DETECTION OF POINT MUTATIONS IN GENES ENCODING GTP BINDING PROTEINS

Abstract: The present invention provides a method for detecting whether a point mutation is present in a nucleic acid encoding a GTP bindingprotein or protein subunit. Methods are also provided for characterizing point mutations, if present. In a preferred embodiment, the method involves amplification of a nucleic acid segment followed by sequence-specific probe hybridization. The method is preferred for nucleic acids which encode a G-protein alpha subunit or p21 ras protein.

Description: DETECTION OF POINT MUTATIONS IN GENES ENCODING TP BINDING PROTEINS This application is a continuation-in-part of copending Serial No.477,260, filed Feburary 7, 1990, which is incorporated herein by*reference. 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.5£: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 -©protein consists of three subunits: α, β, and γ. The specificity of the interaction with the effector molecule is dictated by the α subunit Several G-proteins have been purified and characterized; Gs and Gi are involved in stimulation and inhibition, respectively,* of adenylate cyclase activity. Three Giα subunits Giαl , Giα2, and Giα3 have been identified and cloned (Itoh el al, 1988, 1. Biol. Chem.262:6656-6664). Gt activates cGMP phosphodiesterase in response to photosignal transductiori (Matterael *. 1986. FEBS Lett. 206:36-42. and Didsbury __\., 1987, FEBS Lett 211:160-164). Other G-proteins have been sequenced including Go and Gz (Jones and Reed, 1987, 1. Biol. Chem.262:14241- 14249, and Strathmann £ al, 1989, Proc. Nafl. Acad. £ci. USA: ££7407-7409). Additionally, Gk (Yatani & al, 1987, Science 235:2071 and Golf (Jones and Reed, 1989, Science 244:790) have been identified as G*-proteins. Gs activity raises the level of cAMP in cells by stimulating adenylyl cyclase. In pituitary somatotrophs, cAMP stimulates secretion of human growth hormone and causes cellular proliferation. Recently, a subset of human pituitary tumors were described having elevated levels of growth hormone and cAMP (Vallar ≤£ al., 1987, Nature 3_3_Q:566-568). Landis £t al, 1989, Nature 3_4Q:692-696, proposed that the abnormal cell proliferation observed by Vallar £t al. was the result of a defect in the G-protein responsible for controlling cAMP levels, resulting in an accumulation of cAMP. Landis £t al. identified patients harboring tumors secreting excessive amounts of growth hormone and determined that four tumors had constutively elevated levels of Gs activity. RNA was purified from fresh tissue, reverse transcribed, and cloned. The entire Gsα coding region of the cDNA was sequenced and point mutations within codons 201 and 227 were identified. These mutations are Qs& mutations. 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. (Lyons & al, 1990, Science.242:655-659) Arginine 201 is a major site of ADP-ribosylation of Gsα 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 Gsα equivalent of glutamine 61 in Eas p21 proteins (Landis ≤l al- supra.). Mutational replacement of Gln-61 in 12s p21 produces a protein that promotes malignant transformation (Der ≤£ al-, 1986, Cell 44:167-176). The ias 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 McCoπnick, 1989, £gll 56:5-8). The human ias 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 ias proto-oncogenes into oncogenes. Polymerase chain reaction (PCR) methods have been used to detect known point mutations in ias oncogenes in genomic DNA isolated from tumors (Verlaan-de Vries ejal, 1986,Gen£ Ω:313-320, and Almoguera el al, 1988, £sll :549-554). Farr&al, 1988, Proc. Natl. £____ - Sci. USA 85:1629-1633. have combined PCR with oligonucleotide dot blot methods to examine specific ias gene point mutations in DNA isolated from patients afflicted with acute myeloid leukemia (AML). 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 α subunit, in a sample, that comprises: (a) hybridizing a G-protein α subunit probe to said sample and (b) determining whether hybridization has occurred. * • In another embodiment, the method comprises a method for detecting point mutation, if present, in a nucleic acid encoding a G-protein α subunit in a sample, comprising: (a) treating the sample with a G-protein α subunit primer pair, an agent for polymerization, and deoxynucleoside 5' triphosphates under conditions such that an extension product of each primer can be synthesized, wherein said primers are sufficiently complementary to separate strands of a nucleic acid encoding a segment of a G-protein α subunit to hybridize thereto so that the extension product synthesized from one member of said pair, when separated from its complementary strand, can serve as a template for synthesis of the extension product of the other member of said pair; (b) separating the primer extension products from the templates on which the extension products were synthesized to form single-stranded molecules; (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; (d) repeating steps (b) and (c) at least once to provide amplified DNA; (e) hybridizing a G-protein α subunit probe to said amplified DNA, wherein said probe contains a nucleic acid sequence that will hybridize to a sequence, selected from a wild type and mutant nucleic acid sequence, within said amplified DNA; and (f) determining if hybridization has occurred. The present invention provides novel primers and probes useful for detecting potentially oncogenic point mutations within a nucleic acid encoding a G-protein α subunit. The present invention also provides kits for amplifying and detecting point mutations, if present, within a nucleic acid encoding a G-protein α subunit Figure 1 shows the identification of point mutations in Gsα and Giα2 genes. A region containing the indicated codons of the Gsα or Giα2 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. Analysis of Gsα genes is shown in Figure 1 A and described in Example 2. This analysis identified mutations in Arg 201 and Gin 227 codons of Gsα in 18 biochemically-characterized human growth hormone secreting pituitary tumors. Analysis of Giα2 genes is shown in Figure IB and described in Example 3. This analysis identified mutations in codon 179 of Giα2 in three human adrenocortical tumors and one ovarian granulosa cell tumor. Figure 2 provides the GVA (gel visualization assay) analysis of ia RNA/PCR products from a normal human spleen and the K562 cell line as described in Example 5. 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 ∑ as 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 π filters showing the positions of wild type and mutant _____ 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 α 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. Thus, the present methods, primers, and probes allow one skilled in the art to distinguish between these two types of G-protein genes. In the embodiment of the invention illustrated below, point mutations are detected by oligonucleotide probes. To detect an oncogenic point mutation in a nucleic acid encoding a G-protein, 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 Gsα. The amino acid at 226 may be of interest as well. These single nucleotide changes affect the translation product of the gene. Of course, in some samples, a mutated oncogene may not be present. In such a sample only the wild type probe will hybridize to the sample. Thus, where a point mutation is not detected, the wild type probe serves to verify the-presence of the amplified product in the sample. G-protein α subunits share a high degree of homology. Matsuoka at a , 1988, Proc. Natl. Acad. Sci. USA 55:5384-5388, provide a comparison of the amino acid sequences of several Gα subunits ^ Although the Gα 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 Gsα Gly 49, Gsα Arg 201, Gsα Gly 226, and Gsα Gin 227 can be determined for all Gα subunits. For example, in Giα2, Arg 179, arid Gin 205 correspond to Arg 201 and Gin 227 in Gsα. The present invention also provides probes which detect point mutations in nucleic acid encoding a 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 examples below describe methods to detect the presence of a point mutation within a nucleic add sequence contained in a sample. For example, 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. Alternatively, the methods can be used to detect and classify a point mutation. In this case, 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. According to the method, only the probe comprising the exact nucleic acid sequence contained within the sample nucleic acid will hybridize to the nucleic acid in the sample. Those skilled in the art will recognize that 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 Giα2 is a proto-oncogene. The present invention has led to the discovery of new point mutations that may give rise to cancer. In addition, the invention provides primers and probes that can be used to identify oncogenic point mutations in genes encoding Giαl, Giα3, Goα, and Gzα 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 α subunits such as Gtα, Gkα, and Golfα. These point mutations may well be related to oncogenesis and their identification lays a critical foundation for experimental research in oncogenesis. Regardless of oncogenidty, these point mutations are useful to discriminate between individuals. Methods which discrώinate 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 Gsα and Gi2α. These methods are also suitable for detecting point mutations, if present and classifying those mutations in any nucleic acid encoding a G- protein α subunit Tables 1-4 provide primers and probes suitable for detecting and classifying point mutations in nucleic adds encoding Gsα, Giαl, Giα2, Giα3, Goα, and Gzα. These methods are directly applicable to the detection of point mutations in other Gα subunits, for example, Gtα, Gk, or Golf. Table 4 provides probes which correspond to codons 49, 201, and 227 in the Gsα subunit. In a preferred method for detecting and classifying point mutations within oncogenes or proto-oncogenes, 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 proto- oncogenes. Those skilled in the art will recognize that with the disclosed methods, primers, and probes, new oncogenes can be readily detected. For example, the primers and probes of the invention led to the discovery of novel point mutations and, thus, a novel putative oncogene, Giα2. Novel oncogenic genes encoding Giαl, Giα3, Goα, and Gzα 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 Gsα. In one embodiment of the present invention, primers are selected such that the resultant amplified fragment comprises both codons 201 and 227 (or correspondingly 179 and 205 in Giα2). However, 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-speάfic 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. Amplification of DNA by PCR is disclosed in U.S. Patent Nos.4,683,195 and 4,683,202 (both of which are incorporated herein by reference). Methods for amplifying and detecting nucleic adds by PCR using a thermostable enzyme are disclosed in U.S. Patent No. 4,965,188, which is incorporated herein by reference. PCR amplification of DNA involves repeated cycles of heat-denaturing the DNA, anneahng two oUgonucleotide 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 5 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. In the disclosed embodiment, Taq DNA polymerase is preferred although this is 0 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. The choice of primers for use in PCR determines the specificity of the amplification reaction. In the present invention, primers are used that will amplify G- 5 protein or as 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. For example, the primers may be sufficiently complementary to all types of ias genes to amplify a DNA sequence of any rasDNA present in the sample. Illustrative primers of 0 this type are referred to, for example, as "pan" ias 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- i-QS- Because the "pan" ias primers span large intron sequences, cDNA and RNA templates are preferred. The amplified DNA can therefore be used to classify the ias gene present in the sample. 5 Alternatively, it may be desirable to use 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. Giαl, Giα2, Giα3, Gsα, Goα, or Gzα. In one embodiment, three separate pairs of ras primers are included in one PCR reaction such that each pair will specifically amplify either c-N-∑as, c-Ki-∑as, CM- C- Ha-ras. The primers are designed so that each PCR product has a discrete size. Thus, 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 electrophσresis and size determination. The presence of point mutations and the classification of such mutations can be subsequently determined by the methods provided. When more than one nucleic acid segment is characterized, it is not essential that 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. Alternatively, 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. In another embodiment of the present invention, nested primers are used (Mullis & 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. When nested primers are used, 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. Once a sample has been treated with a primer pair under conditions suitable for PCR, 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. There are a variety of ways to determine whether amplification has occurred. A portion of the reaction mixture can be subjected to gel electrophoresis and the resulting gel stained with ethidium bromide and exposed to ultraviolet light to observe whether a product of the expected size is present. 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 oUgonucleotide probe or mixture of probes designed to hybridize to only the amplified DNA. Alternatively, 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 abiUty to hybridize to one or more specific probes. Due to the enormous amplification possible with the PCR process, small levels of DNA carryover from samples with high DNA levels, positive control templates or from previous amplifications can ϊesult in PCR product, even in the absence of purposefully added template DNA. If possible, all reaction mixes are set up in an area separate from PCR product analysis and sample preparation. The use of dedicated or disposable vessels, solutions, and pipettes (preferably positive displacement pipettes) for RNA DNA preparation, reaction mixing, and sample analysis will minimize cross contamination. See also Higuchi and Kwok, 1989, Nature.222:237-238 and Kwok, and Orrego, in: Innis £l al eds., 1990 PCR Protocols: A Guide to Methods and Applications. Academic Press, Inc., San Diego, CA, which are incorporated herein by reference. One particular method for minimizing the effects of cross contamination of nucleic acid amplification is described in U.S. Serial No. 609,157, filed November 2, 1990, which is incorporated herein by reference. 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. For example, uracil-DNA glycόsylase wiU 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 wiU continue to lead to the discovery of many previously unknown or uncharacterized oncogenes. For example, using the methods, primers, and probes of die invention, four clinical samples examined were discovered to contain two different Giα2 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. In one embodiment, 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 typicaUy without great impact on the suitabiUty of that primer or probe in the present methods. The essential aspect of these probes is their abiUty 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. There are a number of ways to determine whether a probe has hybridized to a DNA sequence contained in a sample. Typically, 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 soUd support, and determination of whether hybridization has occurred simply involves determining whether the label is present on the soUd 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. The hybridization probes disclosed herein are sequence-specific oUgonucleotide probes for each G-protein α subunit codon to be characterized. As described above, sequence-specific probes are similar to allele-specific probes in use and utiUty. Methods for utiUzing ASOs are described in Saiki el al, 1986, Nature 324: 163-166, incorporated herein by reference. The probes may be used individually for detecting, for example, a wild type sequence. Alternatively, 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. Although solution hybridization of a nucleic add probe to a complementary target sequence is clearly within the scope of the present invention, commerdalization of the invention will likely result in the use of immobilized probes and thus a quasi "soUd- phase" hybridization. Li this format the probe is covalently attached to a soUd support and target sequences are hybridized with the probe. A preferred method for immobiUzing probes on soUd supports is disclosed in U.S. patent appUcation S.N. 347,495, filed May 4, 1989, incorporated herein by reference. According to this method, sequence-specific probes are attached to a solid support by virtue of long stretches of T residues which are added during probe synthesis on an automated synthesizer after the hybridizing sequence is synthesized. For example, in a fixed probe format, the foUowing probes are useful for detecting point mutations in a gene encoding Gsα at amino add positions 201 and 227. Table 1 Amino Add Probe Name ≤sausnce Position JFL316 5'TCGCTGCCGTGTCCTGGAC 201 ' JFL317 5 CGCTGCAGTGTCCTGGACT 201 JFL318 5'TCGCTGCGGTGTCCTGGAC 201 JFL319 5'TCGCTGCTGTGTCCTGGACT 201 JFL320 5'TCGCTGCCATGTCCTGGACT 201 JFL321 5 CGCTOCCTTGTCTTGGACT 201 JFL322 5'TGGGTGGCCAGCGGCGATGA 227 JFL323 5'TGGGTGGCCTGCGCGATGA 227 JFL324 5'TGGGTGGCCCGCGCGATG 227 JFL325 5'TGGGTGGCCGGCGCGATG 227 JFL326 5'TGGGTGGCGAGCGCGATGA 227 JFL327 5'TGGGTGGCTAGCGCGATGA 227 JFL328 5'TGGGTGGCCATCGCGATGA 227 JFL329 5 GGGTC 3CAAGCGCGATGA 227 JLF330 5'TGGGTGGCCACCGCGATG 227 A fixed probe format is suitable for detecting other gsp mutations, as weU, and is demostrated in Example 5 for detecting point mutations in as gene PCR products. Many methods for labeling nucleic acid , whether probe or target, are known in the art and are suitable for purposes of the present invention. In one embodiment iUustrated below the probes were labeled with radioactive phosphorous 32 P, by treating the probes with polynucleotide kinase in the presence of radiolabelled ATP. However, other non-radioactive labeling systems may be preferred, i.e., horseradish peroxidase- avidin-biotin systems. Horse-radish peroxidase (HRP) can be detected by its abiUty to covert diaminobenzidine to a blue pigment A preferred method for HRP-based detection uses tetramethyl-benzidine (TMB) as described in Clin. Chem.22:1368 (1987). An alternative detection system is the enhanced chemiluminescent (ECL) detection kit commercially available from Amersham. The kit is used in accordance with manufacturer's instructions. A variety of alternative dyes and chromogens and corresponding labels are available for nucleic acid detection systems (see, e.g., U.S. patent appUcation S.N. 136,166, filed December 18, 1987). Another non-radioactive alternative detection method uses terminal transferase (Tdt) and biotinylated dUTP to add homopolymer tails to the oUgonucleotide probes. Biotin serves as the detectable moiety. FoUowing probe hybridization, 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 visuaUze the biotin-HRP product. Probes are typicaUy labeled with radioactive phosphorous 32 P, by treating the probes with polynucleotide kinase in the presence of radiolabeled ATP. However, for commercial purposes 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 ei al-, 1988, Nuc. Adds Res.16:8719) instead of the probe, then hybridization can be detected by assay for the presence of labeled PCR product. Li a preferred embodiment, probes are biotinylated and detected with the ECL system described above. For example, biotinylated probes are prepared by direct biotinylation of the oUgonucleotide rather than incorporation of biotin-dUTP during PCR. For 5' biotinylation of oUgonucleotides direct soUd phrase synthesis using biotin containing phosphoramidites is done according to Alves ei al, 1989, Tetra. Let 2Q:3098; Cocuzza, 1989, JsύΑ Let- 2Q:6287; and Barabino sL al, 1989, EMBO J_. &4171. Solid phase synthesis of biotinylated oUgonucleotides at any internal or terminal (5' or 3') position is also suitable for preparing biotinylated primers and probes (Pieles ≤I al-, 1989, NAR 1&4355, and Misiura ≤t al, 1989, NAR 11:4345). Alternatively, probes and primers are conjugated to HRP, for example, by the method disclosed in WO89 2932, and Beaucage si al, 1981, Tetra. Lett.22:1859-1862. These references are incorporated herein by reference. Those skiUed in the art wiU recognize that with the above description, primers, and probes for amplifying, detecting, and characterizing new G-protein point mutations can be readily obtained. It wiU also be readily apparent to those skiUed in the art that the specific primers and probes provided in the examples are merely iUustrative 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 specificaUy exemplified herein, for example, codons corresponding to Gsα 49. The method of the present invention is appUcable for detecting a point mutation within a gene encoding a GTP binding protein or the expression product of that gene. Thus, the method can detect specific point mutations in a sample containing RNA, or DNA, or both. If the sample contains RNA, the nucleic acid wiU be reverse transcribed, providing a double-stranded DNA template prior to amplification. Procedures for reverse transcribing RNA are known (see Maniatis ≤J al, 1982, Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, NY). In one embodiment, the methods are used to detect point mutations in RNA. Alternatively, if RNA is abundant, the probes (or the complement of a probe sequence) are suitable for direct detection and characterization of an oncogene or proto- oncogene 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. Li one embodiment of the invention, paraffin-embedded samples are analyzed using G-protein primers and probes. In another embodiment, methods are provided for ias oncogene detection in RNA purified from surgical biopsy samples or cultured ceUs. 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 Giβ, Gi 2 , Gsα, Gii, Gz, and Go. Table 2 Primers and PCR Conditions for G-Protein Analysis Amino Gene Acid Product Position Gi 3 α 201 (178) Gi 3 α 227 (204) Gi 2 α 201 + 227 nested (179 + 205) primer outers set 201 + 227 (179 + 205) inners Gsα 49 Gsα 201 + 227 nested outers primer JFL 70, antisense set CGCAGGGGGTGGGCGGTCACTCCA Table 2 - Continued Primers and PCR Conditions for G-Protein Analysis Amino Number Product Gene Acid Cycling Parameters of Size Product Position Primer Pair 1 2 Cycles Gsα 201 + 227 JFL 135, sense 95°C 57°C 72'C 30 526 nested inners GTGATCAAGCAGGCTGACTATGTG 30 sec 40 sec 45 sec primer JFL 136 antisense set GCTGCTGGCCACCACGAAGATGAT Giiα 201 (173) JFL 223, sense 95'C 53 β C 72°C 40 99 TTG GAC AGA ATA GCT CAA CCA AAT SP 33 antisense GAA AGT AAA ATG GGT TTC AAC Gtι<& 227 (199) JFL 2 , antisense 95°C 53°C 72"C 40 114 TAG AAC CAG GTC GTA GTC ACT SP 34, sense I AAA TGT TTG ATG TGG GAG GT Gi 2 α 49 (42) JFL 235, sense 95°C 55 * C 72 * C 45 63 GCC CTC TGT TCC AGG TGC 30 sec 30 sec 30 sec JFL 237, antisense ACTTACTTCATCTGCTTGACG Gi 2 α 201 (179) JFL 55, sense 95'C 57°C 72'C 50 118 ATT GCA CAG AGT GAC TAC ATC CCC 60 sec 30 sec 30 sec JFL 212, antisense GCT CAC TTG AAG TGT AGG Gi 2 α 227 (205) JFL 215, sense 95 * C 57 * C 72'C 50 -123 CTG CAG GAT GTT TGA TGT GG 60 sec 30 sec 30 sec JFL 56, antisense GGCGCTCAAGGCTACGCAGAA Table 2 - Continued Primers and PCR Conditions for G-Protein Analysis Amino Gene Acid Product Position Goα 201 Goα 227 Gzα 201 + 227 Gsα 201 Gsα 227 JFL 13 , antisense GCTGCTGGCCACCACGAAGATGAT Numbers in parentheses indicate the exact amino acid position within the named G protein subunit. Gi 3 amino add position 178 corresponds to position 201 in Gsα, and position 204 corresponds to position 227 in Gsα. For Giiα the amino acid positions 173 and 199 correspond to positions 201 and 227, respectively in Gsα. For Gi2α amino acid positions 42, 179, and 205 correspond to Gsα 49, 201, and 227, respectively. Li some instances DNA samples are less weU preserved and, consequendy, amplification of long segments is problematic. Example 1 describes primers for amplifying short segments of DNA that include oncogenic regions of gsp. Thus, 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 Remission and recurrence of tumors can be monitored as weU. Comparing the activation of proto-oncogenes into oncogenes at the genomic level, with the expression of activated oncogenes at the mRNA level, provides valuable information for analysis of cardnogenesis and therapies. These methods may also provide information regarding a predipostion to specific maUgnanάes. The invention provides needed tools useful in analysis of cardnogenesis events. Probes are provided for determining aUeUc dominance for a specific oncogene. For example, probes specific for Giα2 aUeles can be used to analyze somatic ceUs as weU as tumor cells. These probes can also distinguish, in the case of a tumor comprising one mutant and one wild type aUele, the relative abundance of the mRNA products of these aUeles. One skilled in the art will recognize the utiUty of such analyses foUowing study of and studying mutagenic events. Li another aspect, the primers and probes described provide a method for phenotyping a ceU. The ceU may be a tumor ceU or a somatic ceU. Analysis using the methods of the invention provides information relating to the proto-oncogene and oncogene profile of a ceU. 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 maUgnancy with a specific oncogene or point mutation. For example, of 306 samples for 15 tumor types analyzed for Gsα, 18 point mutations were detected; aU 18 Gsα oncogenes were detected in pituitary adenomas and thyroid tumors. In another example using Giα2 primers and probes, 254 samples representing 14 different tumor types were analyzed. Four tumors of two types had Giα2 oncogenes; one ovarian granulosa ceU 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 α subunits are activated in endocrine tumors. These studies suggest that distribution of Gsα and Giα2 oncogenes is restricted among specific endocrine target ceUs. An analysis of G-protein oncogenes and tumor specificity provides information of useful in determining the role of G-proteins, where that role is as yet undefined. For example, the data suggests that corticotropin (ACTH), which stimulates cσrtisol secretion via Gs, adenylyl cyclase, and cAMP, does not utiUze Gs and cAMP to stimulate proliferation of ACTH target ceUs. Tumors derived from adrenal cortical ceUs (the target ceU of ACTH) do not harbor the Gsα oncogene. Those skiUed in the art will recognize that the methods of the present invention provide essential tools for identifying new oncogenes, such as Giα2 (alternatively referred to herein as gip2) and exploring the diverse mix of signalling pathways that mediate regulation of prolif eration in endocrine target ceUs. 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. Thus, antibodies enabled by the present invention would also find use in, for example, screening transplantation tissue as an indicator of potential oncogenic compUcations. It wiU be apparent to those skiUed in the art that the method of the present invention is amenable to commercialization as a kit for the quantitation of one or more nucleic acids in a sample. For example, in its simplest embodiment such a kit would provide an oUgonucleotide primer pair for amplification of a G-protein α subunit segment and corresponding wild type oUgonucleotide probe. L another embodiment, a kit may contain an array of G protein probes fixed onto a soUd support and a corresponding primer pair for amplifying and detecting oncogenic point mutations in genes encoding G protein subunits. Li another embodiment, a kit may contain an oUgonucleotide primer pair, corresponding G-protein α subunit wUd 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. Further, the kits may contain a thermostable DNA polymerase; for example, the thermostable DNA polymerase Taq isolated from Thermus aquaticus as an agent of polymerization. To faciUtate the understanding of the invention, brief definitions are provided below. "Proto-oncogene" refers to the wUd 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 α subunit primers" refer to primer pairs which hybridize to complementary strands of a nucleic add encoding a G-protein α subunit and wiU function in a PCR reaction to amplify a nucleic add segment comprising one or more codons suspected of harboring a point mutation. In the disclosed embodiment, these codons encode amino acids corresponding to Gsα amino acids 49, 201, and 227. However, those skiUed in the art wffl recognize that potentiaUy oncogenic point mutations may exist at other positions as weU. G-protein α subunit primers can readily be designed to amplify such regions according to the methods described herein. "G-protein α subunit probe;" "G-protein probe," or "probe" as used herein refers to an oUgonucleotide probe designed to characterize the nucleic add sequence encoding an amino acid at a position suspected of containing a point mutation. In the preferred embodiment of the present invention, 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. In the present examples, the specific codons include for each G-protein α subunit, codons encoding the amino acids corresponding to Goα amino acid positions 49, 102, and 227. For any specific codon and any specific G-protein, one wUd type probe and nine point mutation probes may bfc designed and used in the present methods. However, it is not an essential aspect of the invention that probes be included for practice of these methods. L fact, the use of only one probe may be sufficient to discriminate between two individuals or between the presence and absence of a point mutation. The foUowing examples provide an iUustration of the present invention. They are not a limitation to the scope of the invention. Those skiUed in the art wiU recognize that the primers and probes disclosed can be modified, for example, by altering the length of an oUgonucleotide without altering the purpose and effectiveness of the described invention. Example 1 Materials and Methods A. Source of Tumors Human pituitary tumors specimens were supplied by Charles WUson (University of CaUfomia, San Frandsco) and Anna Spada (MUan, Italy) provided biochemicaUy characterized pituitary samples. Additional samples were provided by Hans Feichtinger and Kurt Grϋnewald at the University of Innsbruch, Austria, and Claudia Landis, Griffith Harsh, Quan- Yang, Dun and Qrlo Clark at the University of California, San Frandsco. B. Sample Preparation For isolation of genomic DNA from paraffin-embedded tissue, 3-5 adjacent 5 mm sections were cut from paraffin blocks and mounted on glass sUdes (Wright ei al- PCR Protocols: A Qm S. iΩ. Methods ami Applications eds. M. Innis, D. Gelfand, J. Sninsky, and T. White, Academic Press, San Diego, pp. 153-158, incorporated herein by reference). One sUde was stained with hematoxylin and eosin and used as a guide to select a region composed entirely of tumor on the other sUdes. With a razor blade, excess paraffin and unwanted tissue were removed from the unstained sUdes, and the remaining tumor tissue was scraped into a sterile 1.5 ml microcentrifuge tube. To remove contaminating paraffin, the sample was incubated with 5 mis of octane (anhydrous, Aldrich) or Hemo-De (Fischer) at room temperature for 30 minutes with shaking. The tissue sample was peUeted by centrifugation (5 minutes, 1000X g) and the supernatant was discarded. The tissue sample was extracted 2X with 500 mis absolute ethanol to remove traces of octane and then vacuum dried. To digest the tissue and release the genomic DNA, 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 ei al., 1986, Gene 50:313. C. Amplification Procedure Nested amplification primers were used in the PCR amplification procedure to improve specificity and yield (MulUs & al, supra " ). Genomic DNA (100-500 ng DNA from fresh tissue or 10 mis of the DNA solution from paraffin-embedded tissue) was first amplified with 30 pmols of the outer primers (see Table 1) in 100 mis of 0.1 mM dNTPs; 50 mM KC1; 20 mM Tris, pH 8.3; 2.5 mM MgC12; 100 g ml BSA; and 1.5 units Taq polymerase (Perkin-Elmer Cetus). A theπnocycler (Peririn-Elmer Cetus) was used for amplification. The amplification program was: 5 minutes at 95 β C foUowed by 30 cycles of 1 minute at 95 β C, 2 minutes at 50"C, and 2 minutes at 72 β C. A second amplification reaction with 30 pmols of the inner primers was done using 2 μls 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 μls of the final product was sized on a 2% Nusieve, 1% Seakem agarose gel and visuaUzed by ethidium bromide staining. A 526 base pair fragment was obtained for Gsα and a 504 bp fragment for Giα2. P. Dot Blot Procedure Nylon filters (PaU 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 β C for 30 minutes. One ng of a 32 P end-labeled oUgonudeotide was added to the prehybridization solution and incubated at 50°C for 45-60 minutes. The hybridized filters were washed briefly in 2X SSPE and 0.1% SDS at room temperature, foUowed 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 Gsα codon 201, 64.5°C; Gsα codon 227, 67'C; Gioώ codon 179, 61.5'Q Giα2 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. In this way, the appropriate wash temperature is determined. Under these conditions, the chosen temperature aUows only fuUy 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 oUgonucleotides, the nylon filters were stripped by boiling the filters for five minutes in 2X SSPE, 0.1% SDS and then processed as described above. E, Sequencing For double-stranded sequencing of PCR product a single band of appropriate size was exdsed from an ethidium bromide-stained agarose gel under UV Ught The excised band was placed into a Costar spin-X 0.22 μm ceUulose acetate filter unit frozen at -70 * C for 15 minutes and spun in an Eppendorf microcentrifuge for 15 minutes at fuU speed. The DNA-containing eluate was transferred to a microcentrifuge tube, 20 μg glycogen was added, and DNA was predpitated with 0.2 volumes of 3 M sodium acetate and 0.3 volumes ISO propanol. DNA was peUeted in a microcentrifuge, washed with 70% ethanol, vacuum dried, and resuspended in 20 μl double distilled water. The sample was sequenced according to the Sequenase (United States Biochemical) protocol using 7.75 μl DNA solution and 2.5 pmol sequencing primer. F. OUgonucleotides 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 α subunit the primer pair is suitable for amplification of either a DNA or cDNA template. The nucleic acid sequence of the G-protein α subunits are pubUshed for Giα (see Bray si al, 1987, Proc. Nad. Acad. £ci. USA 24:5115-5119), Gsα (see Kozasa ei al-, 1988, Proc. Nafl. Acad. Sci. USA £5_:2081-2085), Goα (see Lavu ei al., 1988, Biochem. Biophys. Res. Comm. 150:811-815 ' ). and Gzα (see Fong Si al, 1988, Proc. Nad. Acad. Sci. USA S5_:3066-3070). For each primer pair, the size of the amplification product from a genomic DNA template is shown. Note that for Giαl, Giα2, Giα3, Goα, and Gzα the designation 201 and 227 refers to the codon encoding the amino acid corresponding to position 201 or 227 in amino add sequence of Gsα. Table 3 AmpUfication Primers for Detection of G-Protein Point Mutations Primer Gsα 201/227 Outer Primers Position JFL69 5' GCG CTG TGA ACA CCC CAC GTG TCT intron JFL70 5 ' CGC AGG GGG TGG GCG GTC ACT CCA intron Gsα 201/227 Inner Primers (526 bpt JFL135 5 ' GTG ATC AAG CAG GCT GAC TAT GTG exon JFL136 5 ' GCT GCT GGC CAC CAC GAA GAT GAT exon Primer Gsα -201 (289 h ^ Position JFL228 5' AAG AAA CCA TGA TCT CTG TTA TAT intron JFL135 5' GTG ATC AAG CAG GCT GAC TAT GTG exon Gs -227 (263 h ^ JFL229 5' CCC CAG TCC CTC TGG AAT AAC CAG intron JFL136 5' GCT GCT GGC CAC CAC GAA GAT GAT exon Gsα -49 (171 bp^ JFL226 5' AAC AGC AGA CCT CCC TGC CCA AAG intron JFL227 5' CCC CCC TGC ACA GAT TTG ACA CTT intron Giαl -201/227 JFL223 5' TTG GAC AGA ATA GCT CAA CCA AAT exon JFL224 5' TAG AAC CAG GTC GTA GTC ACT exon Giα2 - 201/227 Outer Primers 5' CCC CCC ATC CCC AGC TAC CT exon/intron 5' TCT CAC CAE«,CTC CTC GTC CTC exon/intron QiαZ - ail Inner famsrs (54 bp) 5' ATT GCA CAG AGT GAC TAC ATC CCC exon 5' GGC GCT CAA GGC TAC GCA GAA exon Giα3- 201 Outer Primers 5' TGT CTT TTA TTT AGT ATC AA exonintron 5' GAT CTG GAT AGA ATA TCC CAG exon/intron Gi«3 - 201 Inner Pπmers (99 bp 5* GAT CTG GAT AGA ATA TCC CAG exon 5' GGT GAA ATG TGT TTC TAC AAT exon Giα3- 227 Outer Primers 5' TTC CCC TTG CGC AGG ATG TTT exonmtron 5' ACA TAC CAT CTC CTC GTC CTC exonintron Giα3 - 207 Inner Primers (120 bp^ 5' CAG AAC AAG GTC ATA ATC ACT exon 5' TTC CCC TTG CGC AGG ATG TTT Goα - 201 (87 bp^ 5' CTG GAC AGC CTG GAT CGG ATT GGG exon 5' GAG GTT CTT GAA TGT GAA GTG GGT exon Goα -227(129 h ^ 5' AAC CTC CAC TTC AGG CTG TTT exon 5' GTG GAG CAC CTG GTC ATA GCC GCT exon Gzα -201/227 (-200^ 5 * TGT GAC GCC CTC GAA GCA GT exon 5' AAC GAC CTG GAG CGC ATC GCC exon 25 Giα2 - 201 5 * TA CGG ACC £Q£ GTA AAG ACC 3' Ajg AGC Ser GGC Gly TGC Cys CAC His * CCC Pro CTC Leu Gc-α - 227 5 ' GTC GGA GGC CΔG CGA TCT GA 3 ' Gin AAG Lys GAG Glu CCG Pro CGG Arg CTG Leu CAT His CAC His Qm - 2v\ 5 ' TG CGC TCC CGG GAC ATG ACC 3 ' Ajg GGG Gly TGG Trp CAG Gin CCG Pro CTG Leu Gzα - 227 5 ' TG GGG GGG CΔG AGG TCA GAG 3 ' Gin AAG Lys GAG Glu CCG Pro CTG Leu CAC His CAT His CGG Arg For each G-protein codon to be characterized, oUgonucleotide probes are shown in Table 4 as foUows. For each position, i.e., Gsα 201, the full probe sequence is shown for the wUd type aUele. The wild type codon at the potentiaUy 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 wUd type except at the codon to be characterized. Thus, for non-wUd 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 wUd type amino acid are shown. Table 5 shows stretches of Gsα sequence surrounding the arginine-201 and glutamine-227 codons which are highly conserved in G-protein α chains of vertebrates, yeast, and slime mold. PubUshed sequences include rat Gsα, Giα2, Giα3, and Goα, human Gzα (Katada and Vi, 1982, Proc. Natl. Acad. Sci. USA 22:3129), Gtα of bovine retinal rod cells (Chambard ≤t al., 1987, Nature 226:800), the α chain of the G- protein (caUed GPAl/SCGl) that mediates pheromone signalling in Saccharomyces cerevisiae (Corven ei al-, 1989, CsJl 52, and Itoh & al-, 1988, J_. Biol. Chem. 263:6656). and an α chain (Gαl) from DictvosteUum discoideum (Zarbl £1 al-, 1985, Nature 315:382). The number is parenthesis foUowing each sequence is the actual amino acid position of the last amino acid in the sequence shown. In the table, a one letter amino acid code is used where: D = Asp K = Lys L = Leu G = Glu R = Arg M = Met C = Cys A = Ala V = Val Q - Gln T- Thr F = Phe S = Ser E = Glu I = Ue Table 5 Conservation of Sequences Among G-Protein α Chains ∞ t Example 2 Identification of G-Protein Point Mutations in Gsα Genes To determine the frequency of mutations in codons 201 and 227 of Gsα genes in pituitary and other tumors, the polymerase chain reaction (PCR) was used to amplify 5 a specific region of genomic DNA and high-stringency hybridization of sequence-specific oUgonucleotides to detect point mutations in the amplified product To detect mutations in the Gsα gene, a single region including both codons 201 and 227 and an intervening intron was- amplified from tumor genomic DNA prepared from fresh frozen or paraffin embedded samples as described in Example 1. The 10 amplification primers shown in Table 3, designated "Gsα 201/227 Outer Primers" and "Gsα 201 227 Inner Primers," were used according to the method described above. OUgonucleotides specific for wUd type or single-base mutations at codon 201 (6 possible missense mutations) or codon 227 (7 possible missense mutations, 1 nonsense mutation) were hybridized to the a pUfied product (Table 4). Genomic DNA from 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 normaUy to stimulatory agents, group 2 tumors had marked elevation of basal adenylyl cyclase activity that responded poorly to stimulatory agents. 20 The hybridization results are shown in Figure 1 A. The hybridization probes shown in Table 4 were used as fottows: R201 indicates the wUd type probe for Gsα 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 25 probe corresponding to Gin 227 containing a point mutation (CAG to CGG) encoding arginine. in aU tumors where a mutant aUele was detected, suggesting that mutations that activate Gs are dominant. Of 42 GH-secreting pituitary tumors, 16 were biochemicaUy characterized in terms of adenylyl cyclase activity. Eight tumors showing elevated adenylyl cyclase were predicted to harbor an activated Gsα; each of these tumors contained a mutation in codon 201 or codon 227 (Figure 1A). No mutations were detected in eight tumors that showed normal adenylyl cyclase activity. Although Gsα mutations in other codons may also inhibit GTPase, the strong concordance between elevated adenylyl cyclase activity and a mutation in codon 201 or codon 227 indicates that activating mutations at other sites are relatively infrequent Table 6 provides a summary of human tumors screened for mutations in codons 201 and 227 for Gsα and codons 179 and 205 for Giα2. DNA for PCR amplification MuUis £t al, supra., was isolated from either fresh tissue, Verlaan-de Vries et al, 1986, Gene 5_Q:313, or paraffin-embedded tissue Kozasa ei al., 1988, Proc. Natl. Acad. Sci. USA 85:2081. Eighteen growth hormone (GH) secreting pituitary adenomas contained a mutation in either Gsα codons 201 or 227. One ovarian granulosa ceU tumor and three adrenal cortical tumors (two adenomas, one carcinoma) contained a mutation in Giα2 codon 179. Table 6 HΠIQΓ Type Pituitary adenoma GH Prolactin TSH ACTH Nonsecretor Ovarian granulosa ceU tumor Adrenal cortical tumori Thyroid tumors 2 Melanoma GUoblastoma Ovarian adenocarcinoma Gastric adenocarcinoma Renal ceU carcinoma w Breast adenocarcinoma Bladder transitional ceU carcinoma Pancreatic adenocarcinoma Acute myelogenous leukemia Squamous cell carcinoma Colonic adenocarcinoma Hepatoma Prostatic tumor3 306 254 1. Adenoma (5 for sα, 4 for iα2); carcinoma (6 for sα, 5 for iα2) 2. Papillary carcinoma 6; foUicular carcinoma 3; follicular adenoma 3; Hurthle cell carcinoma 1; multinodular goiter 3; medullary carcinoma 8 3. Benign hyperplasia 6, adenocarcinoma 6 Table 7 provides a Ust of wUd type codons for the conserved arginine and conserved glutamine in Gsα and Giα2 genes. The table also shows single-nucleotide base changes (in bold) and the resulting amino acid changes. OUgonucleotides specific for wild type and each missense or nonsense single-base change Usted were used to screen human tumors, with the exception of base changes that would be sUent (marked with asterisks). Mutations detected in the tumors Usted in Table 6 are underlined. "Term" indicates termination or stop signal. Table 7 Possible Mutations in Two Key Codons of Gsα and Giα2 Arginine Glutamine sα WT=CGT (Arg 201) WT=CAG (Gin 227) TGT Cvs CAT His CGC Arg* TAG Term CGG Arg CAA Gin* GGT Gly CCT Pro CGG Arg* CAG Glu CCG Leu CAC His AGT Ser CTT Leu CGA Arg* AAG Lys CTG Pro CAI His iα2 WT=CGC (Arg 179) WT=CAG (Gin 205) TGC Cvs CAC His CGT Arg* TAG Term CGG Arg CAA Gin* GGC Gly CCC Pro CGG Arg* GAG Glu CCG Leu CAC His AGC Ser CTC Leu CGA Arg* AAG Lys CTG Pro CAT His t-*l Example 3 Identification of G-Protein Point Mutations in Giα2 Genes In order to investigate the possibiUty that mutational activation of signaling pathways mediated by other G-proteins might lead to abnormal proliferation and tumor formation, a large panel of human tumors was screened for mutations in a Giα gene. In Giα2 the coding sequence and intron between the two codons to be tested, arginine-179 (corresponding to Gsα Arg 201) and glutamine-205 (corresponding to Gsα Gin 227), is short enough to aUow PCR amplification of a single genomic DNA fragment containing both codons (Itoh £i al., 1988, ∑. Biol. Chem.263:6656). The primers and probes used for detection and characterization of Giα2 are shown in Tables 3 and 4. Samples were prepared and analyzed as described in Example 1. The hybridization results are shown in Figure IB. 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 Giα2/201 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 Giα2/227 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 Giα2 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 wUd type aUele 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 ceU types suggests that these mutations converted the Giα2 gene into an oncogene, referred to herein as gip2 (for Gi protein-2). Strikingly, the amino acids that replaced arginine-179 in Giα2, cysteine and histidine, were the same as those that replaced the cognate arginine at position 201 in Gsα oncogene products found in pituitary tumors (Landis ei al). It is possible that, of the six possible missense mutations that can result from single-base changes in these codons of Gsα and Giα2, only these mutant proteins are biologicaUy active. Also, aU mutations found so far, in either Gsα or Giα2, are transition mutations (Table 7); consequently, these mutations may reflect a common mutagenic mechanism. In one tumor of the adrenal cortex, a normal aUele of Giα2 was not detected (Figure IB). Sequence analysis of PCR products revealed a single sequence, corresponding to the codon 179 mutation. This result makes it likely that the normal aUele was missing, although the possibiUty that both aUeles contain the same mutation cannot be excluded. Loss of the normal aUele suggests that its protein product interferes with the oncogenic effect of the mutant protein, so that faUure to express normal Giα2 confers an additional selective advantage on ceUs carrying an activating Giα2 mutation in the other aUele. Example 4 Detection of g.ψ Mntations n Paraffin-Embedded Samples bv Microdissection Changes in the two Gsα codons affected by gsp. mutations were detected in formalin-fixed, paraffin-embedded tissue blocks of human tumors by isolating genomic DNA, amplifying appropriate Gsα sequences with the polymerase chain reaction (PCR), and screening the amplified products for their abiUty to hybridize with aUele- specific oUgonucleotides. In Examples 1-3 DNA from aU ceUs in a 5 mm tissue section was analyzed. DNA from one thyroid tumor had two different gsjj mutations, in addition to the wUd type aUele. To ask whether the two mutations were located in different regions of the 5 m tissue sUce, an adjacent 5 mm section from the same tumor was divided into several smaUer fragments; genomic DNA was isolated from each fragment separately and subjected to PGR amplification and screening with aUele- specific oUgonucleotides. The two gsβ mutations were detected in different fragments; in addition, several fragments contained only the wUd type codons at positions 201 and 227 of Gsα. The latter observation suggested that the heterogeneous distribution of mutations in 5 mm sections may go undetected due to dilution by wild type DNA sequences from ceUs in parts of the sections that contained no mutations. Accordingly, this modified microdissection approach was applied to additional tumors. Identification of a high prevalence of gsp. mutations some in tumors earUer tested as negative indicated that this approach is more sensitive at detecting gsβ mutations. A. Mahgnant Thvroid Tumors To explore the roles of gsp mutations in pathogenesis of maUgnant thyroid tumors, the microdissection technique to tissue blocks of primary tumor or lymph node metastases from 37 patients with differentiated thyroid carcinoma. These tissue fragments were also tested for mutations in specific codons of the three human ras genes. Stained sections from these surgical specimens were examined and each microdissected fragment was classified as maUgnant or benign thyroid tissue; in every case, the pathologic diagnosis appUed to at least 90% of the ceUs in the fragment. Both the pathologic examination and theφNA analysis were performed in a blinded fashion: The pathologist diagnosed histology of tissue fragments without knowledge of the presence or absence of mutations, and the DNA analyses were performed on coded samples. B. Sample Preparation Multiple 5 mm sections were cut from each tissue block and one was stained with hematoxylin and eosin. Regions of the stained sUde, 30-100 mm 2 in area, were demarcated with a pen and designated by number, when possible, demarcations separated histologically distinct regions. Using the stained sUde as a template, an adjacent unstained 5 mm section was divided into corresponding fragments with a razor blade. These fragments were transferred into separate tubes and each was processed for PCR and hybridization screening, exactly as described in Example 1. C. PCR Amplification Using 20-25 base pair (bp) oUgonucleotide primers upstream and downstream of codons 201 and 227 in the human Gsα 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 described in Example 1, however, the oUgonucleotides and cycling temperatures used were as were as foUows. For nested primer amplification of Gsα codons 201-227: Outer sense, (JFL69) 5'GCG CTG TGA ACA CCC CAC GTG TCT; outer antisense (JFL70), 5'CGC AGG GGG TGG GCG GTC ACT CCA; product 1,200 bp; optimal PCR condition, 30 cycles of 1, 2, and 2 min at 95750772°; inner sense (JFL135), 5'GTG ATC AAG CAG GCT GAC TAT GTG; inner antisense (JFL136), 5'GCT GCT GGC CAC CAC GAA GAT GAT; product 526 bp; optimal PCR condition, 30 cycles 30, 40, and 45 sec at 95757772°. For Gsα codon 201: sense (JFL135), 5'GTG ATC AAG CAG GCT GAC TAT GTG; antisense (JFL286),5' TA ACA GTT GGC TTA CTG GAA; product 222 bp; optimal PCR conditions: 40 cycles of 30, 30, and 30 sec at 95755772". For Gsα codon 227: sense (JFL229),5' CCC CAG TCC CTC TGG AAT AAC CAG; antisense (JFL136), 5'GCT GCT GGC CAC CAC GAA GAT GAT; product 165 bp; optimal PCR condition, 50 cycles of 60, 30, and 30 sec at 95755772°. 37 For i s gene ampUcations the foUowing standard PCR conditions of 50 cycles of 60, 30, and 30 sec 95755772° were used. Ras primers were as foUows. 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. For 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; for Ki-ras codon 61: sense (JFL248), 5'GTA ATT GAT GGA GAA ACC TG; antisense (JFL249), 5'ATA CAC AAA GAA AGC CCT C product 112 bp. For N-ias codons 12 andl3: sense (JFL216), 5'CTT GCT GGT GTG AAA TGA CT; antisense (JFL257), 5;GGT GGG ATC ATA TTC ATC TA; product 150 bp. For 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. RepUcate PCR amplifications and analyses of adjacent 5 mm sections produced similar results, indicating that the procedure was accurate and reprodudble as weU as sensitive. To avoid false positives by nonspecific hybridization of mutant probes to the PCR product in the absence of mutations, a level of hybridization was set, below which a sample would be considered negative for a particular mutation. This level was set at 20% of the signal detected by hybridization of the wUd 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 ceUs in the assayed tissue fragment contain the mutation, if — as expected for dominant somatic mutations — each ceU has one wUd type and one mutant aUele. D. Hybridization and Detection of Point Mutations For mutation-specific oUπuc eotide hybridization, the PCR product was spotted (dot-blot apparatus, Bio-Rad) and cdvalently bound to a nylori filter (PaU Biodyne-B, 0.45 um) using UV Ught at the auto-crosslink setting (StrataUnker, Stratagene). Hybridization with P P]-radiolabdid mutation-specific oUgonucleotides 20 bp long were performed exactly as described in Example 1. E. Criterion for Presence of a Mutation To verify the presence of point mutations we scanned aU a s hybridization reactions with an AMBIS radioanalytic imaging system (Ambis Co., San Diego, CA), which measures the emission of radioactivity from each mutant (CPM m ) or wild type 5 (CPM wt ) dot on the filter. The p 2 P] b radioactivity of mutant oUgonucleotides nonspecificaUy bound to the same filters after high stringency washing was termed background activity (CPM b ), and ranged from 5 to 10 % of the wUd type hybridization signal. A dot was considered to represent a gsp mutation if (CPM m - CPM b ) divided by (CPMwt - CPM b ) was greater than or equal to 0.2. This criterion thus required that 0 ampUfied DNA samples judged as positive for a mutation must exhibit a mutant signal 20% of that observed with wUd 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. F. Controls 15 The microdissection procedure gave negative results for Gsα and ias mutations in sections of normal connective tissue and human thyroid removed dining parathyroidectomy. To confirm positive results, PCR products from six fragments for which hybridization results indicated R201C mutations at levels near the demonstrated cutoff point (i.e., 22-35%) were sequenced. Genomic DNA was ampUfied using one 20 biotinylated and one non-biotinylated primer, to generate umlateral biotinylated PCR products. After binding the biotinylated PCR product to 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 aU six cases the sequencing gels showed both the wUd type and the mutant 25 201 codon, confirming the results of dot blot hybridization. G. Number and Distribution of Mutations Gsp mutations were found in surgical spedmens from 24 of 37 patients (65 %) with differentiated thyroid cancer. Gsp mutations in these patients were heterogeneously distributed among microdissected fragments; overaU, 81 of 266 30 thyroid tissue fragments (30.5 %) contained detectable gsp mutations. Among the 24 surgical specimens with at least one g≤B positive fragment, a ≤β mutation was found in 60 of 120 histologically maUgnant fragments (50 %). The mutations were not confined to fragments with obviously maUgnant tissue, however, indeed, in the same tumors 21 of 60 histologicaUy benign fragments (36.2 %) also contained g≤β mutations. Of the 24 patients in whom the microdissection technique revealed gsp mutations, only five were originaUy suspected of harboring gsp on the basis of the previous testing procedure, which used the entire 5 m section as a single sample. In each of these five previously positive cases, more than half of the fragments tested with the new procedure were positive for gs mutations, a frequency found in only one other case. These observations indicate that without microdissection the dilution of samples by DNA from regions without mutations can cause a substantial proportion — perhaps more than 75% — of thyroid gsp mutations to be missed. Specimens from six of the 37 patients harbored two different gsp. mutations, and the tumor of one patient (number 22, Table 8) contained three different gsp mutations. These multiple mutations were found in separate fragments of these specimens: Of the 31 gsp-positive fragments tested in patients with more than one gsp mutation, only one fragment contained two mutations. Multiple 532 mutations in a single tumor did not correlate with clinical aggressiveness or other characteristics of the tumor (Table 8). Of the 37 specimens examined, 15 were obtained from tumor metastases to areas physicaUy separate both from the original primary tumor and from normal thyroid tissue — i.e., spread beyond the strap muscles to lymph nodes in 13 cases and to lung in two cases. Of these 15 specimens, 11 harbored gsp. mutations. In three cases (patients 12, 19 and 24), these metastases revealed two different g p mutations. Occasional reports have documented more than one ias mutation in a tumor (e.g., in human cutaneous melanoma, see Van't Veer fit al-, 1989, Mol. Cell Biol.. 2:3114-3116). The present study is the first to find a high incidence of multiple mutations of a single oncogene in individual tumors. The microdissection technique wiU likely find a siπήlar multipUcity of oncogenes in other tumors as weU. H. Ras Mutations Mutational substitutions of amino acid residues in three key positions of p21 rαy — glycine-12, glycine-13, and glutamine-61 — inhibit GTP hydrolysis and promote its abiUty to trigger neoplastic transformation. Other investigators have reported prevalences of ias mutations in thyroid cancer ranging from 17 to 60 % (LeMoine el al., 1989. Oncogene. 4:159-164 and Wright et al.. 1989. Brit. J. Cancer. 60:576-577 Consequently, the 37 surgical specimens from patients with thyroid cancer were tested for oncogenic mutations affecting t ap three key codons of the three human ias genes. Microdissected fragments from 12 of 37 patients (32 %) 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 weU as maUgnant thyroid tissue (Table 8). Of 117 microdissected fragments tested in the 12 rαs-positive patients, 31 (26 %) contained an N-ras mutation. Two patients had more than one different ras mutation. Detection of ias and gsp mutations in the same microdissected fragment does not mean that both mutations are present in the same ceU; indeed, further microdissection would probably segregate different ceU populations containing each mutation. I. New Gsp Mutations 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. J, Conclusions The 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 ceUs 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 ceUs in the tumor fragment. The results of this analysis are presented in Table 8. Table 8 r i n ffr i Th i an r Table 8 - Continued ft 23 44F Fx20 0/3 0/5 0/3 0/5 Table 8 - Continued Patient Characteristics No. Age/Sex Histol/TNM 24 70F Fx20 25 73F Fx30 26 42M Fχ31 27 49M F411 Table 8 - Continued & K. Table 8 Patients numbered 1-27 and 28-37 (left column) underwent surgery in Dϋsseldorf and San Francisco, respectively. Gap and ias 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 ceUs seen in the corresponding part of the stained section were histologicaU 'maUgnant 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 10 present in the material available for analysis. Although Harvey- and Kirsten-rαs mutations were also sought, only N-ras were found. Clinical characteristics of patients: The "HistoJTNM" column (third from the left) indicates whether the tumor was diagnosed as a papiUary (P) or folUcular (F) thyroid carcinoma, and provides the numerical cUnical classification (TNM) of thyroid 15 cancer devised by the World Health Organization 2 !, in which 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 metastases; M (0 or 1) indicates the absence or presents of distant metastases; "x" indicates that the information was not known to us. Fifteen patients 20 (nos. 11, 12, 13, 15, 16, 17, 19, 21, 24, 25, 26, 27, 32, 34, and 37) had lymph node metastases outside the bed of the thyroid gland, and two (nos. 19 and 26) also had lung metastases. The fifth column (R) indicates whether (+) or not (-) the patient had received therapeutic radiation therapy ( 131 I or external) before the operation. Table 9 provides a summary of pituitary gsp mutations identified in Examples 1-4 Table 9 >. Example Materials and Methods A. Human Tissues Human tissues were obtained from surgical biopsy and either RNA immediately 5 extracted or rapidly frozen to -70 * C until use. AU human tissues were obtained by informed consent in approval with the University of California, Davis Human Subjects Review Committee. Tissues were also suppUed by the Tissue Bank of the Department of Pathology University of California, Davis) under direction of Dr. Robert D. Cardiff. Microscope sUdes with bone marrow smears on them were analyzed. One was 10 hematoxylin and eosin (H+E) stained sUde, wh e the other was merely air dried bone marrow. Both sUdes were stored at room temperature for several months prior to RNA extraction. Other archival samples were either methanol- or ethanol-fixed prior to being embedded in paraffin. 15 B, Cell Lines Human tumor ceU lines previously characterized to have known activating point mutations in various ras aUeles were used. CeU lines EJT24 (bladder transitional ceU carcinoma) (Tabin fit al-, 1982, Nature 200:143-149, and Reddy el al-, 1982, Nature 200:149-152) and SK-N-SH (neuroblastoma) (Taparowsky £taL, 1983, Cell 244:581- 20 586) were a gift of Dr. R. Cardiff. Calu-1 (lung carcinoma), SW 480 (colon carcinoma, and PA-1 (teratocarcinoma) were obtained from the American Type Culture CoUection (ATCC) (Capoh ei al, 1983, Nature 201:507-513, and Tainsky fit al-, 1984. Science 225:643-6451 HL-60 (promyelocytic leukemia) was a gift of Dr. J. Lawrence (Murray sial-, 1983, £gfl 23_;749-757). 25 Other ceU lines were used as negative controls for j s mutations because they are not known to contain activated aUeles. K562 (erythroleukemia) was suppUed by the Cetus Tissue Culture CoUection (CTCC) (Lozzio and Lozzio, 1975, Blood 45:321- 334). CeU line G-2101 (renal clear ceU carcinoma) was originated in our lab (Gumerlock si al-, 1988, In Vitro Cell Devel. Biol.24:429-434). The ceU strain T- 30 3891 (fetal lung) is a normal, nonimmortaUzed fibroblastic culture (Rossitto fit al-, 1988, 1. Virol.14Q:431-435). AU of the above ceU Unes were maintained according to the instructions of the suppUer. C. RNA Extractions from Fresh or Frozen Tissues and CeU Lines Total cellular RNA was extracted from tissues and ceU Unes using modifications 35 of the previously described guanidirύum-isothiocyanate-phenol-chloroform methods (Maniatis fit al-, 1982, In- Molecular Cloning, New York, Cold Spring Harbor Page 190, and Chirgwin £ al, 1979, Biochem.1&5294-5299). Guanidmium isothiocyanate solution (5 M guanidinium isothiocyanate, 25 mM sodium citrate, 0.5% sarcosyl, pH 7.0) (GITC) was prepared to 5% β-mercaptoethanol (GITC-ME) just prior to use. Tissue bits were powdered in Uquid 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 polyaUomer tubes (Beckman Laboratories). 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 peUeted through this density gradient by ultracentrifugation at 40,000 rpm in an SW-50.1 rotor at room temperature for 16 to 19 hours. The RNA peUet was suspended in 50 μl 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. The RNA was precipitated by addition of 5 M NaCl to create a final concentration of 0.3 M NaCl foUowed by addition of two volumes of ice-cold 100% ethanol. This solution was placed at -70 e 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 peUet the RNA predpitate. The supernant was decanted and residual Uquid was removed by vacuum desiccation. When nearly dry, the RNA peUet was redissolved in TE (without SDS) and precipitated a second time. This time the RNA peUet was redissolved in 50- 100 μl 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 μg per ml RNA. D. RNA Extraction from Air-dried Bone Marrow SUdes 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 ceUs 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 ceUs. 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. 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 preάpitation of DNA away from the RNA is 5 modification of that described by Chomezynski and Sacchi, 1987, Δnal. Biochem. 162:156-159. FoUowing the incubation of ice, 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. to each tube was added 750 μl of 0 isopropanol, and the tubes were inverted several times before placing at -20°C for one hour. Precipitated RNA was peUeted by spinning in a microfuge at * C for 20 minutes. The RNA was redissolved in 300 μl of GITC-ME and precipitated a second time by addition of 300 μl of isopropanol and placing at -20 * C for one hour. RNA was peUeted again as above, supernatant was discarded, and the RNA peUets washed with 1 ml of 70% ethanol. Once again, the RNA was peUeted, supernatant discarded, and the peUet dried under vacuum desiccation. The RNA was finaUy redissolved in 50 μl of 0.2 X TE and quantitated. Both sUdes each yielded over 15 μg of RNA. E. RNA Extraction from Alcohol-fixed Paraffin-Embedded Tissues Fifty micron sections of paraffin blocks were cut and deparaffinized in 1 ml of 20 xylenes by vigorous shaking in a microfuge tube for 30 minutes. Tissue bits were peUeted by microfuging for five minutes and the xylenes decanted. Residual xylenes were removed by washing with 100% ethanol andrepeUeting 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. AU 25 subsequent steps in the RNA isolation were the same as those described above for the bone marrow sUdes where DNA was predpitated away from RNA. Each fifty micron section yielded approximately 25 μg of RNA. F. RNA/PCR Procedure The strategy for amplification of mRNA sequences in the polymerase chain 30 reaction (RNA PCR) is based on that previously described (Kawasaki fi &L 1988, Proc. Natl. Acad ≤cj. USA S5_:5698*-5702). Total cellular RNA was converted to a pool of cDNAs by reverse transcription. This'cDNA was then subjected to PCR using gene-specific primer pairs. The primer pairs werd designed to be homologous to exon sequences separated by one or more introns. Primer A, the upstream primer of the 3 5 pair, comprised same sequence (5 * — >3') as that determined for the non-template strand used in RNA transcription of the gene (the same sequence as the RNA itself with thymine substituted for uradl). Primer B, the downstream primer, was designed to be complementary to and anti-paraUel to the non-template strand. Primers were generaUy 21 to 24 bases in length (21- to 24-mers) with a GC content of 40-60%. With the primer pair spanning an intron, PCR amplification of spUced mRNA results in ampUfied products of a predicted length containing only the exon sequences of the gene. Amplification of unspUced RNA or any contaminating genomic DNA in the RNA preparation yields products of a larger size including the intron sequences. The smaUer product predicted from the spUced mRNA sequence wiU only be produced if spUced 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 VisuaUzation Assay (GVA) for gene expression. The predicted ampUfied products were confirmed by use of internal probe hybridization to the PCR products. For this reason, oUgonucleotides were prepared in sets of three: two as the ampUmer pair and a third internal to both ampUmers to be used as a probe for the resulting products of PCR. However, with confirmation of the predicted band, GVA can be used to screen for gene expression in a extremely rapid and sensitive fashion. G. Ras Primers and Probes Seven sets ofras-specific RNA/PCR primers were designed and are Usted in Table 6. An upstream primer specific for exon 1 in each of the three human ias genes, c-N-ias (primer EK 221), c-Ha-ιas-1 (EK 222), and c-Ki-∑as-2 (EK 223) was prepared, these were each used separately in combination with a generic exon 2 downstream primer (EK 225) targeted to a conserved region. In addition, a generic upstream primer (EK 224) was used, paired with EK 225 to detect "pan" -ras expression. The products of each of these primer sets were predicted to be a size of 200 bp. 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 aU three gene expression assays to be run simultaneously in the same reaction mixture and then viewed in a single lane of a gel. The result saved in enzyme expenses and aUowed scale-up of the number of samples to be screened by a factor of three. RNA/PCR products from c-N-ras (primers EK 365 and EK 366), c-Ha-ras- 1 (EK 367 and EK 368), and c-Ki-∑as-2 (EK 369 and EK 370) messages of 299 bp, 259 bp, and 234 bp, respectively. 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. In these experiments, aUele-specific oUgonucleotide (ASO) probes hybridization was used to detect point mutations at the 12th and 61st codons to screen for activating point mutations in ias aUeles. The<20-mer probes used are Usted in Table 7. These probes were used according to the procedure described in Fair fit al-, 1988, Proc. Natl. Δsad. Sfii- 1ZSΔ £5:9268-9272. H. Synthesis of cDNA from RNA Complementary DNA (cDNA) was synthesized from the extracted total ceUular RNA essentiaUy as previously described (Kawasaki ≤t __[., 1988, Proc Natl. Acad. Sd. USA £5:5698-5702). One microgram of total RNA was reverse transcribed with Moloney Murine Leukemia Virus (Mo-MuLV) reverse transcriptase (Bethesda Research Laboratories) in a 20 μl 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 μl 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 oUgo-dT primers was based cm a recent report showing better yield of RNA/PCR products (Noonan and Roninson, 1988, E - Acid Res.16:10366). Reverse transcription reactions were incubated at room temperature for 15 minutes, 42 β C for 30 minutes, and 95 * C for five minutes. The 95 * C incubation was done to heat denature the Mo-MuLV reverse transcriptase enzyme. These cDNAs were stored at 4 β C until use in the PCR reaction. 5 I, PCR Reactions Using cPNA The PCR method was used according to Saiki fit al., as described for DNA using recombinant thermostable DNA polymerase originating from Thermus aquaticus (rTaq) (Perkin Elmer-Cetus). SUght modifications included reducing the amount of primers to 10 pmoles each, reducing the dNTPs to 1 μl of the 10 mM each stock Q described above, and using the rTaq enzyme at 2 U per reaction in a total 100 μl reaction. The substrate for RNA PCR was 2 μl of the 20 μl cDNA described above. This amount corresponds to 100 ng of the initial 1 μg of total cellular RNA used in the reverse transcription reaction. Where three gene reactions were run simultaneously, this corresponds to a minimum of 30 gene expression studies from 1 μg of total cellular 5 RNA. PCR thermal profiles of 95 * C for one minute (denaturing of double strands), 55"C for 30 seconds (annealing of amplimers), and 72 * C for 30 seconds (synthesis of DNA) were performed in a programmable heat block (Perkin Elmer-Cetus) for 30-50 cycles. A 30 second synthesis step at 72 * C was suffident to produce RNA/PCR products of at least 935 bp. J. Gel VisuaUzation Assav fGVA't for mRNA Expression Discrete gene expression was scored by the gel visuaUzation assay (GVA) foUowing RNA/PCR. RNA/PCR products were screened by running 9 μl of the reaction mixture in 2% NuSieve (FMC, Roc land, MD), 1% agarose gels in Tris- 10 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 ceUs (Bio- Rad Laboratories) in TBE at a constant 100 volts for approximately 90 minutes. Gels were stained in an ethidium bromide solution (0.5 μg per ml) for 30 minutes, detained for 30 minutes, and photographed under ultraviolet Ught with a Polaroid Land camera. 15 K. ASO Probing of RNA/PCR Products 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. 2 Q FoUowing transfer, blots were neutraUzed in 2X SSC for 5 minutes. 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 β C for one hour with circular agitation. Hybridization with ASO probes that were kinase- labeled with gamma- 32 P-ATP was done in 5 ml of the TMAC buffer Usted above with 25 2 x 10 6 cpm per ml of probe added. Hybridization continued at 55 β C for one hour. The hybridization buffer and the first wash of 2X SSC with 0.1 % SDS (50 ml) at room temperature were discarded as radioactive waste. A second wash was done at room temperature with 2X SCC with 0.1% SDS. Blots were quickly rinsed in the TMAC buffer minus the Denhardt's solution and extensively washed in the same buffer at 30 61 * C for one hour. This wash was the critical wash for aUowing the discriminatory abiUty of the ASO probes to distinguish point mutations. Blots were then blotted dry with Whatmann 3 MM paper and exposed to Kodak XAR film for at -70 β C for one hour. Film was then developed in an automatic processor and data interpreted. Table 10 OUgonucleotide primers for ias RNA/PCR. AU sequences are Usted in a 5' — >3' direction (in EK 224, K=T or G in EK 225, R=A or G). EK 221: N-ms exon 1 (upstream) GACTGAGTACAAAGTGGTGGTGG EK 222: Ha-ras-1 exon 1 (upstream) GACGGAATATAAGCTGGTGGTGG EK 223: Ki-∑as-2 exon 1 (upstream) GACTGAATATAAACTTGTGGTAG EK 224: generic ias exon 1 (upstream) ATGACTGAATATAAACTGGTGGTGGTKGG EK 225: generic Q exon 2 (downstream) CATGTACTGGTCCCTCATGGCRCTG EK 365: N-∑as exon 1 (upstream) CGGGGTCTCCAACATΠTTCC EK 366: N-o exon 2 (downstream) GTATIXJGTCTCTCATGGCACT EK 367: Ha-ms-1 exon 1 (upstream) AGGAGACCCTGTAGGAGGACC EK 368: Ha-ιas-1 exon 2 (downstream) GTACTGGTCCCGCATGGCGCT EK 369: Ki-ιas-2 exon 1 (upstream) CGGGAGAGAGGCCTGCTGAAA EK 370: Ki-ιas-2 exon 2 (downstream) GTACTGGTCCCTCATTGCACT Table U OUgonucleotides for ias ASO probing. AU sequences are Usted in a 5'™>3' direction (R=A or G; H=C, A, or T; V= A, C, or G; S=G, A, or T). JN 01 : Ha-ias- 1 12 (w.t) GTGGGCGCCGGCGGTGTGGG JN 02: Ha-ms-1 12-1 GTGGGCGCCHGCGGTGTGGG JN 03: Ha-ιas-l 12-2 GTGGGCGCCGHCGGTGTGGG JN 04: Ha-ιas-1 61 (w.t.) TACTCCTCCTGGCCGGCGGT JN 05: Ha-∑as-1 61- 1 TACTCCTCCTHGCCGGCGGT JN 06: Ha-ιas-1 61-2 TACTCCTCCVGGCCGGCGGT JN 07: Ha-ιas-1 61-3 TACTCCTCRTGGCCGGCGGT JN 08: Ki-ιas-2 12 (w.t.) CCTACGCCACCAGCTCCAAC JN 09: Ki-ιas-2 12-1 CCTACGCCACSAGCTCCAAC JN 10: Ki-ιas-2 12-2 CCTACGCCASCAGCTCCAAC JN 11 : Ki-ιas-2 61 (w.t.) TACTCCTCTTGACCTGCTGT JN 12: Ki-ιas-2 61-1 TACrCCTCTTHACCTGCTGT JN 13: Ki-∑as-2 61-2 TACrCCrCTVGACCTGCTGT JN 14: Ki-ιas-2 61-3 TACTCCTCRTGACCTGCTGT JN 15: N-ns 12/13 (w.L) GGAGCAGGTGGTGTTGGGAA JN 16: N-∑as 12-1 GGAGCAHGTGGTGTTGGGAA JN 17: N-ias 12-2 GGAGCAGHTGGTGTTGGGAA JN 18: N-xas 13-1 * GGAGCAGGTHGTGTTGGGAA JN 19: N-ias 13-2 GGAGCAGGTGHTGTTGGGAA JN 20: N-∑as 61 (w.t.) TACTCTTCΓTGTCCAGCTGT JN 21: N-as 61-l TACTCΓTCTTHTCCAGCTGT JN 22: N-ms 61-2 TACΓCTTCTVGTCCAGCΓGT 5 JN 23: N-ιas 61-3 TACTCTTCRTGTCCAGCTGT Example 6 Efi UltS The results of GVA of RNA/PCR amplification of human ias fanήly mRNAs are shown in Figure 2. The samples used were a normal spleen and the ceU Une K562. 10 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" ias primers EK 224 and EK 225 on the RNA from the normal human spleen, the K562 ceU Une, and the negative control of no RNA, respectively. As predicted, die 200 bp ampUfied product is present Lanes 5-7 display 15 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 ceUs. 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 ceUs). No product is seen from RNA isolated from 20 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 utiUty 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 25 369 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 ceU Une. Example 7 30 Utilization of the ras RNA/PCR Products in the Screening for Activating Point Mutations ASO probe hybridization with a different point mutation specific probe was conducted as described in Example 5. The results are shown in Figure 3. The ceU line samples on each blot are the same: lane 1, EJ/T24 RNA amplified 35 with primers EK 222 and EK 225 (c-Ha-ias- 1); lane 2, EJ/T24 RNA amplified with primers EK 224 and EK 225 ("pan" jas); lane 3, PA-1 RNA ampUfied with primers EK 221 and EK 225 (C-N-ias); lane 4, SW-48- RNA ampUfied with primers EK 223 and EK 225 (c-Ki-ιas-2); lane 5, SW-480 RNA amplified with primers EK 224 and EK 225 ("pan" ias); lane 6, HL-60 RNA amplified ^vith primers EK 221 and EK 225 (c-N- ias); lane 7, Calu-1 RNA ampUfied with primers EK 223 and EK 225 (c-Ki-ss-2); lane 8, Calu-1 RNA amplified with primers EK 224 and EK 225 ("pan" ∑as); lane 9, G2101 RNA ampUfied with primers EK 224 and EK 225 ("pan" ias); lane 20, 123 bp DNA ladder. The blot in Panel A has been probed with a pool of oUgonucleotides specific for activating point mutations at the second nucleotide of the 12th codon of c-Ha-ras-1 (JN 03). As predicted, lanes 1 and 2 Containing the EJ/T24 RNA amplified both the c-Ha- ras-1 primers and the "pan" as primers are positive for the characterized EJ/T24 mutation. Panel B blot has been probed with a pool of oUgonucleotides specific for activating point mutations at the second nucleotide of the 12th codon of c-N-ras (JN 5 17). The PA-2 ceU line is known to contain a mutation at this position, and lane 3 is positive as expected. Panel C blot has been probed with oUgonucleotide pool JN 09 targeted to mutations at the first nucleotide of codon 12 in c-Ki-ras-2. The SW-480 ceU Une contains one of those mutations and lanes 4 and 5 containing RNA/PCR products for that ceU line are positive. Because the signal in lane o 5 is quite weak, it may indicate that the mutant aUele's message is in low abundance with respect to aU other ias messages in the ceU as the "pan" cas primers were used for that lane or that the "pan" i s primers are less efficient at amplifying c-Ki-ras-2 messages with respect to the other two ias genes. Panel D blot has been probed with pool JN 22 specific for mutations at the 5 second position of the 61st codon of c-N-ras. CeU Une 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-ias) contain sequences of both the 12th, 13th, and 61st codons of that gene. This is a significant advance for ias point mutation screening in that a single PCR reaction aUows Q one to screen both activating hotsppts in a single product as opposed to the need for two reactions when screening genomic DNA due to those two spots being present in exons 1 and 2 separated by a largeiutron. There is also the additional value of screening for expressed mutations' in mRNA as opposed to the possibiUty of detecting mutations in non-expressed aUeles. Example 8 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- 5 ias: 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-ias- 2: 234 bp). Lanes 2-4 are the negative controls with no RNA added to the reverse transcriptase reaction of RNA/PCR. No products are seen in those lanes other than the primer dimers mentioned above. Lanes 6-8 contain RNA/PCR products from the Calu- 10 1 cell Une and the products corresponding to as messages from aU three genes are present. Lanes 9-11 contain the RNA/PCR products from the ceU Une G-2101. In this case, there is a lack of any signal from c-Ha-ιas-1 messages indicating lack of expression. These reactions were run for 50 cycles of RNA/PCR which increases the 15 presence of background bands, but also the technique for precipitating DNA away from RNA which was used in the preparation of these RNAs is not totaUy effident and can result in contaminating genomic DNA in the RNA preparations. The contaminating genomic DNA can result in specific amplification of the ias genes containing the intron between exons 1 and 2 but it wiU be a much larger size than that predicted for the 20 spliced mRNA. Example 9 The "pan" ias primers were used to amplify reverse transcribed RNA products. Sample preparation and the amplification procedure were as described in Example 4. The results of "pan" ias amplification of RNA isolated from the air-dried stained 25 microscope sUde preparations of human bone marrow are shown in Figure 5. The predicted 200 bp RNA/PCR products using primers EK 224 and EK 225 are shown for the unstained sUde (lane 3) and the stained sUde (lane 4) adjacent to the negative control (no RNA) in lane 2 and the 123 bp DNA ladder in lane 1. These are the results of a 50 cycle RNA/PCR run. 30 Example 10 Detection of ras Mutations by Format II The synthesis of biotinylated primers, poly T tailing of the probes and preparation of the format II filters are as described in commonly assigned, copending U.S. Serial No. 197,000 incorporated herein by reference (also see Chiang fit al., 35 1989. BioTechniques 7(41:3601. Three sets of filters representing 21 mutation specific oUgo probes for N-, H-, and K-ras. respectively, are shown ijji Figure 6. The sequences of the ias probes are Usted in Table 12. AU probes were designed to have approximately the same melting temperature (~50 -52'C) so that hybridization conditions could be standardized for aU the oUgonucleotides. Five pmoles of each taUed probe were spotted onto Biodyne Nylon membranes (Patt Biosupporf, NY) and UV immobilized. The ia oUgonucleotide bound filters were hybridized in 5X SSPE, 0.5% SDS with alkaU 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 μg/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 Ught signal produced was detected by exposing Kodak XRP film to the filters for 20 seconds to one minute. P TU 9100858 59 K-rascodon61 YZ65 AGCAGGTCAAGAGGAG YZ66 AGCAGGTGAAGAGGAG YZ67 AGCAGGTAAAGAGGAGT YZ68 GCAGGTCGAGAGGAG YZ69 AGCAGGTCTAGAGGAG YZ70 GCAGGTCCAG GGAG YZ71 AGCAGGTCATGAGGAG YZ72 GCAGGTCACGAGGAG

Claims: I-n the Claims 1. A method for detecting a G-protein α subunit point mutation in a nucleic add segment present in a biological sample, said method comprising: (a) treating the sample with a G-protein α subunit primer pair, an agent for 5 polymerization, and deoxynucleoside 5' triphosphates under conditions such that an extension product of each primer can be synthesized, wherein said primers are suffidentiy complementary to separate strands of a nucleic acid encoding a segment of a G-protein α subunit to hybridize thereto so that the extension product synthesized from one member of said pair, when separated from its conφlementary strand, can serve as a 0 template for synthesis of the extension product of the other member of said pair, (b) separating the primer extension products from the templates on which the extension products were synthesized to form single-stranded molecules; (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 5 using each of the single-stranded molecules produced in step (b) as a template; (d) repeating steps (b) and (c) at least once to provide ampUfied DNA; (e) hybridizing a G-protein α subunit probe to said ampUfied DNA, wherein said probe contains a nucleic acid sequence that wiU hybridize to a sequence selected from a wild type and a mutant nucleic add sequence within said amplified DNA; and o (f) determining if hybridization has occurred. 2. The method of Claim 1 , wherein steps (b) and (c) are repeated at least five times and said agent of polymerization is a thermostable DNA polymerase. 3. The method of Claim 2, wherein said thermostable DNA polymerase is Taq polymerase. 5 4. The method of Claim 1, wherein the sample is removed from a human tumor. 5. The method of Claim 4, wherein said human tumor is an endocrine tumor. 6. The method of Claim 1 , wherein said primer pair wiU amplify a nucleic acid segment encoding a subsequence of a G-protein α subunit selected from the group 0 consisting of Giαl, Giα2, Giα3, Gzα, Goα, and Gsα. 61 7. The method of Claim 6, wherein said nudeic acid segment encodes at least one amino acid selected from the group consisting of the amino acids corresponding to Gsα 49, 201, and 227. 8. The method of Claim 7, wherein said primer pair is selected from the group consisting of: JFL69 and JFL70; JFL135 and JFL136; JFL228 and JFL135; JFL229 and JFL136; JFL226 and JFL227; JFL223 and JFL224; JL54 and JL57; JFL109 and JLFl 10; JFLl 10 and JfLl 12; JFLl 13 and JFLl 14; JFLl 15 and JFLl 13; SP9 and SP10; JFL139 and SP11; JFL201 and SP15; JL55 and JL56; JFL223 and SP33; and JFL224 and SP34; JFL235 and JFL237; JL55 and JEL212; JFL215 and JL56; and JFL135 and JFL286. 9. The method of Claim 1, wherein said probe comprises a sequence which hybridizes to DNA encoding a subsequence of a G-protein subunit selected from the group consisting of Giαl, Giα2, Giα3, Goα, Gsα, and Gzα. 10. The method according to Claim 1 , wherein said probe hybridizes to a G- protein subsequence encoding the amino acid corresponding to the Gsα amino acid at position 49, 201, or 227. 11. The method of Claim 10, wherein said probe is selected from die group consisting of 5' TA GGT GCT GGA GAA TCT GGT 3' 5' TA GGT GCT AGA GAA TCT GGT 3' 5' TA GGT GCT CGA GAA TCT GGT 3' 5' TA GGT GCT GAA GAA TCT GGT 3' 5' TA GGT GCT GCA GAA TCT GGT 3' 5' TA GGT GCT GTA GAA TCT GGT 3' 5' TT CGC TGC CGT GTC CTG ACT 3' 5' TT CGC TGC TGT GTC CTG ACT 3' 5' TT CGC TGC GGT GTC CTG ACT 3' 5' TT CGC TGC AGT GTC CTG ACT 3' 5' TT CGC TGC CAT GTC" CTG ACT 3' 5' TT CGC TGC CCT GTC CTG ACT 3' 5' TT CGC TGC CTT GTC CTG ACT 3' 5' TGG GTG GCT AGC GCG ATG A 3 * ; 5' TGG GTG GCC ATC GCG ATG A 3'; 5' TGG GTG GCA AGC GCG ATG A 3; and 5' TGG GTG GCC ACC GCG ATG 3". 12. The method of Claim 1, wherein said nucleic acid is RNA and is reverse transcribed to provide a double-stranded cDNA copy prior to step (a). 13. The method of Claim 1, wherein said sample is paraffin embedded tissue. 14. An oUgonucleotide primer pair for the amplification of a subsequence of a nucleic acid encoding a G-protein α subunit wherein said subsequence comprises a nucleic add sequence encoding an amino add corresponding to the Gsα amino acid at a position selected from the group consisting of 49, 201, and 227. 15. An oUgonucleotide primer pair according to Claim 14, wherein said G- protein α subunit is selected from the group consisting of Gsα, Goα, Giαl, Giα2, Giα3, and Gzα. 16. An oUgonucleotide probe for distinguishing between oncogenes and proto- oncogenes encoding G-protein α subunits, wherein said probe hybridizes to a region of nucleic acid encoding an amino add selected from the group consisting amino acids corresponding to the Gsα amino acid at positions 49, 201, and 227. 17. An oUgonucleotide primer pair for the amplification of a subsequence of a nucleic add encoding a G-protein α subunit wherein the primer pair is selected from the group consisting of: JFL69 and JFL70; JFL135 and JFL136; JFL228 and JEL135; JFL229 and JFL136; JFL226 and JFL227; JFL223 and JFL224; JL54 and JL57; JFL109 and JLFl 10; JFLl 10 and JFLl 12; JFLl 13 and JFLl 14; JFLl 15 and JFLl 13; SP9 and SP10; JFL139 and SP11; JFL201 and SP15; JL55 and JL56; JFL223 and SP33; and JFL224 and SP34; JFL235 and JFL237; JL55 and JFL212; JFL215 and JL56; and JFL135 and JFL286. 18. A probe for the detection of a point mutation in a nucleic acid encoding ; segment of a G-protein α subunit wherein the probe is selected from the group consisting of: 5' TGG GTG GCC ACC GCG ATG 3'. 19. A method for detecting a point mutation, if present in a nucleic acid encoding a G-protein α subunit in a sample comprising: (a) hybridizing a G-protein α subunit probe to said sample, and (b) determining whether hybridization has occurred. 20. A kit for detecting point mutations in a nucleic acid encoding a G-protein α subunit, comprising, in a separate container (a) a G-protein primer pair suitable for providing an ampUfied G-protein DNA segment in a PCR reaction; (b) a G-protein probe comprising a wfld type sequence which wUl hybridized to said DNA segment if it is a wUd type; and (c) a G-protdn probe comprising a sequence containing a point mutation for detecting a point mutation if present in said DNA segment.

Inventors: LYONS JOHN F, MCCORMICK FRANCIS P, LYONS, JOHN, F., MCCORMICK, FRANCIS, P.
Applicants: CETUS CORP, CETUS CORPORATION
Application year: 1991
Main classification: G01N33
IPC codes: C12N15, C12Q1