WO1991012343A2 - Detection de mutations ponctuelles dans des genes codant des proteines de liaison de gtp - Google Patents

Detection de mutations ponctuelles dans des genes codant des proteines de liaison de gtp Download PDF

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WO1991012343A2
WO1991012343A2 PCT/US1991/000858 US9100858W WO9112343A2 WO 1991012343 A2 WO1991012343 A2 WO 1991012343A2 US 9100858 W US9100858 W US 9100858W WO 9112343 A2 WO9112343 A2 WO 9112343A2
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ggt
gtg
acc
cgg
tct
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WO1991012343A3 (fr
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Francis P. Mccormick
John F. Lyons
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F Hoffmann La Roche AG
Novartis Vaccines and Diagnostics Inc
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F Hoffmann La Roche AG
Cetus Corp
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/112—Disease subtyping, staging or classification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present invention relates to the identification of point mutations within nucleic acids encoding GTP binding proteins in human samples. Point mutations within GTP binding proteins are associated with malignancies.
  • the invention provides specific primers and probes for the detection and classification of these point mutations and potential oncogenes. The identification of oncogenes is important in the study of cell growth and cardnogenesis.
  • the invention provides methods which relate specific point mutations to specific tumor types. In a preferred embodiment point mutations are described witiiin nucleic acids encoding G-proteins.
  • G-proteins function as intermediates in transmembrane signalling pathways (Gilman , 1987, Ann. Rev. Biochem.56: 615). These pathways consist of receptors, G-proteins and effector molecules and are regulated by the cyclic association of GTP and GDP with G-proteins.
  • Each G-protein consists of three subunits: ⁇ , ⁇ , and ⁇ . The specificity of the interaction with the effector molecule is dictated by the ⁇ subunit
  • Gs and Gi are involved in stimulation and inhibition, respectively, of adenylate cyclase activity.
  • Gi Three Gi ⁇ subunits Gi ⁇ 1, Gi ⁇ 2, and Gi ⁇ 3 have been identified and cloned (Itoh el al, 1988, J. Biol. Chem.263:6656-6664).
  • Gt activates cGMP phosphodiesterase in response to photosignal transductiori (Mattera et al., 1986. FEBS Lett. 206:36-42, and Didsbury et al., 1987, FEBS Lett 211:160-164).
  • Other G-proteins have been sequenced including Go and Gz (Jones and Reed, 1987, J.
  • Gs activity raises the level of cAMP in cells by stimulating adenylyl cyclase.
  • cAMP stimulates secretion of human growth hormone and causes cellular proliferation.
  • a subset of human pituitary tumors were described having elevated levels of growth hormone and cAMP (Vallar et al., 1987, Nature 330:566-568).
  • Gsp mutations are a class of mutations that activate Gs, which normally mediates stimulation by thyrotropin (TSH) of thyrocyte proliferation and production of thyroid hormones.
  • Arginine 201 is a major site of ADP-ribosylation of Gs ⁇ by cholera toxin. This modification allows constitutive adenylyl cyclase activation (Lo and Hughes, 1987, FEBS Lett 224:1-31. Glutamine 227 is predicted to be a Gs ⁇ equivalent of glutamine 61 in Eas p21 proteins (Landis et al. supra.). Mutational replacement of Gln-61 in 12s p21 produces a protein that promotes malignant transformation (Der et al., 1986, Cell 44:167-176).
  • the ras genes encode highly related proteins approximately 21,000 daltons in molecular weight (p21s). While the exact functions of these proteins in cellular signalling pathways remains elusive, the p21s have GTPase enzymatic activities and interact with a GTPase activating protein (GAP) (Bishop, 1983, Ann. Rev. Biochem. 52:301-354, and McCormick, 1989, Cell 56:5-8).
  • GAP GTPase activating protein
  • the human ras gene family which includes the closely related Ha-, Ki-, and N- ras genes, is one of the potential targets for mutational changes that have been implicated in the development of many human malignancies (Bos, 1988, Mutation Research 195:255-271). These alterations are either point mutations in codon 12, 13, or 61, or alternatively a 5- to 50- fold amplification of the wild-type gene. These changes convert the ras proto-oncogenes into oncogenes.
  • PCR Polymerase chain reaction
  • the present invention provides methods for screening nucleic acids encoding G-proteins. Methods for screening ras genes are also provided.
  • the nucleic acids may be RNA or DNA.
  • Primers and probes are provided which aid in the identification of potential oncogenes and characterization of point mutations within an oncogene or potential oncogene.
  • the invention provides primers and probes which are particularly suitable for detection of point mutations in nucleic acids encoding Gz, Gs, Go, Ga, and Gi proteins in endocrine tumors.
  • the present invention provides a method for detecting whether a point mutation is present in a nucleic acid encoding a G-protein ⁇ subunit, in a sample, that comprises: (a) hybridizing a G-protein ⁇ subunit probe to said sample and (b) determining whether hybridization has occurred.
  • the method comprises a method for detecting point mutation, if present, in a nucleic acid encoding a G-protein ⁇ subunit in a sample, comprising:
  • step (c) treating the single-stranded molecules generated in step (b) with the primers of step (a) under conditions such that a primer extension product is synthesized using each of the single-stranded molecules produced in step (b) as a template;
  • the present invention provides novel primers and probes useful for detecting potentially oncogenic point mutations within a nucleic acid encoding a G-protein ⁇ subunit.
  • the present invention also provides kits for amplifying and detecting point mutations, if present, within a nucleic acid encoding a G-protein ⁇ subunit
  • Figure 1 shows the identification of point mutations in Gs ⁇ and Gi ⁇ 2 genes.
  • a region containing the indicated codons of the Gs ⁇ or Gi ⁇ 2 gene was amplified by PCR from genomic DNA isolated from either fresh frozen tissue or paraffin-embedded tissue. Point mutations were detected with high-stringency hybridization of sequence- specific oligonucleotides to the amplified product Each panel represents hybridization with a different oligonucleotide.
  • This analysis identified mutations in Arg 201 and Gin 227 codons of Gs ⁇ in 18 biochemically-characterized human growth hormone secreting pituitary tumors. Analysis of Gi ⁇ 2 genes is shown in Figure 1B and described in Example 3. This analysis identified mutations in codon 179 of Gi ⁇ 2 in three human adrenocortical tumors and one ovarian granulosa cell tumor.
  • Figure 2 provides the GVA (gel visualization assay) analysis of ias RNA/PCR products from a normal human spleen and the K562 cell line as described in
  • Figure 3 shows the results of a southern blot analysis of ias RNA/PCR products using ASO probes specific for activating point mutations in characterized cell lines. The experiment is described in Example 6.
  • Figure 4 provides the GVA analysis of ias RNA/PCR from alcohol-fixed, paraffin-embedded samples as described in Example 7.
  • Figure 5 provides the GVA analysis of ras RNA/PCR from stained and unstained microscope slides of human bone marrow as described in Example 8.
  • Figure 6 is schematic diagram of ras format II filters showing the positions of wild type and mutant ras oligonucleotide probes as described in Example 9.
  • the present invention provides a method for detecting and characterizing point mutations, if present, in a nucleic acid encoding a G-protein ⁇ subunit
  • the point mutations detected by these methods are believed to be involved in oncogenesis.
  • the nucleic acid is a G-protein subunit gene, RNA transcription product, cDNA product or a subsequence thereof.
  • the method involves amplifying by a polymerase chain reaction, a segment of nucleic acid encoding at least one G-protein amino acid of interest For each nucleic acid segment suspected of comprising a potentially oncogenic point mutation, a pair of oligonucleotide primers are provided for amplification in a polymerase chain reaction.
  • the primers will amplify wild type or mutant nucleic acid segments.
  • Genes that encode G-proteins are proto-oncogenes in the normal somatotrophic state and are referred to as wild type. If a point mutation is present the gene is a putative oncogene.
  • the present methods, primers, and probes allow one skilled in the art to distinguish between these two types of G-protein genes.
  • point mutations are detected by oligonucleotide probes.
  • the probes contain either the wild type nucleic acid sequence or single nucleotide changes within codons which correspond to amino acids 49, 201, and 227 in the sequence of Gs ⁇ . The amino acid at 226 may be of interest as well.
  • These single nucleotide changes affect the translation product of the gene.
  • a mutated oncogene may not be present. In such a sample only the wild type probe will hybridize to the sample.
  • the wild type probe serves to verify the-presence of the amplified product in the sample.
  • G-protein ⁇ subunits share a high degree of homology. Matsuoka et al., 1988, Proc. Natl. Acad. Sci. USA 85:5384-5388, provide a comparison of the amino acid sequences of several G ⁇ subunits. Although the G ⁇ subunits vary in length, the degree of homology between the published amino add sequences is sufficiently high such that sequence alignment is possible. In this way, the amino add corresponding to Gs ⁇ Gly 49, Gs ⁇ Arg 201, Gs ⁇ Gly 226, and Gs ⁇ Gin 227 can be determined for all G ⁇ subunits. For example, in Gi ⁇ 2, Arg 179, arid Gin 205 correspond to Arg 201 and Gin 227 in Gs ⁇ .
  • the present invention also provides probes which detect point mutations in nucleic acid encoding as proteins.
  • Such probes contain single base changes as well as the wild type sequences for codons encoding amino acids at p21 positions 12, 13, and 61.
  • the sequence to be examined can be assayed by hybridization using a wild type probe and a pool of probes, each probe containing a point mutation at a specific position.
  • the methods can be used to detect and classify a point mutation.
  • the probes, one comprising the wild type sequence and the others comprising point mutations which affect the translation product at a specific position are used individually.
  • the disclosed methods and probes enable new oncogenes to be detected and classified. These methods led to the discovery that for example, the gene encoding Gi ⁇ 2 is a proto-oncogene. The present invention has led to the discovery of new point mutations that may give rise to cancer.
  • the invention provides primers and probes that can be used to identify oncogenic point mutations in genes encoding Gi ⁇ 1, Gi ⁇ 3, Go ⁇ , and Gz ⁇ if such mutations exist Similarly, these methods may be used to design primers and probes to identify new oncogenic point mutations in other G-protein ⁇ subunits such as Gt ⁇ , Gk ⁇ , and Golf ⁇ . These point mutations may well be related to oncogenesis and their identification lays a critical foundation for experimental research in oncogenesis.
  • these point mutations are useful to discriminate between individuals. Methods which discr ⁇ inate between individuals based on nucleic acid sequence differences are used in, for example, forensic medicine.
  • the disclosed examples of the invention relating to G-proteins, provide methods, primers, and probes to detect and classify point mutations in nucleic adds encoding Gs ⁇ and Gi2 ⁇ . These methods are also suitable for detecting point mutations, if present and classifying those mutations in any nucleic acid encoding a G- protein ⁇ subunit
  • Tables 1-4 provide primers and probes suitable for detecting and classifying point mutations in nucleic adds encoding Gs ⁇ , Gi ⁇ 1, Gi ⁇ 2, Gi ⁇ 3, Go ⁇ , and Gz ⁇ . These methods are directly applicable to the detection of point mutations in other G ⁇ subunits, for example, Gt ⁇ , Gk, or Golf.
  • Table 4 provides probes which correspond to codons 49, 201, and 227 in the Gs ⁇ subunit.
  • the nucleic acid containing the region of interest is amplified by a polymerase chain reaction prior to detection.
  • Primers useful in these methods are suitable for amplification of proto-oncogenes as well as activated protooncogenes.
  • new oncogenes can be readily detected.
  • the primers and probes of the invention led to the discovery of novel point mutations and, thus, a novel putative oncogene, Gi ⁇ 2. Novel oncogenic genes encoding Gi ⁇ 1, Gi ⁇ 3, Go ⁇ , and Gz ⁇ may also be discovered by the methods and compositions provided.
  • Primers of use in the present invention hybridize to genomic DNA at sites such that in a PCR reaction, the primers amplify a specific region of the G-protein DNA.
  • the specific region amplified comprises at least one codon which corresponds to Gly 49, Arg 201, or Gin 227 in Gs ⁇ .
  • primers are selected such that the resultant amplified fragment comprises both codons 201 and 227 (or correspondingly 179 and 205 in Gi ⁇ 2).
  • this is not an essential aspect of the invention. If, for example, a large intron or more than one intron exists between these codons in the genomic DNA, or if the DNA is degraded as in some paraffin embedded tissue, amplification primers are designed for the region comprising codon 201 and separately for the region comprising codon 227. Such amplification reactions are run separately or simultaneously in one reaction vessel.
  • Amplification requires the use of primer pairs that will amplify a discrete region of DNA present in a sample. These primer pairs are oligonucleotides. The PCR products generated from these primers are then analyzed by hybridization with sequence specific probes. Sequence specific probes may also be allele-specific probes. An allele-specific probe (ASO) will hybridize to an allele-specific sequence in a nucleic acid within a sample. An allele-specific sequence is a component of an individual's genotype. A sequence-spe ⁇ fic probe comprises a specific sequence which may or may not exist as an allele. Thus, until a sequence is identified in at least one individual, probes which are sequence-specific are not necessarily allele-specific. However, as the term allele-specific probe is used by those of skill in the art these terms are used interchangeably herein.
  • PCR amplification of DNA involves repeated cycles of heat-denaturing the DNA, anneahng two ollgonucleotide primers to sequences that flank the DNA segment to be amplified, and extending the annealed primers with DNA polymerase.
  • the primers hybridize to opposite strands of the target sequence and are oriented so that DNA synthesis by the polymerase proceeds across the region between the primers, effectively doubling the amount of the DNA segment Moreover, because the extension products are also complementary to and capable of binding primers, each successive cycle essentially doubles the amount of DNA synthesized in the previous cycle. This results in the exponential accumulation of the spedfic target fragment, at a rate of approximately 2n per cycle, where n is the number of cycles.
  • Taq DNA polymerase is preferred although this is not an essential aspect of the invention.
  • Taq polymerase a thermostable polymerase, is active at high temperatures. Methods for the preparation of Taq are disclosed in U.S. Patent No.4,889,818 and incorporated herein by reference.
  • primers are used that will amplify G- protein or ras p21 sequences present in a sample.
  • the primers of the invention can include degenerate primers. These are mixtures of oligonucleotides synthesized to have any one of several nucleotides incorporated at a selected position during synthesis.
  • the primers may be sufficiently complementary to all types of ras genes to amplify a DNA sequence of any rasDNA present in the sample. Illustrative primers of this type are referred to, for example, as "pan" ras primers.
  • the primers are designed to amplify a region of DNA, cDNA, or RNA that contains sequences specific to any ias p21 gene: c-N-ras, c-Ha-ras, or c-Ki-ras. Because the "pan" ras primers span large intron sequences, cDNA and RNA templates are preferred. The amplified DNA can therefore be used to classify the ras gene present in the sample.
  • primer pairs which are specific for each gene to be detected.
  • Such a primer pair will amplify a specific DNA or RNA segment encoding, for example, c-Ki-ras, c-N-ras, c-Ha-ras, Gi ⁇ 1, Gi ⁇ 2, Gi ⁇ 3, Gs ⁇ , Go ⁇ , or Gz ⁇ .
  • three separate pairs of ras primers are included in one PCR reaction such that each pair will specifically amplify either c-N-ras, c-Ki-ras, or C- Ha-ras.
  • the primers are designed so that each PCR product has a discrete size.
  • three primer pairs are used to simultaneously amplify several DNA or RNA segments.
  • the identity of the segments) amplified can then be determined by, for example, gel electrophoresis and size determination.
  • the presence of point mutations and the classification of such mutations can be subsequently determined by the methods provided.
  • primers are designed such that the resulting amplification products are of different sizes.
  • Amplification reactions using different primer pairs can be run independently of one another and analyzed simultaneously, for example, using individual lanes on an acrylamide gel.
  • several primer pairs can be used simultaneously in one reaction, and the amplification products divided and analyzed to characterize the sample by, for example, separate probe hybridizations.
  • nested primers are used (Mullis et al., 1986, Cold Spring Harbor Symposium on Quantitative Biology 51:263, incorporated herein by reference). This method may be preferred when the amount of nucleic add in a sample is extremely limited, for example, where archival, paraffin embedded samples are used.
  • nested primers the nucleic acid is first amplified with an outer set of primers. This amplification reaction is followed by a second round of amplification cycles using an inner set of primers.
  • the method of the invention requires in a preferred embodiment that the amplified product is characterized. It may be preferred, but is not essential in the practice of the invention, to determine whether amplification has occurred.
  • the use of an internal amplification control to assure the competency of a sample for PCR is within the scope of the invention and reduces the likelihood of false negative results.
  • Labeled primers or deoxyribonucleotide 5'-triphosphates can be added to the PCR reaction mixture, and incorporation of the label into the amplified DNA measured to determine if amplification occurred.
  • Another method for determining if amplification has occurred is to test a portion of PCR reaction mixture for ability to hybridize to a labeled ollgonucleotide probe or mixture of probes designed to hybridize to only the amplified DNA.
  • the determination of amplification and characterization of a point mutation can be carried out in one step by testing a portion of the PCR reaction mixture for its abillty to hybridize to one or more specific probes.
  • the method involves the introduction of unconventional nucleotide bases, such as dUTP, into the amplified product and exposing carryover product to enzymatic and/or physical-chemical treatment to render the product DNA incapable of serving as a template for subsequent amplifications.
  • unconventional nucleotide bases such as dUTP
  • dUTP dUTP
  • uracil-DNA glyc ⁇ sylase will remove uracil residues from PCR product containing that base.
  • the enzyme treatment results in degradation of the contaminating carryover PCR product and Serves to "sterflize" the amplification reaction.
  • the present invention has led and will continue to lead to the discovery of many previously unknown or uncharacterized oncogenes.
  • oncogenes For example, using the methods, primers, and probes of die invention, four clinical samples examined were discovered to contain two different Gi ⁇ 2 oncogenes. These new oncogenes are an important aspect of the present invention, as are the point mutations that distinguish these from wild type, and the probes that hybridize to these gene sequences in specific fashion.
  • the present invention provides a number of probes for use in detecting and characterizing potential oncogenes. These probes are set forth in Table 2, below. Those skilled in the art will recognize that although the specific primers and probes of the invention exemplified herein have a defined number of nucleotide residues, one or more nucleotide residues may be added or deleted from a given primer or probe typically without great impact on the suitability of that primer or probe in the present methods. The essential aspect of these probes is their abillty to discriminate between wild type and mutant sequences. When a portion of the PCR reaction mixture contains DNA that hybridizes to a probe, the sample contains DNA comprising the wild type or mutant sequence according to the specific sequence of the probe.
  • An important aspect of the present invention relates to detecting the novel probes provided for use in the present methods.
  • a probe There are a number of ways to determine whether a probe has hybridized to a DNA sequence contained in a sample.
  • the probe is labeled in a detectable manner, the target DNA (i.e., the amplified DNA in the PCR reaction buffer) is bound to a solld support, and
  • determination of whether hybridization has occurred simply involves determining whether the label is present on the solld support This procedure can be varied, however, and works just as well when the target is labeled and the probe is bound to the solid support.
  • hybridization probes disclosed herein are sequence-specific ollgonucleotide probes for each G-protein ⁇ subunit codon to be characterized. As described above, sequence-specific probes are similar to allele-specific probes in use and utillty.
  • the probes may be used individually for detecting, for example, a wild type sequence.
  • the probes may be used in a panel format for characterizing a tumor genotype.
  • the tumor genotype may be compared to, for example, somatic tissue or other tumor types.
  • the probes can be used in a variety of different hybridization formats.
  • the following probes are useful for detecting point mutations in a gene encoding Gs ⁇ at amino add positions 201 and 227.
  • a fixed probe format is suitable for detecting other gsp mutations, as well, and is demostrated in Example 5 for detecting point mutations in as gene PCR products.
  • nucleic acids whether probe or target, are known in the art and are suitable for purposes of the present invention.
  • the probes were labeled with radioactive phosphorous 32 P, by treating the probes with polynucleotide kinase in the presence of radiolabelled ATP.
  • other non-radioactive labeling systems may be preferred, i.e., horseradish peroxidase- avidin-biotin systems.
  • Horse-radish peroxidase (HRP) can be detected by its abillty to covert diaminobenzidine to a blue pigment
  • TMB tetramethyl-benzidine
  • ECL enhanced chemiluminescent
  • Another non-radioactive alternative detection method uses terminal transferase (Tdt) and biotinylated dUTP to add homopolymer tails to the oligonucleotide probes.
  • Biotin serves as the detectable moiety.
  • the filters are washed as usual.
  • Hybridized biotin is detected with strep-avidin conjugated HRP (Se-eqence ® available from Cetus) according to manufacturer's instructions.
  • the ECL system is then used to visualize the biotin-HRP product.
  • Probes are typically labeled with radioactive phosphorous 32 P, by treating the probes with polynucleotide kinase in the presence of radiolabeled ATP.
  • non-radioactive labeling systems may be preferred, such as, horseradish peroxidase-avidin-biotin or alkaline phosphatase detection systems.
  • HRP can be used in a number of ways. For example, if the primer or one or more of the dNTPs utilized in a PCR amplification is labeled (for instance, the biotinylated dUTP derivatives described by Lo et al., 1988, Nuc.
  • probes are biotinylated and detected with the ECL system described above.
  • biotinylated probes are prepared by direct biotinylation of the ollgonucleotide rather than incorporation of biotin-dUTP during PCR.
  • biotinylation of ollgonucleotides direct solld phrase synthesis using biotin containing phosphoramidites is done according to Alves et al., 1989, Tetra.
  • primers, and probes for amplifying, detecting, and characterizing new G-protein point mutations can be readily obtained. It will also be readily apparent to those skilled in the art that the specific primers and probes provided in the examples are merely illustrative of the invention. Primers and probes of the invention can also be prepared to amplify and detect sequence variations within areas G-protein sequences other than those specifically exemplified herein, for example, codons corresponding to Gs ⁇ 49.
  • the method of the present invention is appllcable for detecting a point mutation within a gene encoding a GTP binding protein or the expression product of that gene.
  • the method can detect specific point mutations in a sample containing RNA, or DNA, or both. If the sample contains RNA, the nucleic acid will be reverse transcribed, providing a double-stranded DNA template prior to amplification.
  • RNA reverse transcribing
  • the probes are suitable for direct detection and characterization of an oncogene or protooncogene mRNA suspected of being present in the sample or in the reverse transcribed cDNA product Similarly, if DNA is abundant, for example, as in a fresh tissue sample, the probes are useful for direct detection of gene sequences. Thus, amplification by PCR is not an essential component of the present invention.
  • Samples suitable for analysis by the methods described may be fresh or archival.
  • Fresh samples may be, for example, biopsied tumor, tissue samples, or blood.
  • Archival samples may be, for example, frozen or paraffin embedded.
  • paraffin-embedded samples are analyzed using G-protein primers and probes.
  • methods are provided for ias oncogene detection in RNA purified from surgical biopsy samples or cultured cells. Methods are also provided for extracting RNA from air-dried bone marrow sides and alcohol-fixed paraffin embedded tissues. For paraffin embedded tissues containing intact DNA Table 2 provides primers and conditions for amplifying potential oncogenic sites in Gi 3 , Gi 2 , Gs ⁇ , Gi 1 , Gz, and Go.
  • Example 1 describes primers for amplifying short segments of DNA that include oncogenic regions of gsp.
  • the present invention provides methods for detecting activated oncogenes at the genomic (DNA) level.
  • the methods are also suitable for monitoring the expression of proto-oncogenes and oncogenes.
  • the level of oncogene expression can be monitored during and following, for example, chemotherapeutic treatment
  • Probes are provided for determining allellc dominance for a specific oncogene.
  • probes specific for Gi ⁇ 2 alleles can be used to analyze somatic cells as well as tumor cells. These probes can also distinguish, in the case of a tumor comprising one mutant and one wild type allele, the relative abundance of the mRNA products of these alleles.
  • One skilled in the art will recognize the utillty of such analyses following study of and studying mutagenic events.
  • the primers and probes described provide a method for phenotyping a cell.
  • the cell may be a tumor cell or a somatic cell.
  • Analysis using the methods of the invention provides information relating to the proto-oncogene and oncogene profile of a cell. In this way, events related to the presence of, for example, more than one G-protein point mutation, may be discerned which were previously undetectable.
  • These methods provide a means for associating a specific mallgnancy with a specific oncogene or point mutation. For example, of 306 samples for 15 tumor types analyzed for Gs ⁇ , 18 point mutations were detected; all 18 Gs ⁇ oncogenes were detected in pituitary adenomas and thyroid tumors. In another example using Gi ⁇ 2 primers and probes, 254 samples representing 14 different tumor types were analyzed. Four tumors of two types had Gi ⁇ 2 oncogenes; one ovarian granulosa cell tumor and three adrenal cortical tumors. Analyses using the methods and probes provided has led to the discovery that the proto-oncogenes encoding G-protein ⁇ subunits are activated in endocrine tumors. These studies suggest that distribution of Gs ⁇ and Gi ⁇ 2 oncogenes is restricted among specific endocrine target cells.
  • G-protein oncogenes and tumor specificity provides information of useful in determining the role of G-proteins, where that role is as yet undefined.
  • corticotropin which stimulates c ⁇ rtisol secretion via Gs, adenylyl cyclase, and cAMP, does not utillze Gs and cAMP to stimulate proliferation of ACTH target cells.
  • Tumors derived from adrenal cortical cells (the target cell of ACTH) do not harbor the Gs ⁇ oncogene.
  • the methods of the present invention provide essential tools for identifying new oncogenes, such as Gi ⁇ 2 (alternatively referred to herein as gip2) and exploring the diverse mix of signalling pathways that mediate regulation of prolif eration in endocrine target cells.
  • Gi ⁇ 2 alternatively referred to herein as gip2
  • the present invention also provides a number of previously unknown point mutations. These sequences encode corresponding mutant proteins and can be used to synthesize novel, mutant, proteins. Such proteins, or protein subunits or
  • subsequences can be used to generate antibodies useful in the detection of mutant G- proteins. These antibodies would provide important tools for screening, for example, biopsied tissue to detect mutant G-proteins, and thus provide important information regarding the genetic make-up of an individual or the carcinogenic state of the sampled tissue.
  • a mutant G-protein may be dominated in vivo by a normal G-protein unless carcinogenesis is triggered by other events.
  • antibodies enabled by the present invention would also find use in, for example, screening transplantation tissue as an indicator of potential oncogenic compllcations.
  • kits for the quantitation of one or more nucleic acids in a sample For example, in its simplest embodiment such a kit would provide an ollgonucleotide primer pair for amplification of a G-protein ⁇ subunit segment and corresponding wild type ollgonucleotide probe.
  • a kit may contain an array of G protein probes fixed onto a solld support and a corresponding primer pair for amplifying and detecting oncogenic point mutations in genes encoding G protein subunits.
  • kits may contain an ollgonucleotide primer pair, corresponding G-protein ⁇ subunit wlld type and mutant probes, a DNA polymerase, a RNA polymerase, a reverse transcriptase, nucleotide triphosphates, restriction enzymes, and buffers for carrying out cDNA synthesis, restriction enzyme digests, and amplification by PCR.
  • the kits may contain a thermostable DNA polymerase; for example, the thermostable DNA polymerase Taq isolated from Thermus aquaticus as an agent of polymerization.
  • Proto-oncogene refers to the wlld type form of gene encoding a protein in which a point mutation affecting the amino add sequence encodes a protein which may have a carcinogenic or tumorigenic effect.
  • Oncogene refers to a proto-oncogene containing a point mutation and encoding protein which may have a carcinogenic or tumorigenic effect. Oncogenes may alternatively be referred to herein as "activated proto-oncogenes.”
  • G-protein ⁇ subunit primers refer to primer pairs which hybridize to complementary strands of a nucleic add encoding a G-protein ⁇ subunit and will function in a PCR reaction to amplify a nucleic add segment comprising one or more codons suspected of harboring a point mutation.
  • these codons encode amino acids corresponding to Gs ⁇ amino acids 49, 201, and 227.
  • G-protein ⁇ subunit primers can readily be designed to amplify such regions according to the methods described herein.
  • G-protein ⁇ subunit probe refers to an ollgonucleotide probe designed to characterize the nucleic add sequence encoding an amino acid at a position suspected of containing a point mutation.
  • individual probes comprise the wild type nucleic acid sequence, or a nucleic add containing a point mutation, within a specific codon.
  • Specific codons include any codon within a nucleic add encoding a G- protein, which when replaced with a non-wild type sequence results in a G-protein oncogene.
  • the specific codons include for each G-protein ⁇ subunit, codons encoding the amino acids corresponding to Go ⁇ amino acid positions 49, 102, and 227.
  • one wlld type probe and nine point mutation probes may bfc designed and used in the present methods.
  • probes be included for practice of these methods.
  • the use of only one probe may be sufficient to discriminate between two individuals or between the presence and absence of a point mutation.
  • slldes For isolation of genomic DNA from paraffin-embedded tissue, 3-5 adjacent 5 mm sections were cut from paraffin blocks and mounted on glass slldes (Wright et al. PCR Protocols: A Guide to Methods and Applications eds. M. Innis, D. Gelfand, J. Sninsky, and T. White, Academic Press, San Diego, pp. 153-158, incorporated herein by reference). One sllde was stained with hematoxylin and eosin and used as a guide to select a region composed entirely of tumor on the other slldes.
  • the sample was treated with 0.2 mg/ml proteinase K in 100 mis digestion buffer (50 mM Tris, pH 8.5; 1 mM EDTA; 0.5% Tween 20) at 37°C overnight The sample was centrifuged to remove undigested debris and the
  • DNA-containing supernatant was incubated at 95°C for 8 minutes to denature proteolytic enzymes and nucleases.
  • Fresh frozen samples were prepared according to Verlaan-de Vries et al., 1986, Gene 50:313.
  • Nested amplification primers were used in the PCR amplification procedure to improve specificity and yield (Mullls et al., supra).
  • Genomic DNA 100-500 ng DNA from fresh tissue or 10 mis of the DNA solution from paraffin-embedded tissue
  • a thermocycler Peririn-Elmer Cetus was used for amplification.
  • the amplification program was: 5 minutes at 95°C followed by 30 cycles of 1 minute at 95°C, 2 minutes at 50°C, and 2 minutes at 72°C.
  • a second amplification reaction with 30 pmols of the inner primers was done using 2 ⁇ ls of the initial amplification mixture in the same dNTP and buffer conditions as above and 0.5 units Taq polymerase.
  • the program for the 2nd amplification reaction was: 30 seconds at 94°C, 40 seconds at 57°C and 45 seconds at 72°C.
  • Five ⁇ ls of the final product was sized on a 2% Nusieve, 1% Seakem agarose gel and visuallzed by ethidium bromide staining. A 526 base pair fragment was obtained for Gs ⁇ and a 504 bp fragment for Gi ⁇ 2.
  • Nylon filters (Pall Biodyne-B, 0.45 mm) were briefly rinsed in water and mounted on a Bio-RAD dot-blot apparatus. Four mis of each final amplification product were denatured in 0.4 N Na OH and 25 mMEDTA for 5 minutes and spotted on the filter. The DNA was crosslinked to the filter using a Stratalinker (Stratagene) set at auto crosslink. The filters were prehyhridized in 5X SSPE and 0.5% SDS at 50°C for 30 minutes. One ng of a 32 P end-labeled ollgonudeotide was added to the prehybridization solution and incubated at 50°C for 45-60 minutes.
  • Stratalinker Stratagene
  • hybridized filters were washed briefly in 2X SSPE and 0.1% SDS at room temperature, followed by a 10 minute incubation in 3 M tetramethyl ammonium chloride, 0.2% SDS, and 50 mM Tris, pH 8.0 (TMACI) at the following temperatures: for Gs ⁇ codon 201, 64.5°C; Gs ⁇ codon 227, 67°C; Gi ⁇ 2 codon 179, 61.5°C; Gi ⁇ 2 codon 205, 67.5°C. When other probes are employed, hybridization is carried out as described above. The filters are then washed in TMACI at 58°C for 10 minutes. The wash temperature is adjusted in 1°C increments until only the wild type and mutant signals can be detected.
  • the appropriate wash temperature is determined. Under these conditions, the chosen temperature allows only fully complementary hybrids to stay formed, resulting in a positive dot on a filter.
  • the filters were exposed to Kodak X-AR film for 2-6 hours at -70°C with intensifying screens. For subsequent hybridization with different ollgonucleotides, the nylon filters were stripped by boiling the filters for five minutes in 2X SSPE, 0.1% SDS and then processed as described above.
  • Table 3 provides primer pairs for amplification of nucleic acid segments possibly containing oncogenic point mutations. If primer sequence is within the coding region (exon) of the ⁇ subunit the primer pair is suitable for amplification of either a DNA or cDNA template.
  • the nucleic acid sequence of the G-protein ⁇ subunits are publlshed for Gi ⁇ (see Bray et al., 1987, Proc. Nad. Acad. Sci. USA 84:5115-5119), Gs ⁇ (see Kozasa et al., 1988, Proc. Natl. Acad. Sci. USA 85:2081-2085), Go ⁇ (see Lavu et al., 1988, Biochem. Biophys. Res. Comm. 150:811-815).
  • ollgonucleotide probes are shown in Table 4 as follows. For each position, i.e., Gs ⁇ 201, the full probe sequence is shown for the wlld type allele. The wild type codon at the potentially oncogenic site is underlined, and the translated amino add is shown to the right A set of probes is provided where the sequence is identical to the wlld type except at the codon to be characterized. Thus, for non-wlld type probes only that codon and the translated amino acid product is shown in the table. Only those point mutations encoding amino adds different from the wlld type amino acid are shown.
  • Table 5 shows stretches of Gs ⁇ sequence surrounding the arginine-201 and glutamine-227 codons which are highly conserved in G-protein ⁇ chains of vertebrates, yeast, and slime mold. Publlshed sequences include rat Gs ⁇ , Gi ⁇ 2, Gi ⁇ 3, and Go ⁇ , human Gz ⁇ (Katada and Vi, 1982, Proc. Natl. Acad. Sci.
  • Gt ⁇ of bovine retinal rod cells (Chambard et al., 1987, Nature 326:800), the ⁇ chain of the G- protein (called GPAl/SCGl) that mediates pheromone signalling in Saccharomyces cerevisiae (Corven el al., 1989, Cell 59, and Itoh et al., 1988, J. Biol. Chem.
  • V Val Q - Gln
  • PCR polymerase chain reaction
  • tumors 15 more than 300 tumors was analyzed either in the form of high molecular weight DNA prepared from fresh tissue or as obtained from paraffin-embedded tissue.
  • Group 1 tumors had low basal adenylyl cyclase activity thatresponded normally to stimulatory agents, group 2 tumors had marked elevation of basal adenylyl cyclase activity that responded poorly to stimulatory agents.
  • the hybridization results are shown in Figure 1 A.
  • the hybridization probes shown in Table 4 were used as fottows: R201 indicates the wlld type probe for Gs ⁇ Arg 201; R201C indicates that the probe used contained a point mutation (CGT to TGT) encoding cysteine; and R201H indicates that the probe used contained a point mutation (CGT to CAT) encoding histidine.
  • the fourth panel was probed with the
  • Table 6 provides a summary of human tumors screened for mutations in codons 201 and 227 for Gs ⁇ and codons 179 and 205 for Gi ⁇ 2.
  • Eighteen growth hormone (GH) secreting pituitary adenomas contained a mutation in either Gs ⁇ codons 201 or 227.
  • One ovarian granulosa cell tumor and three adrenal cortical tumors contained a mutation in Gi ⁇ 2 codon 179.
  • Table 7 provides a llst of wlld type codons for the conserved arginine and conserved glutamine in Gs ⁇ and Gi ⁇ 2 genes.
  • the table also shows single-nucleotide base changes (in bold) and the resulting amino acid changes. Oligonucleotides specific for wild type and each missense or nonsense single-base change listed were used to screen human tumors, with the exception of base changes that would be silent (marked with asterisks). Mutations detected in the tumors listed in Table 6 are underlined. "Term" indicates termination or stop signal.
  • Gi ⁇ 2 the coding sequence and intron between the two codons to be tested, arginine-179 (corresponding to Gs ⁇ Arg 201) and glutamine-205 (corresponding to Gs ⁇ Gin 227), is short enough to allow PCR amplification of a single genomic DNA fragment containing both codons (Itoh et al., 1988, J. Biol. Chem.263:6656).
  • the primers and probes used for detection and characterization of Gi ⁇ 2 are shown in Tables 3 and 4. Samples were prepared and analyzed as described in Example 1. The hybridization results are shown in Figure 1B. In the figure, the first two rows of each panel were probed with the wild type probe for codon Arg 179 (R179). The third and fourth rows were hybridized to the Gi ⁇ 2/201 probe of Table 2 containing a point mutation (CGT or TGT) encoding cysteine (R179C). The last two rows of each panel were hybridized to the Gi ⁇ 2/227 probe shown in Table 4 comprising a point mutation (CGT or CAT) encoding histidine (R179H). The amplification methods are described in Example 1.
  • Table 6 summarizes the hybridization results. Mutations in codon 179 of Gi ⁇ 2 were detected in two different endocrine tumor types 3 of 11 tumors of the adrenal cortex and one of 6 ovarian granulosa cett tumors. The adrenal tumor lacking a wlld type allele was an adenocarcinoma; the other 2 adrenal tumors were adenomas. No mutations were found in codon 205. The high frequency of codon 179 mutations in tumors of two related cell types suggests that these mutations converted the Gi ⁇ 2 gene into an oncogene, referred to herein as gip2 (for Gi protein-2).
  • tissue blocks of primary tumor or lymph node metastases from 37 patients with differentiated thyroid carcinoma were also tested for mutations in specific codons of the three human ras genes.
  • ollgonucleotide primers upstream and downstream of codons 201 and 227 in the human Gs ⁇ gene, templates ranging from 165 to 1,200 bp in length.
  • Primers chosen for PCR amplification of regions around codons 12, 13 and 61 of the human H-ras, Ki-ras and N-ras genes yielded products of 112-117 bp.
  • Amplification of formalin-preserved, paraffin-embedded tissues was most effective with relatively short PCR products.
  • Amplification with nested primers was required when the first PCR of larger DNA fragments produced an unsatisfactory amount of amplified DNA, as judged by agarose gel electrophoresis and ethidium bromide staining.
  • PCR conditions were as generally discribed in Example 1, however, the oligonucleotides and cycling temperatures used were as were as follows.
  • Ha-ras codons 12 and 13 sense (JFL243), 5'AGA CCC TGT AGG AGG ACC CCG GGC C; antisense (JFL244), 5'ATA GTG GGG TCG TAT TCG TCC ACA A; product 150 bp.
  • Ha-ras codon 61 sense (JFL252), 5'GTC ATT GAT GGG GAG ACG TG; antisense (JFL253), 5'ACA CAC ACA GGA AGC CCT CC; product 112 bp; for Ki-ras codons 12 and 13: sense (EK371), 5'CCT GCT GAA AAT GAC TGA ATA TAA A; antisense (EK372), 5'T ATT GTT GGA TCA TAT TCG TCC ACA; product 118 bp;
  • Ki-ras codon 61 sense (JFL248), 5'GTA ATT GAT GGA GAA ACC TG; antisense (JFL249), 5'ATA CAC AAA GAA AGC CCT CQ product 112 bp.
  • N-ras codons 12 and13 sense (JFL216), 5'CTT GCT GGT GTG AAA TGA CT; antisense (JFL257), 5;GGT GGG ATC ATA TTC ATC TA; product 150 bp.
  • N-ras codon 61 sense (JFL218), 5'GTT ATA GAT GGT GAA ACC TG; antisense (JFL242), 5'GGC AAA TAC ACA GAG GAA GCC TTC; product 112 bp.
  • Hybridization dot blots like that shown were scored by counting in an AMBIS radioanalytic imaging system as described above. Repllcate PCR amplifications and analyses of adjacent 5 mm sections produced similar results, indicating that the procedure was accurate and reprodudble as well as sensitive.
  • a level of hybridization was set, below which a sample would be considered negative for a particular mitation. This level was set at 20% of the signal detacted by hydridization of the wild type probe to unmutated DNA in the sample. Hybridization signals were determined to be reproducible at this level, but not below it. Consequently, a positive result (20% or more of the amplified DNA contains a mutation) indicates that at least 40% of the cells in the assayed tissue fragment contain the mutation, if— as expected for dominant somatic mutationseach cell has one wlld type and one mutant allele. D. Hybridization and Detection of Point Mutations
  • the PCR product was spotted (dot-blot apparatus, Bio-Rad) and cdvalently bound to a nylon filter (Pall Biodyne-B, 0.45 um) using UV light at the auto-crosslink setting (Stratallnker, Stratagene).
  • a dot was considered to represent a gsp mutation if (CPM m - CPM b ) divided by (CPM wt - CPM b ) was greater than or equal to 0.2.
  • This criterion thus required that ampllfied DNA samples judged as positive for a mutation must exhibit a mutant signal 20% of that observed with wlld type. Applying this criterion also minimized the chance that a spuriously positive result could result from contamination of a gsp-negative sample by DNA from a gsp-positive sample.
  • PCR products from six fragments for which hybridization results indicated R201C mutations at levels near the demonstrated cutoff point were sequenced.
  • Genomic DNA was ampllfied using one biotinylated and one non-biotinylated primer, to generate umlateral biotinylated PCR products.
  • streptavidin coated beads Dynabeads, Dynal
  • the -complementary strand was denatured and aspirated, leaving single stranded DNA.
  • Sequencing was performed using the Sequenase kit (Sequenase, USB). In all six cases the sequencing gels showed both the wlld type and the mutant 201 codon, confirming the results of dot blot hybridization.
  • Gsp mutations were found in surgical spedmens from 24 of 37 patients (65 %) with differentiated thyroid cancer. Gsp mutations in these patients were
  • Microdissected fragments from 12 of 37 patients contained N-ras mutations. These included 12 fragments with codon-61 mutations (Q61R), 18 codon-13 mutations (nine G13C, nine G13D), and a single fragment with a codon-12 mutation (G12C).
  • N-ras mutations resembled gsp mutations in several respects: Both were heterogeneously distributed, sometimes multiple in a single patient, and present in benign as well as mallgnant thyroid tissue (Table 8). Of 117 microdissected fragments tested in the 12 ras-positive patients, 31 (26 %) contained an N-ras mutation. Two patients had more than one different ras mutation.
  • the thyroid tumors exhibited four previously unreported mutations, including substitutions of protine or serine for arginine-201 and substitutions of histidine or proline for glutamine-227.
  • microdissection technique for finding point mutations greatly extends the practical resolution for detecting certain oncogenes, to the point that it can detect a mutation present in 40% or more of the few thousand cells in a 5 m x 30-100 mm 2 fragment of tissue.
  • Microdissection may serve to uncover heterogeneously distributed oncogenes in non-thyroid tumors also.
  • the increased sensitivity of this technique also shows that a conclusion that a particular oncogene mutation is not present in a tumor can be wrong. If based upon PCR amplification of large fragments of tumor, such a negative conclusion must be qualified; in fact, a negative result only indicates that the mutation is not present in a substantial proportion of the cells in the tumor fragment.
  • Table 8 The results of this analysis are presented in Table 8.
  • Gap and ras mutations are listed as present or absent in microdissected fragments of thyroid tissue; fragments containing 5 no thyroid tissue are not included. Each fragment tested was classified as "carcinoma” or "benign”; for each, 90% or more of the cells seen in the corresponding part of the stained section were histologicall 'mallgnant or benign, as indicated. Mutations are enumerated as a fraction of n mutations detected in N fragments tested; ND indicates that no fragment of the particular classification (carcinoma or benign thyroid) were
  • the "HistoJTNM” column indicates whether the tumor was diagnosed as a papillary (P) or folllcular (F) thyroid carcinoma, and provides the numerical cllnical classification (TNM) of thyroid
  • T, N, and M are numbers referring to different characteristics: T (varying from 1 to 4) indicates increasing size and extent of the tumor mass; N (varying from 1 to 3) indicates increasing numbers of lymph node m
  • Microscope slldes with bone marrow smears on them were analyzed. One was hematoxylin and eosin (H+E) stained sllde, whlle the other was merely air dried bone marrow. Both slldes were stored at room temperature for several months prior to RNA extraction.
  • Calu-1 (lung carcinoma), SW 480 (colon carcinoma, and PA-1 (teratocarcinoma) were obtained from the American Type Culture Collection (ATCC) (Capoh et al., 1983, Nature 304:507-513, and Tainsky et al., 1984. Science 225:643-6451 HL-60 (promyelocytic leukemia) was a gift of Dr. J. Lawrence (Murray et al., 1983, Cell 33:749-757).
  • ATCC American Type Culture Collection
  • K562 erythroleukemia
  • CCC Cetus Tissue Culture Collection
  • G-2101 renal clear cell carcinoma
  • the cell strain T- 3891 (fetal lung) is a normal, nonimmortallzed fibroblastic culture (Rossitto et al., 1988, J. Virol.140:431-435). All of the above cell llnes were maintained according to the instructions of the suppller.
  • Guanidmium-isothiocyanate-phenol-chloroform methdds Maniatis et al., 1982, In. Molecular Cloning, New York, Cold Spring Harbor Page 190, and Chirgwin et al., 1979, Biochem.18:5294-5299.
  • isothiocyanate solution (5 M guanidinium isothiocyanate, 25 mM sodium citrate, 0.5% sarcosyl, pH 7.0) (GITC) was prepared to 5% ⁇ -mercaptoethanol (GITC-ME) just prior to use.
  • Tissue bits were powdered in liquid nitrogen in a mortar, further ground in the mortar upon additional of GITC-ME and 1.5 ml of the slurry was layered onto a cesium chloride (CSCL) density gradient in 13 x 51 mm polyallomer tubes (Beckman Laboratories).
  • CSCL cesium chloride
  • the CsCl density gradient was prepared by layering 1.5 ml of a 40% CsCl density gradient was prepared by layering 1.5 ml of a 40% CsCl solution in 20 mM Tris-HCl, 2 mM EDTA, pH 7.5 (TE) onto 2.0 ml of 5.7 M CsCl in TE.
  • RNA was pelleted through this density gradient by ultracentrifugation at 40,000 rpm in an SW-50.1 rotor at room temperature for 16 to 19 hours.
  • RNA pellet was suspended in 50 ⁇ l TE-SDS (10 mM Tris-HCl, 1 mM EDTA, pH 7.4 wit 0.5% SDS) in a microcentrifuge tube for phenol-chloroform extraction.
  • TE saturated phenol was mixed 1:1 (v/v) with a chloroform:isoamyl alcohol (24:1) solution.
  • An equal volume of this phenol:chloroform solution was added to the RNA solution, vortexed vigorously for 10 seconds and phase separated in a microfuge for two minutes. This extraction was repeated, and the aqueous phase containing the RNA was placed in a 2 ml microcentrifuge tube for predpitation.
  • RNA was precipitated by addition of 5 M NaCl to create a final concentration of 0.3 M NaCl followed by addition of two volumes of ice-cold 100% ethanol. This solution was placed at -70°C for a minimum of one hour. The tube was then warmed to room temperature to melt the ice and spun in a microfuge at 4°C for 15 minutes to pellet the RNA predpitate. The supernant was decanted and residual llquid was removed by vacuum desiccation. When nearly dry, the RNA pellet was redissolved in TE (without SDS) and precipitated a second time. This time the RNA pellet was redissolved in 50- 100 ⁇ l of 0.2X TE. RNA concentrations were determined by reading optical density at 260 mm in a spectrophotometer and calculated by setting 1.0 O.D. equivalent to 40 ⁇ g per ml RNA. D. RNA Extraction from Air-dried Bone Marrow Slides
  • Microscope slides of human bone marrow were extracted for RNA. One was stained with H+E while the other was merely air-dried and left unstained. The cells on these slides were scraped with razor blades into microcentrifuge tubes. To the tubes was added 1 ml of GITC-ME buffer, and the tubes were shaken vigorously on a rotary shaker for 60 minutes to dissolve the cells. The solution was then put in a 2 ml microcentrifuge tube. To precipitate DNA away from the RNA in solution, 0.1 ml of 2 M sodium acetate (pH 4.8) was added to each tube.
  • pH 4.8 2 M sodium acetate
  • the DNA precipitation and an extraction were performed by adding 1 ml of phenolxhloroform to the tube, inverting the tubes multiple times, and placing the tubes on wet ice for 15 minutes.
  • This method of quick RNA extraction including the precipitation of DNA away from the RNA is modification of that described by Chomezynski and Sacchi, 1987, Anal. Biochem. 162:156-159.
  • DNA remains at the interface between the organic and aqueous phases.
  • the tubes were spun in a microfuge at 4°C for 20 minutes, and the aqueous phase containing the RNA was removed and transferred to a new 2 ml tube for precipitation of the RNA.
  • RNA samples Fifty micron sections of paraffin blocks were cut and deparaffinized in 1 ml of xylenes by vigorous shaking in a microfuge tube for 30 minutes. Tissue bits were pelleted by microfuging for five minutes and the xylenes decanted. Residual xylenes were removed by washing with 100% ethanol andrepelleting the tissue bits. To the tissue 1 ml of the GTTC-Me solution described above was added. Tubes were vigorously shaken on a rotary shaker for one hour to dissolve the tissues. All subsequent steps in the RNA isolation were the same as those described above for the bone marrow slldes where DNA was predpitated away from RNA. Each fifty micron section yielded approximately 25 ⁇ g of RNA.
  • RNA/PCR polymerase chain reaction
  • PCR amplification of spllced mRNA results in ampllfied products of a predicted length containing only the exon sequences of the gene.
  • Amplification of unspllced RNA or any contaminating genomic DNA in the RNA preparation yields products of a larger size including the intron sequences.
  • the smaller product predicted from the spllced mRNA sequence will only be produced if spllced mRNA transcribed from the gene of interest is present Therefore, the present of the predicted band on an ethidium bromide stained gel is an unequivocal assay for that gene's transcription or expression. This assay is referred as the Gel Visuallzation Assay (GVA) for gene expression.
  • GVA Gel Visuallzation Assay
  • Primer pairs were also designed for each of the three genes to yield different- sized RNA/PCR products. Using GVA to score gene expression, the production of different-sized products allowed all three gene expression assays to be run
  • RNA/PCR products from c-N-ras (primers EK 365 and EK 366), c-Ha-ras- 1
  • Each upstream primer was designed to have a greater than six base mismatch with the other two upstream primers to prevent cross amplification of the other ias messages. These three sets of primers were able to be used simultaneously in the same reaction mixture.
  • cDNA Complementary DNA
  • RNA was synthesized from the extracted total cellular RNA essentially as previously described (Kawasaki et al., 1988, Proc Natl. Acad. Sci. USA 85:5698-5702).
  • Mo-MuLV Moloney Murine Leukemia Virus
  • Bethesda Research Laboratories in a 20 ⁇ l reaction in 1 X PCR buffer (50 mM KCl, 20 mM Tris-HCl pH 8.3, 2.5 mM MgCl 2 , and 0.01% BSA) containing 20 U RNAsin (Promega), 1 ⁇ l of a 10 mM each stock of nucleotide triphosphates (dATP, dCTP, dGTP, and dTTP), and 100 pmoles of random hexamer primers.
  • the change to random hexamer primers rather than ollgo-dT primers was based cm
  • the PCR method was used according to Saiki et al., as described for DNA using recombinant thermostable DNA polymerase originating from Thermus aquaticus (rTaq) (Perkin Elmer-Cetus). Sllght modifications included reducing the amount of primers to 10 pmoles each, reducing the dNTPs to 1 ⁇ l of the 10 mM each stock described above, and using the rTaq enzyme at 2 U per reaction in a total 100 ⁇ l reaction.
  • the substrate for RNA/PCR was 2 ⁇ l of the 20 ⁇ l cDNA described above. This amount corresponds to 100 ng of the initial 1 ⁇ g of total cellular RNA used in the reverse transcription reaction.
  • RNA/PCR products were screened by running 9 ⁇ l of the reaction mixture in 2% NuSieve (FMC, Rockland, MD), 1% agarose gels in Tris- borate EDTA buffer (TBE). For size markers, the 123 bp DNA ladder (Bethesda Research Laboratories) was used. Gels of 75 ml were run in wide mini-sub cells (Bio- Rad Laboratories) in TBE at a constant 100 volts for approximately 90 minutes.
  • GVA gel visuallzation assay
  • RNA/ PCR products to be probed were run in 2% agarose gels in a Tris-borate EDTA electrophoresis buffer (TBE) in a mini-gel system, alkaline transfer to Zeta- Probe nylon filters (Bio-Rad Laboratories) in a wick-action transfer was done with a 0.4 N NaOH solution in water. Transfers were allowed to proceed for 90 minutes. Following transfer, blots were neutrallzed in 2X SSC for 5 minutes.
  • TBE Tris-borate EDTA electrophoresis buffer
  • Blots were prehy bridized in a solution of 3 M tetra-methyl ammonium chloride (TMAC), 50 mM Tris-HCl pH 7.5, 2 mM EDTA, 5X Denhardt's solution, and 0.3% SDS at 55°C for one hour with circular agitation.
  • TMAC tetra-methyl ammonium chloride
  • ASO probes that were kinase- labeled with gamma- 32 P-ATP was done in 5 ml of the TMAC buffer llsted above with 2 x 10 6 cpm per ml of probe added. Hybridization continued at 55°C for one hour.
  • RNA/PCR amplification of human ras family mRNAs are shown in Figure 2.
  • the samples used were a normal spleen and the cell llne K562.
  • Lanes 1 and 14 contain the 123 bp DNA ladder.
  • Negative controls (no RNA) for each reaction are shown in lanes 4, 7, 10, and 13.
  • Lanes 2-4 display the RNA/PCR products utilizing the "pan" ras primers EK 224 and EK 225 on the RNA from the normal human spleen, the K562 cell llne, and the negative control of no RNA, respectively.
  • Lanes 5-7 display the results using the c-N-ras-specific primers EK 365 and EK 336. The samples are displayed in the same sequence and as predicted, a 299 bp product is present indicating expression a the c-N-ras gene is both samples of human cells. c-Ha-ras- 1 expression is shown in lanes 8-10. The primers EK 367 and EK 368 produce a 259 bp product and that is clearly seen in lane 9 (K562 cells). No product is seen from RNA isolated from the normal human spleen (lane 8).
  • the lack of a product is interpreted to reflect lack of c-Ha-ras-2 mRNA or levels of the message below that detectable after 30 cycles of RNA/PCR in the normal human spleen. This result shows the utillty of the GVA for the detection of mRNA after RNA/PCR.
  • Lanes 11-13 contain the RNA/PCR products using the c-Ki-ras-2 primers EK 369 and EK 370.
  • the predicted product of 234 bp is present in both lanes 11 (normal human spleen RNA) and 12 (K-562 RNA) indicating expression of the gene in both samples; however, the abundance of message is less in the normal human spleen from that in the K562 cell llne.
  • the cell line samples on each blot are the same: lane 1, EJ/T24 RNA amplified with primers EK 222 and EK 225 (c-Ha-ras- 1); lane 2, EJ/T24 RNA amplified with primers EK 224 and EK 225 ("pan” jas); lane 3, PA-1 RNA ampllfied with primers EK 221 and EK 225 (C-N-ras); lane 4, SW-48- RNA ampllfied with primers EK 223 and EK 225 (c-Ki-ras-2); lane 5, SW-480 RNA amplified with primers EK 224 and EK 225 ("pan” ras); lane 6, HL-60 RNA amplified with primers EK 221 and EK 225 (c-N- ras); lane 7, Calu-1 RNA ampllfied with primers EK 223 and EK 225 (c-Ki-ras-2); lane 8, Calu-1
  • lanes 1 and 2 containing the EJ/T24 RNA amplified both the c-Ha- ras-1 primers and the "pan" ras primers are positive for the characterized EJ/T24 mutation.
  • Panel B blot has been probed with a pool of ollgonucleotides specific for activating point mutations at the second nucleotide of the 12th codon of c-N-ras (JN 17).
  • the PA-2 cell line is known to contain a mutation at this position, and lane 3 is positive as expected.
  • Panel C blot has been probed with ollgonucleotide pool JN 09 targeted to mutations at the first nucleotide of codon 12 in c-Ki-ras-2.
  • the SW-480 cell llne contains one of those mutations and lanes 4 and 5 containing RNA/PCR products for that cell line are positive. Because the signal in lane 5 is quite weak, it may indicate that the mutant allele's message is in low abundance with respect to all other ras messages in the cell as the "pan" cas primers were used for that lane or that the "pan” ras primers are less efficient at amplifying c-Ki-ras-2 messages with respect to the other two ras genes.
  • Panel D blot has been probed with pool JN 22 specific for mutations at the second position of the 61st codon of c-N-ras.
  • Cell llne HL-60 has a mutation at the position and is positive in lane 6.
  • This panel in combination with Panel B, illustrates that the RNA/PCR products amplified by the primers EK 221 and EK 225 (c-N-ras) contain sequences of both the 12th, 13th, and 61st codons of that gene.
  • RNA/PCR products from alcohol-fixed paraffin-embedded samples were analyzed by GVA ( Figure 4).
  • Lanes 1, 5, and 12 contain the 123 bp DNA ladder.
  • Samples in lanes 2, 6, and 9 have been amplified with primers EK 365 and 366 (c-N- ras: 299 bp), those in lanes 3, 7, and 10 with primers EK 367 and EK 368 (c-Ha-ras- 1: 259 bp), and those in lanes 4, 8, and 11 with primers EK 369 and EK 370 (c-Ki-ras- 2: 234 bp).
  • Lanes 2-4 are the negative controls with no RNA added to the reverse transcriptase reaction of RNA/PCR.
  • Lanes 6-8 contain RNA/PCR products from the Calu- 1 cell llne and the products corresponding to as messages from all three genes are present. Lanes 9-11 contain the RNA/PCR products from the cell llne G-2101. In this case, there is a lack of any signal from c-Ha-ras-1 messages indicating lack of expression.
  • the "pan” ras primers were used to amplify reverse transcribed RNA products. Sample preparation and the amplification procedure were as described in Example 4.
  • the ias ollgonucleotide bound filters were hybridized in 5X SSPE, 0.5% SDS with alkall denatured PCR products for 60 minutes at 42°C. Washing was done in 3M tetramethylammonium chloride to minimize the influence of base composition among the various nucleotides.
  • the filters were briefly rinsed with 2X SSPE, 0.1% SDS, then incubated in the same buffer wtth 2 ⁇ g/ml srreptavidin-horse radish peroxidase conjugate ("Sequence," Cetus Corporation) for 30 minutes at room temperature. The filters were then washed for five minutes with the same buffer without the conjugate.
  • Reagents of the ECL gene detection system (Amerrsham) were added and incubated for one minute at room temperature. Filters were then wrapped in Saran wrap and the light signal produced was detected by exposing Kodak XRP film to the filters for 20 seconds to one minute.

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Abstract

Procédé de détection de la présence d'une mutation ponctuelle dans un acide nucléique codant une protéine ou une sous-unité protéique de liaison de GTP. L'invention concerne également des procédés de caractérisation de mutations ponctuelles, le cas échéant. Selon un mode de réalisation préféré, le procédé consiste en l'amplification d'un segment d'acide nucléique suivie par l'hybridation d'une sonde spécifique de la séquence. Le procédé s'adresse de préférence à des acides nucléiques codant une sous-unité α de protéine G ou une protéine p21 ras.
PCT/US1991/000858 1990-02-07 1991-02-07 Detection de mutations ponctuelles dans des genes codant des proteines de liaison de gtp Ceased WO1991012343A2 (fr)

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JP91508291A JPH05506151A (ja) 1990-02-07 1991-02-07 Ｇｔｐ結合蛋白質をコードする遺伝子における点突然変異の検出

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Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0511559A1 (fr) * 1991-04-30 1992-11-04 F.Hoffmann-La Roche & Co. Aktiengesellschaft Réactif pour essai d'oligonucleotide
WO1993006248A1 (fr) * 1991-09-16 1993-04-01 The United States Of America Represented By The Secretary, Department Of Health & Human Services Procede de detection de genes c-raf-1
WO1995030748A3 (fr) * 1994-05-04 1995-12-21 Gene Shears Pty Ltd Sequences u14 d'acide nucleique de vegetaux et leurs derives
WO1996013608A3 (fr) * 1994-10-28 1996-07-11 Innogenetics Nv Sequences d'acide polynucleique utilisees dans la detection et la differenciation d'organismes procaryotes
WO1996021042A3 (fr) * 1995-01-04 1996-09-12 Univ Boston Amorces destinees a l'amplification pcr de sequences metastatiques
US5821062A (en) * 1994-03-29 1998-10-13 Sumitomo Chemical Company, Limited Oligonucleotide for use in checking presence or absence of mutation in human-derived cytochrome P450IIC18 gene
WO1998048052A3 (fr) * 1997-04-18 1999-01-21 Abbott Lab Detection de mutations fondee sur l'amplification
US5869308A (en) * 1988-08-26 1999-02-09 The United States Of America As Represented By The Department Of The Health And Human Services Detection method for C-RAF-1 genes
WO2002010447A3 (fr) * 2000-08-01 2003-10-23 Giesing Michael Procede pour detecter des acides nucleiques par hybridation, utilisation dudit procede, kit d'analyse correspondant, oligomeres d'acides nucleiques et utilisation de ces derniers
EP1302547A3 (fr) * 1992-06-17 2003-11-12 City Of Hope Procédé de détection de séquences d'acide nuclèique et de discrimination entre ces séquences
US12366508B2 (en) 2015-07-30 2025-07-22 Qiagen Gmbh Method of preparing a frozen biological sample

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011157678A1 (fr) * 2010-06-14 2011-12-22 Qiagen Gmbh Procédé de détermination de cellules ou de tissu cibles pour l'extraction de biomolécules à partir d'échantillons biologiques fixés

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4871838A (en) * 1985-07-23 1989-10-03 The Board Of Rijks Universiteit Leiden Probes and methods for detecting activated ras oncogenes

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5869308A (en) * 1988-08-26 1999-02-09 The United States Of America As Represented By The Department Of The Health And Human Services Detection method for C-RAF-1 genes
EP0511559A1 (fr) * 1991-04-30 1992-11-04 F.Hoffmann-La Roche & Co. Aktiengesellschaft Réactif pour essai d'oligonucleotide
WO1993006248A1 (fr) * 1991-09-16 1993-04-01 The United States Of America Represented By The Secretary, Department Of Health & Human Services Procede de detection de genes c-raf-1
EP1302547A3 (fr) * 1992-06-17 2003-11-12 City Of Hope Procédé de détection de séquences d'acide nuclèique et de discrimination entre ces séquences
US5821062A (en) * 1994-03-29 1998-10-13 Sumitomo Chemical Company, Limited Oligonucleotide for use in checking presence or absence of mutation in human-derived cytochrome P450IIC18 gene
WO1995030748A3 (fr) * 1994-05-04 1995-12-21 Gene Shears Pty Ltd Sequences u14 d'acide nucleique de vegetaux et leurs derives
WO1996013608A3 (fr) * 1994-10-28 1996-07-11 Innogenetics Nv Sequences d'acide polynucleique utilisees dans la detection et la differenciation d'organismes procaryotes
US6221582B1 (en) 1994-10-28 2001-04-24 Innogenetics N.V. Polynucleic acid sequences for use in the detection and differentiation of prokaryotic organisms
WO1996021042A3 (fr) * 1995-01-04 1996-09-12 Univ Boston Amorces destinees a l'amplification pcr de sequences metastatiques
WO1998048052A3 (fr) * 1997-04-18 1999-01-21 Abbott Lab Detection de mutations fondee sur l'amplification
WO2002010447A3 (fr) * 2000-08-01 2003-10-23 Giesing Michael Procede pour detecter des acides nucleiques par hybridation, utilisation dudit procede, kit d'analyse correspondant, oligomeres d'acides nucleiques et utilisation de ces derniers
US12366508B2 (en) 2015-07-30 2025-07-22 Qiagen Gmbh Method of preparing a frozen biological sample

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EP0514501A1 (fr) 1992-11-25
WO1991012343A3 (fr) 1991-10-31
AU7758991A (en) 1991-09-03
AU642739B2 (en) 1993-10-28
JPH05506151A (ja) 1993-09-16
CA2075053A1 (fr) 1991-08-08

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