG-la cell equivalents.
  • Samples in lanes 1 & 4, 2 & 5, and 3 & 6 contain 125, 62.5, and 12.5 ⁇ l cell equivalents, respectively.
  • Samples in lanes 4-6 were processed with the addition of methylcellulose medium, while samples in lanes 1-3 were processed without the addition of methylcellulose medium.
  • Fig. 4B is an ethidium bromide stained agarose gel showing the effect of residual methylcellulose medium on PCR sensitivity in a lysate consisting of ⁇ l cells in a background of KG-la cells.
  • Samples in lanes 1-6 all contain 375 KG-la cell equivalents.
  • 5A is an ethidium bromide stained agarose gel of direct lysate PCR from single methylcellulose colonies of ⁇ l ( ⁇ positive) and KG-la ( ⁇ negative) cells using only the ⁇ primers F1/B5 (top row) , or the ⁇ and 0-globin primers (multiplex PCR - bottom row) .
  • An arrow indicates the position of the 134 bp ⁇ PCR product and/or the 250 bp 3-globin product.
  • " ⁇ Buffer” is a negative PCR control of the buffer used to process the colonies, and "L” is a 100 bp ladder.
  • Fig. 5B is a Southern blot of the agarose gel of Fig. 5A hybridized to the ⁇ PCR product internal probe, BK29.
  • Fig. 6A is an ethidium bromide stained agarose gel of eighty 14-day methylcellulose colonies derived from hematopoietic stem/progenitor cells growing in methylcellulose medium which were harvested and directly lysed in the 96-well format; a sample of the lysate from each colony was amplified using the F1/B5 ⁇ primers (#'s 1-80) , positive colonies are numbered above the lane; also amplified in the same plate were 10 hematopoietic colonies of Mock transduced cells ("Mocks"); positive controls, (SV29) ("+”) ; and negative PCR controls: "-” (PCR master mix without lysing buffer; "Buf” (PCR master mix with lysing buffer) ; "L” is an 100 bp ladder, and "+” is a positive control (134 bp band) loaded on each side of the gel. For each panel an arrow indicates the location of the F1/B5 ⁇ PCR product.
  • Fig. 6B is the Southern blot of the agarose gel described in Fig. 6A above, hybridized with the internal probe BK29 (B) .
  • Fig. 7A is a representation of the results obtained by ethidium bromide staining (slanted bars) and fluorescence (solid circle) in a 96 well plate.
  • Samples #1-80 represent colonies derived from hematopoietic stem/progenitor cells exposed to retroviral supernatant
  • samples #81-90 are colonies of Mock-transduced cells
  • "+*' represents single SV29 ( ⁇ positive) colonies
  • "KG-la” is a single ⁇ negative colony
  • Buffer” is a negative PCR control.
  • Fig. 7B is a graph showing the results of a direct comparison of an ethidium bromide stained gel and the TaqManTM assay for a subset of the samples of Fig. 7A (#55-90) .
  • the dashed line represents the threshold ⁇ RQ above which samples are positive at the 99.95% confidence level.
  • TaqManTM positive samples are indicated by shaded bars.
  • the present invention features a rapid and highly sensitive assay for detecting gene transfer into cells, for example, Moloney-Murine leukemia virus (MMLV) -based gene transfer.
  • MMLV Moloney-Murine leukemia virus
  • the assay of the present invention is widely applicable since the majority of retroviral vectors used in current gene transfer protocols are MMLV- based (Jolly (1994) Cancer Gene Therapy 1:51) and the assay is specific to all known MMLV-based vectors.
  • references to “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise.
  • references to “a cell” or “a cell colony” include mixtures of such cells or cell colonies.
  • Gene transfer is meant the insertion of a exogenous fragment of nucleic acid into a cell and the subsequent recombination of part or all of the exogenous DNA into the cell's genome. Gene transfer includes the insertion of one or more exogenous nucleotide sequences into a cell by transduction or transfection.
  • the nucleic acid transferred may include all or part of a gene of interest, for example, a nucleic acid sequence encoding a protein or a therapeutic gene.
  • the nucleic acid transferred into a cell may be a therapeutic gene which confers resistance to chemotherapeutic agent, thereby protecting the cells and their progeny from chemotherapeutic treatment.
  • a therapeutic gene may be transferred which confers resistance to an infectious agent.
  • Therapeutic genes also includes antisense or ribozyme sequences, which interfere with viral infection or replication in the transformed cell.
  • the introduced nucleic acid sequences may serve as "decoys", e.g., products which bind essential viral proteins, thereby interfering with the normal viral life cycle and inhibiting replication.
  • detection of gene transfer is meant a method which is able to determine the presence of an exogenous fragment of DNA in a cell.
  • Means of detection include amplification of the specific DNA fragment of interest, e.g. , MMLV DNA, and visualization or quantitation of the exogenous DNA fragment by gel electrophoresis, or by labelling of the amplified PCR product, e.g., with radioactivity, fluorescence, chemiluminescence, etc.
  • amplifica ion is meant any method by which a specific segment of nucleic acid is preferentially synthesized in a detectable amount.
  • PCR is used to amplify a segment of DNA complementary to the vector ⁇ packaging sequence.
  • the method of the invention may also be used with other methods of amplification currently available or under development.
  • Amplification methods suitable for use in the method of the invention include the following.
  • packaging sequence is meant a gene or fragment of a gene encoding proteins required for packaging of viral RNA into infectious virus particles.
  • Packaging sequence ⁇ include a wide number of vector- specific packaging genes and portions of genes known to the art.
  • the packaging sequences useful in the invention include genes and gene fragments of retroviral packaging systems, such as the PA12, ⁇ am, PA317, ⁇ CRIP, DAN, HX, Gibbon Ape Leukemia-based, VSVg, and kat systems (Jolly (1994) Cancer Gene Therapy 1:51-64).
  • a preferred packaging nucleotide sequence is a portion of the MMLV ⁇ packaging sequence.
  • DNA polymerase uses single-stranded DNA as a template for the synthesis of a complementary new strand; reverse transcriptase uses RNA as a template to produce a complementary new strand (cDNA) .
  • cDNA complementary new strand
  • These single-stranded DNA templates can be produced by simply heating double- stranded nucleic acids to temperatures near boiling. A small section of double-stranded template is required to initiate (“prime") synthesis. Therefore, the starting point for nucleic acid synthesis can be specified by supplying an oligonucleotide primer that anneals to the template at that point.
  • the DNA polymerase can be directed to synthesize a specific region of DNA.
  • Both DNA or RNA strands can serve as templates for synthesis provided an oligonucleotide primer is supplied for each strand.
  • a pair of primers are chosen to flank a region of the nucleic acid segment to be amplified so that the newly synthesized strands of DNA, starting at each primer, extend beyond the position of the primer on the opposite strand. Therefore, new primer binding sites are generated on each newly synthesized DNA strand.
  • the reaction mixture is again heated to separate the original and newly synthesized strands, which are then available for further cycles of primer hybridization, DNA synthesis, and strand separation.
  • the net result of a PCR is that by the end of n cycles, the reaction contains a theoretical maximum of 2 n double- stranded DNA molecules that are copies of the DNA sequence between the primers, i.e., amplification of the selected DNA sequence.
  • the methods of the present invention may be used to develop assays universal for any vector used to deliver exogenous genetic sequences to a target cell.
  • the methods of the present invention may be used to develop assays universal to any vector including, for example, adenovirus vectors, adeno-associated virus vectors, MMLV vectors, Harvey sarcoma virus (HSV) vectors, and the like.
  • the assay is specific for MMLV vectors.
  • the MMLV provirus genome was examined for regions which would be found in most or all MMLV-based vectors, and which would not be found in the target cell genome.
  • Any vector-specific sequence may be used in the present invention as long as it is retained in the vector backbone of any given retroviral vector, and its amplification by PCR does not conco itantly amplify sequences from the host genome of transduced cells. Thus, it is desirable to use as target sequences for amplification, vector sequences required for gene delivery or expression and, therefore, not likely to be replaced or modified in any given vector.
  • the ⁇ packaging sequence is a 300-350 bp region downstream of the 5' LTR which extends into the gag coding sequence (Bender et al. (1987) J. Virol. 61:1639) . This region is required for efficient packaging of retroviral genomic RNA molecules.
  • retroviral vectors which contain 3' deletions in this region (Armentano et al. (1987) J. Virol. 16:1647) .
  • Alternative vector sequences may be used in the present invention, including LTRs and tRNA primer binding sites, although care must be taken to ensure the sequences are universal to all known vectors since some of these regions are modified occasionally in different vector backbones. LTR sequences are less desirable for use in the present invention since there appear to be many similar sequences within the human genome.
  • the sequences detected in the present invention are from the ⁇ packaging region.
  • PCR primers that amplify regions of the ⁇ packaging sequence from various MMLV retroviral vectors were selected (shown in Table 1) (SEQ ID NO: 1-8) , and candidate primer combinations were tested in exploratory PCR analyses to identify an ideal universal primer pair (Example 1) .
  • a primer pair is designed to be universal for all retroviral backbones, producing a band of the expected size with little or no spurious background bands; by comparison, non-transduced cells should be free of the expected PCR product.
  • multiple primer combinations are designed to amplify the vector sequence of interest. Each primer combination is used, in separate PCR reactions, to amplify DNA sequences from cells, typically human cells having no vector sequence ⁇ , to test for non-specific background bands.
  • Each primer combination is also tested in PCR reactions with cells carrying different vector backbones to test the universality of the primer pairs. Sensitivity of potential primer pairs is tested in mixtures of cells having different proportions of vector- positive and vector-negative cells.
  • the primer combination F1/B5 (SEQ ID NO-.i/SEQ ID NO:8) was found to meet the criteria for a universal primer pair for MMLV-based retroviral vectors.
  • the F2/B5 primer pair was also shown to meet the criteria of amplifying all MMLV-based vectors tested without spurious background bands. Sensitivity and detection level studies (Example 2) established that the F1/B5 ⁇ primer combination could routinely detect transduction efficiencies of down to 0.6% positive cells in a semi- quantitative assay (2,500 cells total).
  • the primers identified by the methods of the present invention can be used to detect gene transfer into target cells of any tissue type.
  • the assays will be used to detect gene transfer into human cells.
  • Many primary human cell types are targets for gene therapy and can be assayed in the present invention including hematopoietic cells, neural cells, fibroblasts, liver cells, and tumor cell ⁇ .
  • the vector backbone sequences will not be expressed in the transduced cells and, therefore, the integrated DNA sequences will be detected by DNA PCR.
  • the PCR assay may be used to detect vector sequences in the transduced cells anytime from immediately after the transduction to several days or weeks after the transduction, as long as the cells are viable, or indefinitely if the cells are cryopreserved.
  • Transduction may be assessed by PCR on a bulk cell population after transduction or on colonies grown from single cells.
  • retroviral vectors it is preferable to allow the target cells to cycle, allowing integration of the retroviral vector into the host cell DNA, permitting a more accurate measure of transduction frequency.
  • a sample portion of the transduced cell population may be plated in a semi-solid media such as methylcellulose.
  • the resulting colonies each representing single cells from the original transduction mix, can be picked and either pooled or analyzed individually for presence of the vector sequences.
  • methylcellulose medium which can contaminate the lysate during the processing of hematopoietic stem/progenitor cells, can interfere with the PCR (Bregni et al. (1992) J. Clin. Invest. 82:1017) .
  • Residual methylcellulose medium can reduce the sensitivity of the PCR when using alternative primer sites to detect transduction (e.g., lacZ, neomycin targets; data not shown) .
  • An experiment was conducted to determine the effect of residual methylcellulose media on ⁇ PCR sensitivity in a lysate consisting of ⁇ l cell ⁇ ( ⁇ positive) in a background of KG-la cells ( ⁇ negative) (Example 3).
  • Example 4 describes a single colony direct lysi ⁇ PCR protocol which can be used to detect transduced human cells in clonogenic methylcellulose assays. Single colonies of clonogenic cells growing in methylcellulose medium (Brandt et al. (L1988) J. Clin. Invest. 82:1017) are routinely analyzed by PCR (Dick et al. (1991) Blood 78:624) . Cassel et al.
  • the single-colony direct lysate technique described here involves no rinsing of the colonies, and the lysate i ⁇ used directly in the PCR reaction without protein extraction or DNA precipitation.
  • PCR was performed with ⁇ primers or a mix of ⁇ and /3-globin primers as an internal control. All ⁇ l and SV29 colonies were positive for both ⁇ and y3-globin, and KG-la colonies were positive only for 3-globin. Po ⁇ itive colonies detected by ethidium bromide stained agarose gels were confirmed by Southern blot analysis (Figs. 5A and 5B) .
  • Example 5 describes the determination of gene transfer in transduced human CD34 + Thy-1 + Lin " hematopoietic stem cells isolated from cytokine mobilized human peripheral blood.
  • the cells were sorted (Becton Dickinson, FACStar Plus) , transduced with a Moloney-based LMTNL retroviral vector, and plated in methylcellulose medium to assess transduction of the stem/progenitor cell population.
  • Eighty 14-day methylcellulo ⁇ e colonies were harvested individually and lysed in the 96-well format; a sample of the lysate from each colony was amplified using the F1/B5 ⁇ primers.
  • Fig. 6A shows the positive samples detected by ethidium bromide staining. Southern blots of the positive samples probed with the internal probe BK29 (shown in Fig. 6B) verified the specificity of the ⁇ positive samples.
  • the direct lysate technique described in Example 5 permits the effective processing of a large number of samples in the conventional and convenient 96-well format.
  • An important advantage of single-colony direct lysate PCR analysis carried out in a 96-well format is the reduction in processing time. Methylcellulose colonies are harvested directly into the PCR lysing buffer, thereby omitting the PBS rinse step. Decreased handling of the lysate further reduces the potential for contamination and sampling error due to multiple pipetting steps. These time-saving steps allow proces ⁇ ing of greater number ⁇ of ⁇ amples, while utilizing the 96-well format permits the use of multichannel pipetters to expedite the addition of reagents, lysates, etc.
  • the PCR products of the present invention may be detected by any method known in the art including, for example, by ethidium bromide gel electrophoresis and/or Southern blot analysis.
  • any method known in the art including, for example, by ethidium bromide gel electrophoresis and/or Southern blot analysis.
  • an alternative detection system such as a fluore ⁇ cence-based po ⁇ t-PCR detection assay, which can take advantage of the 96-well format of the PCR, would be a step forward in expediting an already improved and efficient assay.
  • the TaqManTM fluorescent system for post-PCR analysis was found to be fast, convenient, and a ⁇ sen ⁇ itive a ⁇ an ethidium-bromide ba ⁇ ed detection ⁇ ystem, as determined by comparison with detection of the PCR product amplified with the F1/B5 ⁇ primers (Example 6) . Furthermore, the specificity of the internal probe makes a hybridization assay of PCR product unnecessary.
  • the TaqManTM system of PCR product detection uses the 5' ⁇ 3' exonuclease activity of Thermus aquaticus DNA polymerase (Holland et al. (1991) Proc. Natl. Acad. Sci.
  • Post-PCR product analysis merely involves placing the 96-well tray/retainer assembly containing the open PCR reaction tubes into the specially designed fluorescent plate reader, and within minutes, the spectrophotometer reads the fluorescence signal from all 96 wells and summarizes the results in spreadsheet format. Since the assay does not consume any part of the reaction volume, the samples can be saved and used for other purposes (e.g. , gel verification) .
  • PCR reaction tubes be designed such that the fluore ⁇ cence can be read directly through the cap, in which case, data acquisition would not require opening the PCR reaction tube, and the potential for PCR product contamination of subsequent amplifications would be significantly reduced.
  • the detection capability of the TaqManTM assay depends largely on the design of the fluorescently-labeled probe and the efficient amplification of the target DNA with the PCR primer pair.
  • Guideline ⁇ for designing an appropriate fluorescently- labeled probe are provided in the manufacturer's instructions (Perkin-Elmer) .
  • the internal probe i ⁇ preferably that identified as SEQ ID NO: 11.
  • Table 1 shows the sequence and position of the 8 candidate ⁇ primers selected for testing - 3 upstream and 5 downstream oligonucleotides. Their position is based on the ⁇ extended packaging signal of the MMLV (Fig. 1) . All sequences are referenced according to the sense strand. TABLE 1. Candidate PCR Pri er ⁇ Used to Amplify a Reg:Lon in the ⁇ Packaging Signal.
  • the human acute myelogenous leukemia CD34 + cell line, KG-la (Koeffler et al. (1980) Blood 56:265), was used as a negative control for the ⁇ primers.
  • the KG-la/ ⁇ l cell line (hereafter referred to as " ⁇ l” ) is a KG-la clone transduced with MFG-Iac-- " and selected by the FACS-Gal procedure (Nolan et al. (1988) Proc. Natl. Acad. Sci. USA 85:2603).
  • KG-la/SV29 cell line (hereafter referred to as "SV29") is a KG-la clone transduced with the MMLV-SV-NLS-lacZ retrovirus which was also cloned via FACS-Gal (Bonnerot et al. (1987) Proc.
  • the Sp6- ⁇ cell line (Hwu et al. (1993) J. Exp. Med. 178:361) is a producer cell line (PA317) containing the LXSN retroviral vector (Miller & Rosman (1989) BioTechniques 7:980) which is a derivative of the LNL6 retroviral vector (Bender et al. (1987) supra) .
  • PCR PCR Conditions. All PCRs were conducted in a total volume of 25-28 ⁇ l using the Perkin Elmer/Applied Biosy ⁇ tems 9600 thermal cycler. Unless otherwise stated, the 40 cycle PCR is as follows: initial denaturation of 30 seconds at 95°C followed by 40 cycles of 10 second denaturation at 95°C, 15 seconds annealing at 64°C, and 15 seconds extension at 72°C. The protocol finishes with a final 5 minute extension at 72°C and a 4°C soak. PCR product was visualized by loading 15 ⁇ l on a 4% NuSieve 3:1 agarose gel (FMC Bioproducts) containing ethidium bromide (Sigma) .
  • FMC Bioproducts NuSieve 3:1 agarose gel
  • PCR cell lysing buffer consists of IX PCR buffer (as above) with the addition of 0.5% NP-40 (Sigma), 0.5% Tween-20 (Sigma), and 0.91 mg/ml of proteinase K (Boehringer Mannheim) .
  • results A complete matrix of 15 possible primer combinations were investigated for their ability to amplify ⁇ packaging sequence DNA. Results are summarized in Table 2 and representative examples of each possible outcome are shown in Fig ⁇ . 2A-2D: XP, exce ⁇ sive non ⁇ specific product formation; IP, insufficient product formation; NP, no product with an LNL6-based vector; and OK, specific product formation of the expected size, with little or no non-specific products.
  • lanes 1-4 represent 250 cell equivalents of the five cell lysates as described above: ,97 ( ⁇ positive) , KG-la ( ⁇ negative) , SV29 ( ⁇ po ⁇ itive) and Sp6- ⁇ ( ⁇ positive) , respectively; "-" is a negative PCR buffer control. This initial PCR was conducted at an annealing temperature of 55°C in order to permit maximum primer-template annealing.
  • Primer combinations using F3 (SEQ ID NO:3) as the upstream primer, although they produced the expected PCR product with the ,97 and SV29 cell lysates, did not produce a target-specific band with Sp6- ⁇ /KG-la cell lysate ⁇ .
  • the F3 primer has a two nucleotide mismatch with the LNL6 vector ⁇ equence at the crucial 3' end of the oligonucleotide and this difference most likely prevents annealing and extension of the target in the Sp6- ⁇ cell lysate.
  • Example 2 Sen ⁇ itivity and specificity of the F1/B5 ⁇ primers.
  • the absolute sensitivity of the F1/B5 ⁇ primer configuration wa ⁇ determined following the procedure described in Example 1, against a 2-fold dilution series of ⁇ l ( ⁇ positive) cell lysates at 200, 100, 50, 25, 12.5, 6.3, 3.1, 1.6, and 0.8 cell equivalents. "-" designates the negative PCR buffer control. PCR amplification of this dilution series, shown in Fig. 3A, showed that the 40 cycle PCR procedure was able to detect a single transduced cell by amplification of the F1/B5 ⁇ sequence. This result was repeated by performing PCR on single ⁇ l cells deposited by FACS into individual wells of a Terasaki plate.
  • the re ⁇ ult ⁇ verified that the F1/B5 ⁇ primer combination had single cell detection sensitivity in a 40 cycle reaction at a frequency of 50% (4/8; data not shown) .
  • the 250 bp ⁇ - globin PCR product was detectable at a level of 8% (1/12; data not shown) .
  • Lysates contained 10% (lysate A), 5% (lysate B) , 2.5% (ly ⁇ ate C) , 1.3% (ly ⁇ ate D) , 0.6% (lysate E) , and 0.3% (lysate F) SV29 cells, respectively. Lysate "0" contain ⁇ 100% KG-la cell ⁇ . The total number of cell equivalent ⁇
  • Assay Sensitivity To determine the level of detection obtainable with the ⁇ primers when multiple clonogenic colonies growing in methylcellulose are pooled and processed for PCR, ⁇ l and KG-la methylcellulose colonies were pooled and processed according to the following ratio: 4:1, 3:2, and 2:3 KG-la ⁇ l colonies. The five colonies were harvested using a P-10 pipet set at 3.0 ⁇ l, rinsed in 200 ⁇ l of PBS, vortexed and centrifuged for 30 seconds at 10K RPM in a benchtop centrifuge (TOMY) . PBS was removed and 100 ⁇ l of lysing buffer (Example 1) was added.
  • cell lysates were made which simulate the conditions of a pooled clonogenic methylcellulose colony assay using ,97 cells ( ⁇ positive) in a background of KG-la cells ( ⁇ negative) , with and without the addition of methylcellulose medium.
  • Duplicate sets of standard ⁇ were made up which included 500, 250, and 50 ⁇ l cell ⁇ per 1500 KG-la cell ⁇ in 200 ⁇ l of PBS.
  • Sample ⁇ in lane ⁇ 1-6 all contain 375 KG-la cell equivalents; 15 ⁇ l of methylcellulose media was added to one set of standards after the colonies were picked but before the PBS was replaced with PCR lysing buffer. All samples were then processed as above.
  • Example 4 Direct lysate PCR from single methylcellulose colonies in a 96-well format.
  • a repeating pipetter was used to fill 0.2 ml thin- wall PCR tubes with 50 ⁇ l of cell lysing buffer (Example 1) . Tubes were held in a 96-well format by a MicroAmp tray/retainer assembly (Perkin Elmer/Applied Biosystems) . Single clonogenic colonies growing in methylcellulose were harvested using a P-10 pipet set at 3.0 ⁇ l and placed directly into the tubes containing cell lysing buffer; while picking colonies the tubes were kept on ice. Several tubes, or "wells", were reserved for PCR and/or tran ⁇ duction control ⁇ (e.g., Mock transduced colonies) and at least one tube contained lysing buffer only as a negative template control.
  • the plate was then covered and transferred to either a specially designed heating block (USA Scientific Plastics) or a thermal cycler (PE 9600, Perkin Elmer/Applied Biosystems) where it was incubated for one hour at 60°C, followed by a 5- minute incubation at 95°C (10 minutes if heating block is used) .
  • a specially designed heating block USA Scientific Plastics
  • a thermal cycler PE 9600, Perkin Elmer/Applied Biosystems
  • lysates were transferred to a second plate containing the PCR master mix.
  • ⁇ primers were used alone or multiplexed with primers which amplify a region of the human 0-globin sequence, BG1, 5'-CAA-CTT-CAT-CCA-CGT-TCA-CC-3 ' (SEQ ID N0:9); BG2, 5'-GAA-GAG-CCA-AGG-ACA-GGT-AC-3' (SEQ ID NO: 10) .
  • the /3-globin primers were used at a concentration of 0.16 ⁇ M.
  • the appropriate single-target or multiplex PCR master mix can be aliquoted into each well of a fresh 96-well plate, before the addition of the methylcellulose colony lysate, using a repeating pipetter.
  • the cell ly ⁇ ate wa ⁇ found to be efficiently transferred to the tubes containing the PCR master mix (on ice) using a multichannel pipetter and filter barrier pipet tips to prevent aerosol contamination. Once all lysates were transferred to the second plate, and the appropriate PCR controls added, the tube ⁇ were covered and the plate transferred to the thermal cycler for amplification.
  • PCR products were analyzed by ethidium bromide visualization of agarose gels.
  • DNA from agarose gels was transferred to Zeta-Probe GT blotting membranes (Bio-Rad) in 20X SSPE and hybridized overnight at 68°C with the ⁇ product oligonucleotide probe BK29 (5'-CTT-CGG-GGG-CCG- TTT-TTG-TGG-CCC-GAC-CT-3') (SEQ ID NO: 11) that was end- labeled with [ ⁇ 32 P]-ATP using standard protocols (Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed.. Cold Spring Harbor Laboratory Pres ⁇ , NY) .
  • Blots were rinsed briefly in 2X SSPE/0.1% SDS, followed by 2x30 minute washes at 68°C in 0.1X SSPE/0.1% SDS, and then exposed 18-hours to a phosphor screen (Molecular Dynamics) .
  • Fig. 5A is the ethidium bromide-stained gel of the single target PCR ( ⁇ only - top row) and multiplex PCR ( ⁇ and 3-globin - bottom row) for single methylcellulose colonies harvested as de ⁇ cribed above. All ⁇ l and SV29 colonies are positive for both the ⁇ sequence and 3-globin, whereas the single KG-la colonies are PCR positive only for 3-globin. There is a faint band in one of the KG-la colonies that runs slightly above the 50 bp ladder. This smaller band, presumably the result of the increase in the incidence of primer di erization when there is no specific primer template available, is the result of non-specific amplification (as shown in Fig.
  • Fig. 5B is an 18-hour exposure after hybridization of the southern blot with the end-labeled internal probe BK29. Only the ⁇ specific PCR product remains hybridized to BK29 after standard high stringency wash conditions.
  • DMEM Dulbecco Modified Eagle Medium
  • fetal calf serum Hyclone Laboratories
  • penicillin JRH Biosciences
  • 50 ⁇ g/ml streptomycin JRH Biosciences
  • 2 mM L-glutamine JRH Biosciences
  • Transduction was terminated after 72 hours and cells were resuspended in PBS containing 1% human serum albumin (alpha-Therapeutic
  • methylcellulo ⁇ e medium 80% MethoCult H4230, Stem Cell Technologies
  • cytokines 10 ng/ml c-kit ligand (R&D System ⁇ )
  • 25 ng/ml GM-CSF 25 ng/ml G-CSF
  • Amgen 10 ng/ml IL-3
  • rhEPO 1.2 U/ml of rhEPO (R&D Systems) .
  • Cells were plated in four 35 X 10 mm tissue culture dishes at 1.1 ml/dish (Nunc, Inc.) and incubated at 37°C/5.0% C0 2 for 14 days after which single hematopoietic colonies were picked according to the protocol above and one-fifth (10 ⁇ l) of the lysate from an individual colony was amplified using the F1/B5 ⁇ primers.
  • the 96 wells of the PCR plate contained: (a) 80 randomly picked colonies from the experimental tran ⁇ duction, (b) 10 colonies from the Mock transduction, (c) 3 colonies of SV29 cells ( ⁇ positive controls) , (d) 1 colony of KG-la cells ( ⁇ negative control) , and (e) 2 wells of PCR lysing buffer (negative PCR control) .
  • ⁇ positive samples were analyzed by Southern blot using the internal probe, BK29. For post- PCR analysis, 15 ⁇ l of the reaction was loaded onto a 4% agarose gel and stained with ethidium bromide.
  • Fig. 6A shows ⁇ the po ⁇ itive colonies (numbered above the lane) as detected by ethidium bromide staining. Positive colonies were analyzed by Southern hybridization using the internal probe, BK29 (Fig. 6B) . Southern blot analysis verified the specificity of the ⁇ positive samples. Result ⁇ indicated that 19% (15/80) of the cells clonogenic in methylcellulose medium were transduced, as determined by amplification of the ⁇ PCR product, whereas none of the 10 mock colonies were positive, further indicating that this assay can be carried out in a 96-well format without aerosol contamination of negative samples by positive samples.
  • Detection Assay Single colonies of clonogenic cells from an experimental transduction were picked according to the protocol above and 10 ⁇ l of the lysate from an individual colony wa ⁇ amplified a ⁇ above except that a 24-mer oligonucleotide probe (5'-CCG-TTT-TTG-TGG-CCC-GAC-CTG- AGT-3') (SEQ ID N0:12) , internal to the F1/B5 ⁇ PCR product, was included in the PCR a ⁇ ter mix at a final concentration of 0.2 ⁇ M, and the total reaction volume wa ⁇ increa ⁇ ed to 60 ⁇ l.
  • a 24-mer oligonucleotide probe 5'-CCG-TTT-TTG-TGG-CCC-GAC-CTG- AGT-3'
  • SEQ ID N0:12 internal to the F1/B5 ⁇ PCR product
  • the 24-mer fluorescent probe (TaqManTM, synthesized by Perkin-Elmer Corporation) was labeled at the 5' end with the reporter dye molecule, TET (6-carboxy-tetrachloro-fluorescein; emi ⁇ sion ⁇ 538 nm) , while the quencher dye molecule, TAMRA (6- carboxytetramethyl-rodamine; e i ⁇ ion ⁇ 582nm) , wa ⁇ added to the 3' end of the probe via a linker arm nucleotide (LAN) .
  • LAN linker arm nucleotide
  • a phosphate was added to the 3' end to prevent extension of the probe during the PCR.
  • a positive result is determined as any value greater than the threshold ⁇ RQ.
  • the threshold ⁇ RQ is determined by calculating both the mean and standard deviation of the Reporter:Quencher ratios for all no template controls (RQ ⁇ ) and then multiplying the standard deviation by a multiplier as determined by a table of ⁇ tudent's t-distribution values.
  • Fig ⁇ . 7A and 7B show the result ⁇ of an experiment to compare the sensitivity and specificity of an ethidium bromide stained gel versus the TaqManTM fluorescence assay.
  • Fig. 7A displays the results for the entire plate.
  • Samples #1-80 represent single methylcellulose colonies from the experimental transduction, whereas samples #81-90 are single methylcellulose colonies of Mock transduced cells.
  • the 96-well plate also includes 3 po ⁇ itive control ⁇ (single SV29 colonies; indicated by a "+" in the last column of the plate) , a negative colony control (KG-la) ; and two negative PCR buffer controls.
  • the TaqManTM assay reports a positive result (sample ⁇ RQ>thre ⁇ hold ⁇ RQ) .
  • sample ⁇ RQ>thre ⁇ hold ⁇ RQ a positive result which i ⁇ not detectable from loading 10 ⁇ l of the PCR product on an ethidium bromide ⁇ tained gel (99.95% confidence level) .
  • Figure 7B shows a direct comparison of a sub ⁇ et of the samples (#55-90) .
  • the dashed line represents the threshold ⁇ RQ above which sample ⁇ are positive at the 99.95% confidence level.
  • TaqManTM positive samples are indicated by shaded bars. Note that non-specific bands that are slightly ⁇ maller than the 134 bp PCR product ( ⁇ ample #'s 59, 77) , primer-dimers (vi ⁇ ible a ⁇ a band at the bottom of each lane) , and other artifacts of the PCR are not positive with the TaqManTM assay and attest to the specificity of the fluorescent probe. It should be noted, however, that for each of the four experiments 10 no-template controls were used to calculate the threshold ⁇ RQ.
  • threshold ⁇ RQ is a function of the statistical power of the assay, it is possible that analyzing fewer no-template controls could increase the threshold ⁇ RQ and reduce the ability to stati ⁇ tically distinguish the faint positive sample ⁇ from the no-template controls.
  • MOLECULE TYPE other nucleic acid
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Abstract

Ce titrage rapide permet d'estimer des transferts géniques dans ds cellules à l'aide d'une PCR à 96 cupules. On peut continuer avec un système de détection par fluorescence afin d'éviter de devoir utiliser des gels d'agarose. On décrit un titrage et des amorces permettant de détecter des vecteurs à base du virus de la leucémie murine de Moloney.
PCT/US1996/019495 1995-12-08 1996-12-05 Titrage rapide concernant l'estimation de transferts geniques dans des cellules WO1997020951A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7972778B2 (en) 1997-04-17 2011-07-05 Applied Biosystems, Llc Method for detecting the presence of a single target nucleic acid in a sample

Citations (2)

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US5512441A (en) * 1994-11-15 1996-04-30 American Health Foundation Quantative method for early detection of mutant alleles and diagnostic kits for carrying out the method
US5545528A (en) * 1993-04-15 1996-08-13 Hitachi Chemical Research Center Rapid screening method of gene amplification products in polypropylene plates

Patent Citations (2)

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Publication number Priority date Publication date Assignee Title
US5545528A (en) * 1993-04-15 1996-08-13 Hitachi Chemical Research Center Rapid screening method of gene amplification products in polypropylene plates
US5512441A (en) * 1994-11-15 1996-04-30 American Health Foundation Quantative method for early detection of mutant alleles and diagnostic kits for carrying out the method

Non-Patent Citations (1)

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Title
HUMAN GENE THERAPY, 1991, Volume 2, COURNOYER D. et al., "Gene Transfer of Adenosine Deaminase Into Primitive Human Hematopoietic Progenitor Cells", pages 203-213. *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7972778B2 (en) 1997-04-17 2011-07-05 Applied Biosystems, Llc Method for detecting the presence of a single target nucleic acid in a sample
US8067159B2 (en) 1997-04-17 2011-11-29 Applied Biosystems, Llc Methods of detecting amplified product
US8257925B2 (en) 1997-04-17 2012-09-04 Applied Biosystems, Llc Method for detecting the presence of a single target nucleic acid in a sample
US8278071B2 (en) 1997-04-17 2012-10-02 Applied Biosystems, Llc Method for detecting the presence of a single target nucleic acid in a sample
US8551698B2 (en) 1997-04-17 2013-10-08 Applied Biosystems, Llc Method of loading sample into a microfluidic device
US8563275B2 (en) 1997-04-17 2013-10-22 Applied Biosystems, Llc Method and device for detecting the presence of a single target nucleic acid in a sample
US8822183B2 (en) 1997-04-17 2014-09-02 Applied Biosystems, Llc Device for amplifying target nucleic acid
US8859204B2 (en) 1997-04-17 2014-10-14 Applied Biosystems, Llc Method for detecting the presence of a target nucleic acid sequence in a sample
US9506105B2 (en) 1997-04-17 2016-11-29 Applied Biosystems, Llc Device and method for amplifying target nucleic acid

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