CA2075053A1 - Detection of point mutations in genes encoding gtp binding proteins - Google Patents

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

prT~Ils 9 1 / 0 0 8 5 8 DETECTION OF POINT MUTATIONS IN GENES
ENCOD~NG GTP BINDING PE~OTEINS
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 dctection and classification of these point mutations and potential oncogenes. The identification of oncogenes is important in the study of 10 cell growth and carcinogenesis. The invention provides methods which relate specific point mutations to specific tumor types. In a preferred embodiment point mutations are described within nucleic acids encoding G-proteins.
G-proteins function as interrnediates in transmembrane signalling pathways (Gilman, 1987, Ann. ~y. Biochem. ~:615~. These pathways consist of receptors, 15 G-proteins and effector molecules and are regulateA by the cyclic association of GTP
and GDP with G-proteins. Each G-protein consists of three subunits: a, ~, and y.The specificity of the interaction with the effector molecule is dictated by the ~ subunit.
Several G-prdteins have been purified and characteriæd; Gs and Gi are ~ -~
involved in stimulation and inhibition, respectively, of adenylate cyclase activity. Three 20 Gia subunits Gial, Gia2, and Gia3 have been identified and cloned (Itoh çt al., 1988, ~ kçm ~:6656-6664). Gt activates cGMP phosphodiesterase in response to photosignal transduction (Mattera çt.al-, 1986, ~ 1~ ~Q~:36-42, and Didsbury et al., 1987, E~. L~ ;~.: 160- 164). Other G-proteins have been sequenced including Go and Gz (Jones and Reed, 1987, I. ~ 14241-25 14249, and Stratllmann ~., 1989, BQS~. ~. ~a51 ~. I~: ~fi:7407-7409).
Additionally, Gk (Yatàni ~ ~., 1987, Science ~:207) and Golf (Jones and Reed, 1989, Science ~:790) have been identified as G-proteins.
Gs activity raises the level of cAMP in cells by stimulating adenylyl cyclase. In pituitàry somatotrophs, cAMP stimulates secretion of human growth horrnone and 30 causes cellular proliferation. Recently, a subset of human pituitary turnors were descnbcd having elevated levels of growth horn~ne and cAMP (Vallar ~ ~.,1987, Nature 33Q:566-568). Landis t ~., 1989, ~a~ ~Q:692-696, proposed tha~ .the abnorrnal cdl proliferation obsa~ed by Vallar ~ al. was the result of a defcct in the G-protein responsible for controlling cAMP levels, resulting in an accumulation of 35 cAMP. Landis ~.al identified patients harboring tumors secreting excessive arnounts of growth honnone and dete~nined that four tumors had constutively elevated levels of Gs activity. RNA was purified from fresh tissue, reverse transcribed, and cloned. The . . .: . , : , . , -. ~ - ., . -, , ~ ~ - -. .. ..

~ PCTIUS 9~ ~ Ql 59~

entire Gsa coding region of the cDNA was sequenced and point mutations within codons 201 and 227 were identified. These mutations are ~S~ mutations. Gs~
mutations are a class of mutations that activate Gs, which normally me~iates stimulation by thyrotropin (TSH) of thyrocyte proliferation and production of thyroid hormones.
(Lyons ~ ~., 1990, ~, ~2:655-659) Arginine 201 is a major site of ADP-ribosylation of Gs by cholera toxin. This modification allows constitutive adenylyl cyclase activation (Lo and Hughes, 1987, E~ h~ ~: 1 -3). Glutamine 227 is predicted to be a Gs equivalent of glutamine 61 in ~ p21 proteins (Landis ~. ~.). Mutational replacement of Gln-61 in p21 produces a protein that promotes malignant transformation (Der ~ ~., 1986, 44: 167- 176).
The ra~ genes encode highly related proteins approximately 21,000 daltons in molecular weight (p21s). While the exact functions of these proteins in cellularsignalling pathways remains elusive, the p21s have GTPase enzyrnatic activities and interact with a GTPase activating protein (GAP) (Bishop, 1983, e~nn. Rev. Biochem.
~2:301 -354, and McCorrnick, 1989, ~ ~:5-8).
The human ~ gene family, which includes the closely related Ha-, Ki-, and N-genes, is one of thre potential targets for mutational changes that have been irnplicated in the development of rnany human rnalignancies (Bos, 1988, M~a~Qn Research 195:25S-271). These alterations are either point mutations in codon 12, 13, --or 61, or altematively a 5- to 50- fold arnplification of the wild-type gene. These changes convert the T.~ pro~oncogenes into oncogenes.
Polymerase chain reaction (PCR) methods havc been used to detect known point mutations in ~a~ oncogenes in genomic DNA isolatcd from tumors (Verlaan-deVries~al-. 1986,Gene~:313-320,andAlmoguera~1., 1988,~:549-554).
Farr~., 1988, Proc. ~. ~a~l. ~. ~ ~:1629-1633, have eombined PCR
with oligonucleotide dot blot methods to examine speeifie ~ gene point mutations in DNA isolated from padents afflieted with aeute myeloid leukemia (AML), The present invention provides methods for scrcening nueleie acids encoding - 30 G-proteins. Methods for sereening ~ genes are also provided. The nucleie acids may be RNA or DNA. Primers and probes are provided which aid in the identification of potential oncogenes and eharaeterizadon of point mutadons within an oneogene or potentdal oneogene. The invention provides prirners and probes whieh are par~ieularly suitable for detectdon of point mutations in nucleie aeids eneoding Gz, Gs, Go, Ga, and 35 Gi proteins in endocrine tumo;s.
The present invention provides a method for detec~ing whether a point mutation is presen~ in a nucleie acid eneoding a G-protein subunit, in a sample, ehat eomprises:

PCTillS 91/008 58 1 3 M~Y ~

(a) hybridizing a G-protein a subunit probe to said sarnple and (b) deterrnining 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 a subunit in a sarnple, 5 comprising:
(a) treating the sample with a G-protein a subunit primer pair, an agent for polyrnerization, and deoxynucleosidc 5' triphosphatcs under conditions such that an extension product of each primer can be synthcsized, wherein said primers are sufficiently complementary to separate strands of a nucleic acid encoding a segment of a 10 G-protcin c~ subunit to hybridize thereto so that the extension product synthesized from one member of said pair, when separa~ed ~om i~s complemcntary strand, can serve as a template for synthesis of the extension product of the other member of said pair, (b) separating the prirner extension products f~om the templates on which the extension products were synthesized to fo~n 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 produccd in step (b) as a template;
(d) repeating steps (b) and (c) at least once to provide amplified DNA;
(e) hybridizing a G-protein a subunit probe to said arnplified DNA, 20 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 prirners and probes useful for detecting 2S potentially oncogenic point mutations within a nucleic acid encoding a G-protein a subuniL
The prcsent invention also provides kits for amplifying and detecting point mutations, if prcsent, within a nucleic acid encoding a G-protein a subunit.
Figure 1 shows the idendfication of point mutadons in Gsa and Gic~2 genes. A
30 Tegion containing the indicated codons of the Gs or Gia2 gene was amplified by PCR
from genomic DNA isolated from either fresh frozen dssue or paraffin~mbedded tissue. Point mutations were detected with high-stnngency hybridization of sequence-specific oligonucleotides to the amplified producL Each panel represents hybridization with a different oligonucleotide.
Analysis of Gs~ genes is shown in Figure lA and described in Exarnple 2.
This analysis identified mutadons in Arg 201 and Gln 227 codons of C;sa in l 8 biochemically-characterized human gro~nh ho;mone sec~eting pituitary turnors.

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P~T 'JS ~ 1 / 00 ~ 5 8 .

Analysis of Gi2 genes is shown in Figu~e lB and described in Exarnple 3.
This analysis identified mutations in codon 179 of Gic~2 in three hurnan adrenocortical turnors and one ovarian granulosa cell tumor.
Figure 2 provides the GVA (gel visualization assay) analysis of ~ RNAIPCR
5 products from a normal human spleen and the K562 cell line as described in Exarnple 5.
Figure 3 shows the results of a southern blot analysis of ~ RNA/PCR
products using ASO probes specific for activating point mutations in characterized cell lines. The experiment is described in Exarnple 6.
Figure 4 provides the GVA analysis of ~a~ RNA/PCR from alcohol-fixed, paraffin-embedded samples as deseribed in Example 7.
Figure 5 provides the GVA analysis of B~ RNA/PCR from stained and unstained microscope slides of human bone marrow as deseribed in Exarnple 8.
Figure 6 is schematic diag~am of ~ forrnat n filters showing the positions of 15 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 a subuniL The point ~ -mutations detected by these methods are believed to be involved in oncogenesis. The nucleic acid is a G-protein subunit gene, RNA transeription product, cDNA product, or 20 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 eaeh nucleic aeid segment suspected of comprising a potentially oncogenic point mutation, a pair of oligonucleotide prirners are p~vided for arnplification in a : - -polymerase chain reaction. The primers will amplify wild type or mutant nucleic acid 2s segments. Genes that encode G-proteins are proto-oncogenes in the normal somatotrophie 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 sldlled in the art to disdnguish between these two types of G-protein genes.In the embodiment of the invention illust~ted below, point mutadons are 30 detected by oligonueleotide probes. To deteet an oneogenic point mutation in a nucleic acid eneoding a G-protein, the pro~es eontain either the wild type nucleic acid sequence or single nueleodde ehanges within codons whieh eorrespond to amino aeids 49, 201, and æ7 in the sequenee of Gsct. The amino acid at 226 may be of interest as well.
These single nueleotide ehanges affcct the translation p~duet of the gene. Of eourse, 35 in some samples, a mutated oncogene may not be p~sent. In sueh a sarnple only the wild type probe will hybridize to the sample. Thus, where a point mutation is not . . . .

~: : . - - . . . ,- : --` ` P~T'I IS 9 1 / 0 0 8 5 8 AY 13~1 detected, the wild type probe serves to verify the presence of the arnplified pr~duct in the sarnple.
G-protein a subunits share a high degree of homology. Matsuoka ~ ~., 1988, ~. ~- ~a~. ~. ~ ~:538~5388, provide a comparison of the amino acid 5 sequences of several G subunits. Although the G subuni~s vary in length, thedegree of homology between the published amino acid sequences is sufficiently high such that sequence alignment is possible. In this way, the amino acid corresponding to Gs Gly 49, Gsa Arg 201, Gs Gly 226, and Gsa Gln 227 can be determined for all Ga~ subunits. For example, in Gia2, Arg 179, and Gln 205 correspond to Arg 201 10 and Gln 227 in Gsa.
The present invention also pr~vides probes which detect point mutations in nucleic acid encoding ~ proteins. Such pr~bes contain single base changes as well as the wild type sequences for codons encoding amino acids at p2 1 positi~ns 12, 13, and 61 .
The examples below describe methods to detect the presence of a point mutation within a nucleic acid sequence contained in a sarnple. For exarnple, the sequence to be examined can be assa~yed by hybridization using a wild type probe and a pool of probes, each probe containing a point mutation at a specific position. Altematively, the methods can be used to detect and classify a point mutation. In Ihis case, the probes, 20 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 exac~ nucleic acid sequence contained within the sample nucleic acid will hy~idize to the nucleic acid in the sample.

Tho~e s~ ed in the art will recognize that the disclosed methods and probes 25 enable new oncogenes to be detected and classified. These methods led to the discovery that, for example, the gene oncoding Gia2 is a proto-oncogcne. 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 cncoding Gial, Gia3, Goa, and Gza if such 30 mutadons exist. Similarly, these methods may ~e used to design primers and probes to identify new oncogenic point mutations in other G-protein a subunits such as GtcL, -- -Gl~, and Golfa~ These point mutadons may well be related to oncogenesis and their iden~ification lays a critical foundation for expçrimental rescarch in oncogenesis.
Regardless of oncogenicity, these point mutations are useful to discriminate be~ween 35 individuals. Methods which discnrnina~ bctween individuals based on nucleic acid seqùencc differences are used in, for example, forensic medicine.

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-` PCTIUS 9 1 / 00 8 5 8 1 3 ~ r;v ~

The disclosed examples of the invenion, relating to G-proteins, provide methods, prirncrs, and probes to detect and classify point mutations in nucleic acids encoding Gs and Gi2a. Thesc mcthods ale also suitable for detecting point mutations, if present, and classifying those mutations in any nucleic acid encoding a G-S protein o~ subuniL Tables 1-4 provide primers and probes suitable for detecting and classifying point mutations in nucleic acids encoding Gs, Gial, Gi2, Gia3, Goc~, and GzoL These methods are directly applicablc to the detection of point mutations in other Ga subunits, for example, Gt, Gk, or Golf. Table 4 provides probes which ~-corres~ond to codons 49, 201, and 227 in the Gsa subunit.
In a preferred method for detecting and classifying point mutations vithin ~ - -oncogenes or proto-oncogenes, the nucleic acid containing the region of interest is arnplified by a polyrnerase chain reaction prior to detecion. Primers useful in these methods are suitable for amplification of pr~to-oncogenes as well as activated proto~
oncogenes. Those skilled in the art will recognize that with the disclosed methods, 15 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, Gia2. Novel oncogenic genes encoding Gi~1, 5ia3, GooL, and Gza rnay 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
20 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 Gln 227 in Gs.
In one embodirnent of the present invention, primers are selected such that the resultant arnplified ~ragrnent comprises both codons 201 and 227 (or correspondingly 25 179 and 205 in Gia2). However, this is not an essential aspect of the invention. If, for cxarnple, a large intron or rnore than one intron exists between these codons in the genomic DNA, or if the DNA is degradcd as h somc paraffin embedded tissue, amplification pr~rners are designed for the rcgion comprising codon 201 and separately for the region comprising codon 22~. Such amplification rcactions are run separately 30 or simultaneously in one xaction vessel.
Amplification ~quires the use of prirner pairs that will amplify a discrete region of DNA present in a sample. These prirner pairs are oligonucleotides. The PCR
products generated from these pnsners are then analyzed by hybridization with sequence specific probes. Sequence specific probes may also be allele-specific probes.
35 An allele-specific probe (ASO) will hybridize to an allele-specific sequence in a nucleic acid within a sarnple. An allele-specific sequence is a component of an individual's genotype. A sequence-specific probe comprises a specific sequence which may or may .
- , ~. . . . . .

- PCTIUs 9113/M4 8~5q8 no~ 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 terrn allele-specific probe is used by those of skill in the art, these terms are used interchangeably herein.
S 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 acids 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~enaturing the DNA, 10 annealing two oligonucleotide prirners to sequences that flank the DNA segment to be amplified, and extending the annealed primers with DNA polymerase. The prirners hybridize to opposite strands of the target sequence and are oriented so that DNA
synthesis by the polyrnerase proceeds across thc region between the prirners, effectively doubling the amount of the DNA segrnent. Moreover, because the extension 15 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 specific 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 20 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 incolporated herein by reference.
The choice of primers for use in PCR determines the specificity of the amplification reaction. In the present invention, pr~mers are used that will amplify G~
25 protein or ~ p21 sequences present in a sample. The pnmers of the invention can include degenerate pnmers. These are mixtures of oligonucleotides synthesized to have any one of several nucleotides incorporatcd at a selected position during synthesis. For example, the primers may be sufficiently complementary to all types of ~aa genes to amplify a DNA sequence of any ~ DNA prcsent in the sample. IllustratiYe prirners of 30 this type are referred to, for example, as "pan" ~aS prirners. The primers are designed to arnplify a region of DNA, cDNA, or RNA that contains sequences specific to any p21 gene: c-N-~, c-Ha-E~, or c-Ki-~. Because the "pan" ~ prirners span large intron sequences, cDNA and RNA templates are preferred. The arnplified DNA can therefore be used to classify the ~ genc present in the sample.
Altemadvely, it may be desirable to use pnrner pairs which are specific for eachgene to be detected. Such a prirner pair will arnplify a specific DNA or RNA segment - encoding, for example, c-Ki-~, c-N-~, e-Ha-ra~, Gial, Gia2, Gia3, Gs~, Go, - .. . .. .. .
~ ~ - . - . . . . .
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or Gza. In one embodiment, three separate pairs of ~ primers are included in onePCR reaction such that each pair will specifically amplify either c-N-~, c-Ki-~, or c-Ha-ra~. The primers are designed so that each PCR product has a discrete size. Thus, three primer pairs are used to simultaneously arnplify several DNA or RNA segrnents.
5 The identity of the segment(s) amplified can then be determined by, for ~xample, gel electrophoresis and size deterrnina~ion. The presence of point mutations and theclassification of such mutations can be subsequently determined by the methods provided.
When more than one nucleic acid segment is charactelized, it is no~ essential that 10 primers are designed such that the resulting atnplification products are of different sizes. Amplification reactions using different prirner pairs can be run independently of one another and analyzed sirnultaneously, for exarnple, using individual lanes on an acrylamide gel. Alternatively, several primer pairs can be used simultaneously in one reaction, and the amplification pr~ducts divided and analyzed to characterize the sample 15 by, for example, separate probe hybridizations.
In another embodiment of the present invention, nested plimers are used (Mullis Çl ~-, 1986, ~QLd ~ }la~ SymDosil~m Qn Ouantitatiy_ Biolo~y 51:263, incorporated herein by reference). This method may be preferred when the arnount of nucleic acid in a sample is extremely limited, for example, where archival, paraffin 20 embedded samples are used. When nested primers are used, the nucleic acid is first arnplified with an outer set of primers. This amplification reaction is followed by a second round of asnplification 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 arnplified ~ -25 product is characterized. It may be preferred, but is not essential in the practice of the invention, to deterrnine whether amp ification has occu~d. The use of an internal amplification control to assure the competency of a sample for PCR is within thc scope of the invention and reduccs the likelihood of false negative results. There are a variety of ways to detertmine whether arnplification has occurred. A portion of the reaction 30 rnixture can be subjected to gel electropho~sis 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 deoxynbonucleotide S'-triphosphates can be addcd to the PCR reaction rnixture, and inc~rporadon of the label into the amplified DNA measured to delerrnine if amplification occur~ed. Another rnethod for detennining 35 if amplification has occurred is to test a pordon of PCR reaction rnixture for ability to hybridize to a labeled oligonucleodde probe or mixture of pr~bes designed to hybridize to only the amplified DNA. Alternatdvely, the detcm~ination of amplificatdon and .

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PCTIIJS 9 1 / O u 8 5 8 characterization of a point mutation can be carried out in one step by testing a portion of the PCR reaction mixture for its ability to hybridize to one or more specific probes.
Due to the enonnous 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 result in PCR product, even in the absence of purposefully added template DNA. If possible, all reaction rnixes 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 rnLxing, and sample analysis will minimize cross contamination. See also Higuchi and Kwok, 1989, tur~ 237-238 and Kwolc, and O~rego, in: Innis ~ al- eds., l990 ~otQ~Qls: A G~lj~ to MethQ~s and Ap~liç~ons, Academic Press, Inc., San Diego, CA, which are incorporated herein by reference.
One particular method for minimizing the effe~ts of cross contam~nation of nucleic acid arnplification is desclibed in U.S. Serial No. 609,lS7, filed November 2, l990, which is incorporated herein by referenee. The method involvesthe introduction of unconventional nucleotide bases, such as dUTP, into the amplified product and exposing~carryover product to enzyrnatic and/or physical-chemieal treatment to render the product DNA incapable of serving as a template for subsequent arnplifications. For example, uracil-DNA glycosylase will remove uracil residues ~om PCR product eontaining that base. The enzyme treatment results in degradation of the contaminating carryover PCR produet and serves to "sterilize" the amplification -reaction.
The present invention has led and will continue to lead to the discovery of many2s previously unknown or uncharacterized oncogenes. For example, using the methods, psimers, and probes of the invention, four clinical satnples examined were discovered to eontain nvo different Gia2 oneogenes. These new oneogenes ax 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 spccifie fashion.
In one embodiment, the present invention provides a number of probes for use in detecting and charaeterizing potential oneogenes. These probes are set forth in Table 2, below. Those sldlled in the art will rccognize that although the sp cific primers and probcs of the invention exemplified herein have a defined number of nucleotide residues, onc or more nuclcotide residues may be added or deleted from a given pnmer or probe typically without gleat impact on the suitability of that primer or probc in the present n~thods. The cssential aspect of these probes is their ability to discriminate between wild type and mutant sequences. Wllen a portion of the PCR
'~

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`` PCTIUS91/00858 I ` .'! Y l ~ ~ l ......
reaction rnixture contains DNA that hybridizes to a probe, the sample contains DNA
comprising the wild typc or mutant se~uence according to the specific sequence of the probe.
An important aspect of thc present invention relates to detecting the novel 5 probes provided for use in the prcsent methods. There are a number of ways to determine whether a probe has hybridized to a DNA scquencc contained in a sample.
Typically, the probe is labeled in a detectablc manner, the target DNA (i.e., the amplified DNA in the PCR reaction buffer) is bound to a solid support, and determination of whether hybridization has oscurred sirnply involves determining10 whether the label is present on the solid supp~ This procedure can be varied,however, and worl~s just as well when the target is labeled and the probe is bound to the solid support.
The hybridization probes disclosed hercin are sequence-specifc oligonucleotide probes for each G-protein subunit codon to be characterized. As described above, : -15 sequence-s~ecific probes are similar to allele-specific probes in use and utility.
Methods for utilizing ASOs arc dcscribed in Saiki ~ al., 1986, ~ 163-166, incorporated herein by reference. The prohes rnay be used individually for detecting, for example, a wild type sequence. Altematively, the probes may be used in a panel -;
format for characterizing a tumor genotype. The tumor genotype may be compared to, 20 for example, sornatic tissue or other tumor types.
The probes can be used in a variety of different hybridization formats.
Although solution hybridization of a nucleic acid probe tO a complementaTy target sequence is clearly within the scope of the present invention, comrnercialization of the invention will likely result in the use of immobilized probes and thus a quasi "solid-25 phase" hybridization. In this format, thc probe is covalently attached to a solid supportand target sequences are hybrid;ized with the probe. A preferred method for immobilizing probes on solid supports is disclosed in U.S. patent application S.N.
347,495, filed May 4, 1989, incorporated herein by reference. According to this method, sequence-specific probes are a~tached to a solid support by virtue of long 30 stretches of T residues which are added during probe synthesis on an automated synthesizer after the hybridizing sequence is synthesiæd.
For exarnple, in a fixed probe fonnat, the following probes are useful for detecting point mutations in a gene cncoding Gsa at amino aad positions 201 and 227.

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~ P(~T'US 91/00858 Tab~
Amino Acid Pr~bc Nam~ Sequen~ePosi~ion ~316 (SEQ ~ NO:I) 5'TCGCTGCCGTGTCCTGGAC 201 ~317 (SEQ ~ NO:2) 5'TCGCTGCAGTGTCCTGGACT 201 ~318 (SEQ ~ NO:3) 5'TCGCTGCGGTGTCCTGGAC 201 ~319 (SEQ ~ NO:4) 5'TCGCTGCTGTGTCCTGGACT 201 ~320 (SEQ ~ NO:5) 5'TCGCTGCCATGTCCTGGACT 2QI
~321 (SEQ ~ NO:6) 5'TCGCTGCCTTGTCTTGGACT 201 ~322 (SEQ ~ NO: 7) 5'TGGGTGGCCAGCGGCGATGA 227 ~323 (SEQ ~ NO:8) 5'TGGGTGGCCTGCGCGATGA 227 ~L324 (SEQ ~ NO:9) 5'TGGGTGGCCCGCGCGATG 227 ~325 (SFQ ~ NO:I0) 5'TGGGTGGCCGGCGCGATG 227 ~326 (SEQ ~ NO:II) 5'TGGGTGGCGAGCGCGATGA 227 ~327 (SEQ ~ NO:12) 5'TGGGTGGCTAGCGCGAT~A 227 ~328 (SEQ ~ NO:13) 5'TGGGTGGCCATCGCGATGA 227 ~329 (SEQ ~ NO:14) 5'TGGGTGGCAAGCGCGATGA 227 ~F330 (SEQ ~ NO:15) 5'TGGGTGGCCACCGCGATG 227 A fixed probe fonnat is suitable for detecting other ~ mutations, as well, and is 20 demostrated in Exarnple 5 for detecting point mutations in ~ gene PCR products.
Many mcthods for labeling nucleic acids, whether probe or target, are known in the art and are suitable for purposes of the present invention. In one embodirnent illustrated bclow the probes were labeled with radioactive phosphorous 32p, by treating the probes with polynucleotide kinase in the presence of radiolabelled ATP. However, 25 other non-radioactive labeling systems may be prefe~ed, i.e., horseradish peroxidase-avidin-biotin systems. Horsc-radish pcroxidase (HRP) can be detected by its ability to coYcrt diaminobenzidine to a blue pigrncnt. A preferrcd method for HRP-bascd dctection uses tctramethyl-bcnzidine (-I~MB) as described in ~. ~ 1368 (1987). An alte2native detcction system is the enhanced chernilurninescent (E~CL) -30 detecdon kit comlslercially available ~om Ame~;ham. The ki~ is used in accordance - with manufacturer's instructdons. A variety of alternadve dyes and chromogens and corrcsponding labels are available for nucleic acid detection systems (see, e.g., U.S.
patent applicadon S.N. 136,166, filed December 18, 1987).
Another non-~dioactdve alternadve detection method uses term~nal transferase 35 (Tdt) and biotinylated dUTP to add homopolymer tails ~o the oligonucleotide probes.
Biotin serves as the detectable moiety. Following probe hybridization, the filters are .. . . ~ , .

?i T l~S ? 1 / 0 0 8 5 8 1 ~ MA`~

washed as usual. Hybridized biotin is detected with strep-avidin conjugated HRP
(Se eqence~ available from Cetus) according to manufacturer's instructions. The ECL
system is then used to visualize the biotin-HRP product.
Probes are typically labeled with ~adioactive phosphorous 32p, by treating the 5 probes with polynucleotide kinase in the presence of radiolabeled ATP. However, for cornmercial 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 utiliz~d in a PCR amplification is labeled (for instance, the biotinylated dUTP
10 derivatives desc~ibed by Lo ~ ~1.. 1988, ~. ~i~ ~. 16:8719) instead of the probe, then hybridi7ation can be detected by assay for the presence of labeled PCR
product. In a preferred embodiment, probes are biotinylated and detected with the ECL
system described above. For example, biotinylated probes are prepared by direct bio~inylation of the oligonucleotide rather than incorporation of biotin-dUTP during 15 PCR. For 5' biotinylation of oligonucleotides direct solid phrase synthesis using bioin containing phosphoramidites is done according to Alves ~ ~., 1989, :~a. I&~
30:3098; Cocuzza,1989, ~ L~. ~Q:6287; and Barabino et ~., 1989, EMB0 1 ~:4171. Solid phase stynthesis of biotinylated oligonucleotides at any internal or terminal (5' or 3'~ position is also suitable for preparing biotinylated primers and 20 probes (Pieles ~ al . 1989, NAR 1 ~:435~, and Misiura ~ ~., 1989, N!'~,18:4345).
Alternatively, probes and prirners are conjugated to HRP, for example, by the method disclosed in W089/2932, and Beaucage ~ al-.1981, ~. L~. 72:1859-1862. These references are incoIporated herein by reference.
Those skilled in the art will recognize that with the above description, primers, 25 and probes for arnplifying, detecdng, and characterizing new G-protein point mutations can be reaclily obtained. It will also be readily apparent to those skilled in the art that the specific primers and probes p~vided in the examples are me~ely illustradve of the invcndon. Primers and probes of the invention can also be prepared to amplify and detect sequence variations within areas G-protein sequences other than those 30 specifically exemplified herein, for exarnple, codons corresponding to Gsc~ 49.
Thc rnethod of thc present invcntion is applicable for detecdng a point mutationwithin a gene cncoding a GTP binding protein or the expression product of that gene.
Thus, the method can detect specific point mutations in a sarnple containing RNA, or DNA, or k)th. If the sample contains RNA, the nucleic acid will be reverse 35 transcrib~ providing a doublç-stranded DNA template prior to amplificanon.
Procedures for reverse transcribing RNA are known (sec Maniatis ~ ~., 1982, . - ~ .
, . , .
-, . - . , ., : -- . ~ , ~ -~ ~CTIUS9110~858 Molecular Clonin~: A Laborato~ ~an~-, Cold Spring Harbor, NY). In one embodimen~, the rnethods are used to detect point mutations in RNA.
Alternatively, if RNA is abundant, the probes (or the complemen~ of a probe sequence~ arc suitable for dir~t detection and characterization of an oncogene or proto-5 oncogene rnRNA suspected of being present in the sample or in the reverse transcribedcDNA pr~duct. Sirnilarly, if DNA is abundant, fa exarnple, as in a fresh tissue sarnple, the probes are useful for direct detcction of gene sequences. Thus, amplification by PCR is not an cssential component of the present invention.
Samples suitable for analysis by the mcthods described may be fresh or 10 archival. Fresh samples may be, for exarnple, biopsied tumor, tissue samples, or blood. Archival samples may be, for example, frozen or paraffin embedded. In oneembodirnent of the invention, paraffin-embedded samples arc analyzed using G-protein primers and probes. In another embodiment, methods are provided for ~ oncogene detection in RNA purified from surgical biopsy samples or cultured cells. Methods are 15 also provided for extracting RNA from air-dri~ 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 Gi3, Gi2, Gsc~, Gil, Gz, and Go.

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~ PCTIuS 911/ 00~,8 5 8 In some instances DNA samples are less well preserved and, consequently, amplification of long segments is problematic. Example 1 describes primers for amplifying short segments of DNA that include oncogenic regions of ~.
Thus, the present invention provides methods for detecting activated oncogenes 5 at the genomic (DNA) level. The rnethods are also suitablc for monitoring the expression of prot~oncogenes and oncogenes. The level of oncogene expression canbe rnonitored during and following, for example, chemotherapeutic treatment.
Rer~ussion and recurrence of turnors can be monitored as well. Companng the activation of proto-oncogenes into oncogenes at thc genornic level, with the expression f activated oncogenes at the mRNA level, provides valuable information for analysis - -of carcinogenesis and therapies. These methods may also provide information regarding a predipostion to specific malignancies.
The invention provides needed tools useful in analysis of carcinogenesis events.Probes are provided for determining allelic dominance for a specific oncogene. For example, probes specific for Gi2 alleles can be used to analyze somatic cells as well as tumor cells. These probes can also distinguish, in the case of a tumor comprising one mutant and one wild type allele, the relative abundance of the rnRNA products of these alleles. One sk~led in the art will recognize the utility of such analyses following study of and studying mutagenic events.
In another aspect, the primers and probes described provide a method for phenotyping a cell. The cell may be a turnor cell or a sornatic cell. Analysis using the methods of the invendon provides information relating to the proto-oncogene and oncogene profile of a cell. In this way, events related to the presence of, for example, more than one G-protein point mutation, may be discerned which were previously - -undetectable.
These methods provide a means for associating a specific malignancy with a specific oncogenc orpoint mutadon. For cxample, of 306 samples for lS tumor types analyzed for Gsa, 18 point mutadons were detected; all 18 Gsa oncogenes were detected h pituitary adenomas and thyroid turnors. In another examplc using Gia2primers and probes, 254 samples representing 14 different tumor types were analyzed.
Four tumors of two typcs had Giat2 oncogenes; one ovarian granulosa cell tumor and three adrenal cortical turnors. Analyses using the methods and probes provided has led to the discovery that the pro~oncogenes cncoding G-protein a subunits are acdvated in endocrine tumors. These studies suggest that distribution of Gsct and Gio~2 35 oncogenes is restricted among specific endocrine target cells.
An analysis of G-protein oncogenes and turnor specificinf provides infomnation of usefi~l in determining the rolc of G-proteins, where that role is as yet undefined. For CTiUS 91~00858 ,''V 1 example, the data suggests that corticotropin (ACI'H), which stimulates cortisolsecretion via Gs, adenylyl cyclase, and cAMP, does not utilize Gs and cAMP to stimulate proliferation of ACIH target cells. Tumors denved from adrenal cortical cells (the target cell of ACIH) do not harbor the Gsa oncogene. Those skilled in the art will recognize that the methods of the present invention provide essential tools for identifying new oncogenes, such as Gia2 (alternatively referred to herein as gip2) and exploring the diverse mix of signalling pathways that rnediate regulation of proliferation in endocrine target cells.
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 irnportant information regarding the genetic make-up of an individual or the carcinogenic state of the sarnpled tissue.
A mutant G-pr tein may be domina~ed in vivo by a noTTnal G-protein unless carcinogenesis is triggered by other events. Thus, antibodies enabled by the present invention would also find use in, for example, screening ~ansplantation tissue as an indicator of potential oncogenic complications.
It will be apparent to those skilled in the a;t that the method of the present invenion is amenable to eommercialization as a kit for the quantitation of one or more nucleic acids in a sample. For example, in its simplest embodiment, sucb a kit would provide an oligonucleotide pnmer pair for amplification of a G-pT~tein cc subunit segrnent and corresponding wild type oligonucleotide probe. In another embodiment, a kit may eontain an alray of G protein probes fixod onto a solid support and a eorresponding prirner pair for amplifying and detecting oncogenic polnt mutations in genes eneoding G pT~tein subunits. In another embodiment, a kit may eontain an oligonueleotide pTimer pair, eorresponding G-protein a subunit wild type and mutant probes, a DNA polymerase, a RNA polymeTæ, a reverse transcriptase, nueleotide tTiphosphates, restTiction cnzymes, and buffers for carrying out cDNA synthesis,restTietion enzyme digests, and amplifieation by PCR. Further, the kits may contain a thermostable DNA polymerase; for example, the thermostable DNA polymerase Taq isolated from ~ ~ as an agent of polymeriza~ion.
To faeilitale the understanding of the invention, bTief definiTions aT~ pT~videdbelow.

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PC~ U~ ? 1 '008 58 1 ;, ~.lAY 1 ~1 "Proto-oncogene" refers to thc wild type forrn of gene encoding a protein in which a point mutation affecting the amino acid sequence encodes a protein which may have a carcinogenic or tumorigenic effect.
"Oncogene" refers to a prot~oncogene containing a point mutation and 5 encoding protein which may havc a ca~:inogenic or tumorigenic effect. Oncogenes may alternatively be referred to herein as "activated proto-oncogenes."
"G-protein subunit primers" refer to primer pairs which hybridize to complementary strands of a nucleic acid encoding a G-protein a subunit and will function in a PCR reacion to amplify a nucleic acid segment comprising one or more codons suspected of harboring a point muta~ion. In the disclosed embodirnent, these - ~
codons encode arnino acids corresponding to Gsc~ amino acids 49, 201, and 227. -However, those skilled in the art will recognize that po~eneially oncogenic point mutations may exist at other positions as well. G-protein a subunit primers can readily be designed to arnplify such regions according to the methods described herein.
"G protein subunit probe," "G-protein probe," or "probe" as used herein refers to an oligonucleotide probe designed to characterize the nucleic acid sequence encoding an amino acid at a position suspected of containing a point mutation. In the preferred embodimen~ of the present invention, individual probes comprise the wild type nucleic acid sequence, or a nucleic acid containing a point mutation, within a specific codon. Specific codons include any codon within a nucleic acid encoding a G-protein, which when replaced with a non-wild ype sequence results in a G-proteinoncogene. In the present examples, the specific codons include for each G-protein a subunit, codons encoding the arnino acids corresponding to Go amino acid positions 49, 1-02, and 227.
For any specific codon and any specific G-protein, one wild type probe and ninc point mutation probes may be designed and used in the present methods.
However, it is not an essential aspect of the invention that probes be included for pracdce of these methods. In fact, the use of only one probe may be sufficient to discliminate between two individuals or between the presence and absence of a point mutadon.
The following examples provide an illustration of the present invendon. They are not a lirnitadon to the scope of the invcndon. ~hose sl~lled in the art will recognize that the prirne~s and probes disclosed can be Iwdificd, for exarnple, by altering the length of an oligonucleodde without altering thc purpose and effectiveness of thc desc~ibedinvention.
~ .
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PCT!US 9 1 / ~ 9~8 Ex~ 1 A. ~c~
Hurnan pituitary turnors specimens were supplied by Charles Wilson 5 (University of California, San F~ncisco) and Anna Spada (Milan, Italy) provided biochemically characterized pituitary samples~ Additional samples were provided by Hans Feichtinger and Kurt Griinewald at the University of Innsbruch, Austna, andClaudia Landis, Griffith Harsh, Quan-Yang, Duh and Orlo Clark at the University of California, San Francisco.

10 B,~m~ Prepa-~Qn For isolation of genomic DNA from paraffin-embedded tissue, 3-5 adjacent 5 mm sections were cut from paraffin blocks and mounted on glass slides (Wright et al-E~B, ProtQcols: A g~ IQ Methods ~ ~QD5 eds. M. Innis, D. Gelfand, ~ -J. Sninsky, and T. White, Academ~c Press, San Diego, pp. 153-158, incorporated 15 herein by reference). One slide was stained with hematoxylin and eosin and used as a guide to select a region composed entirely of tumor on the other slides. With a razor blade, excess parafflr~ and unwanted tissue were removed from the unstained slides, and the remaining tumor tissue was scraped into a sterile 1.5 rnl microcentnfuge tube.
To remove contaminating paraff~n, the sample was incubated with 5 mls of octane 20 (anhydrous, Aldrich) or Hemo-De (Fischer) a~ room temperature for 30 minutes with shaking. The tissue sample was pelleted by centrifugation (5 minutes, 1000X g) and the supematant was discarded. The tissue sample was extracted 2X vith 500 mls absolute ethanol to remove traces of octane and then vacuum drie~ To digest the tissue and release the genomic DNA, the sarnple was treated with 0.2 mg/ml proteinase K in -25 100 rnls digestion buffer (50 mM Tris, pH 8.5; 1 mM EDTA; 0.5% Tween 20) at 37-C
ovemight. The sample was cen~ifuged 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 sarnples were prepared according to Verlaan-de Vries ~t ~., 1986, 30 Gen~ 313.

C. ~ifis~Q~roced~
Nested amplification plimers we~e used in the PCR amplification procedure to improve specificin~r and yield (Mullis ~ ~., ~). Genomic DNA (100-500 ng DNA ~ :
from fresh tissuc or 10 mls of the DNA soludon from pa~affin~mbedded tissue) was- 35 firstamplifiedwith30pmolsoftheouterpritners(seeTable l)in lOOmlsofO.l mM

' '. . . , ~ ' . . ' .

` `~ PCTIIJ.S 9 1 / 0 0 8 5 13MAYl^

dNTPs; 50 mM KCl; 20 rnM Tris, pH 8.3; 2.5 rnM MgC12; 100 g/ml BSA; and 1.5 units Taq polymerase (Pe*in-Elmcr Cetus). A thertnocycler (Perkin-Elmer Cetus) was used for amplification. The amplification program was: S minutes at 95 C followed by 30 cycles of 1 minute at 95-C, 2 minutes at 50 C, and 2 rninutes at 72 C. A second S amplification reaction with 30 pmols of the inner prirners was done using 2 ,uls 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 arnplification reaction was: 30 seconds a~ 94-C, 40 seconds at 57 C, and 45 seconds at 72 C. Five ~lls of the final produc~ was sized on a 2% Nusieve, 1% Seakem agarose gel and visualizeid by ethidium bromide staining. A 526 base pair fragrnent was obtained for Gs and a 504 bp fragment for Gia2.

D. Dot E~ pmcedur~
Nylon filters (Pall Biodyne-B, 0.45 mm) were briefly rinsed in water and rnounted on a Bio-R~D dot-blot apparatus. Four mls of each final arnplification i -product werei denatured in 0.4 N Na OH and 25 mM EDTA 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 prehybridized in 5X SSPE and 0.5% SDS at 50 C
for 30 minutes. One ng of a 32p end-labeled digonucleodde was added to the ~ -prehybridizadon soludon and incubated at 50C for 45-60 minutes. The hybridized filters were washed briefly in 2X SSPE and 0.1% SDS at room temperature, followed --by a 10 minutc incubadon in 3 M tetramethyl ammonium chloride, 0.2% SDS, and 50 mM Tris, pH 8.0 ~lMACI) at the following temperatures: for Gs codon 201, 64.5C;
Gs~c codon 227, 67'C; Gi~2 codon 179, 61.5-C; Gi2 codon 205, 67.5C. When other probes are employed, hybridization is canied out as desc~ibed above. The filters are then washed in TMACI at S8-C for 10 minutes. The wash temperature is adjusted in l-C increments until only thc wild type and mutant signals can be detected. In this way, thc appropriate wash temperature is dctcrmined. Undcr these conditions, thechosen temperatwc allows only fully complementary hybrids to stay formc~, resulting in a posidve dot on a filter. The filtcrs were exposed to Kodak X-AR film for 2-6 hours at -70-C with intensifying screens. For subsequent hybridi~ation with different oligonuclcotides, the nylon filters werc slripped by boiling the filters for fivc minutes in -~
2X SSPE, 0.1% SDS and then processed as describcd above.
E. Se~uel~
FM double-stranded scquencing of PCR product, a single band of appropriate 35 size was exciscd from an ethidium brt)mide-suinéd agarosc gel under W light. The excised band was placcd into a Costar spin-X 0.22 ~m cellulose acetate filter unit, ... - . . ................................... .

- . . , .. : . . : : :

P"T!IIS 91 / 008 58 13 MAY lC91 .

frozen at -70-C for 15 rninutes and spun in an Eppendorf microcentrifuge for 15 minutes at full speed The DNA-containing eluate was transferred to a microcentrifuge tube, 2011g glycogen was added, and DNA was precipitated with 0.2 volumes of 3 Msodium acetate and 0.3 volumes ISO propanol. DNA was pelleted in a 5 microcentrifuge, washcd with 709'o ethanol, vacuum dried, and resuspended in 20 ~LI
double disilled water. The sample was sequenced according to ~he Sequenase (IJnited Sta~es Biochemical) protocol using 7.75 111 DNA solution and 2.5 pmol sequencingprimer.

F. Oli~onu~otides Table 3 provides prirncr pairs for amplification of nucleic acid segments possibly containing oncogenic point mutations. If primer sequence is within the coding region (exon) of the a subunit, the primer pair is suitable for arnplification of either a DNA or cDNA template. The nucleic acid sequence of ~he G-protein subunits are published for &ia (sec Bray ~ al.. 1987, oc. ~ - ~. ~ 84:5115-S 119.~, 15 Gsa (see Kozasa et al~ 1988, Proc. ~a~l. e~. ~. lI~ ~:2081-2085), Goa (see Lavu ~ ~., 1988, Biocherr~. ~hY~ Qmm. 150:811-815), and Gza (see Fong ~ ~., 1988, ~. ~ ~. ~. ~ ~:3066-3070). For each primer pair, the size of the amplification product from a genomic DNA template is shown. Note that for Gial, Gia?" Gia3, Goa, and Gza the designation 201 and 227 refers to the codon 20 encoding the arnino acid corresponding to position 201 or 227 in amino acid sequence of Gsa.

Table 3 i~a~ Primer~ for Detection of Q-Protein Point Mutatio~
Primer Gs~201t~l Outer Prjm~ ~Q~iiQn 25 JFL69 (SEQIDNO:26) S' GCG CTG TGA ACA CCC CAC GTG TCT intron JFL70 (SEQIDNO:27) 5' CGC AGG GGG TGG GCG GTC ACT CCA intron Gs~201n27 Inner Primers(S26b~) JFL135(SEQIDNO:28) 5' GTG ATC AAG CAG GCT GAC TAT GTG exon JFL136(SEQIDNO:29) 5' GCT GCT GGC CAC CAC G M GAT GAT exon .-:: - ., . , ~, . .,, , ~ . - , . ~ - - , . -~ PCTIIJS ~ 0~85,8 Pli ner Gsa - 201 (2~ bD) E~Q~Qn JFL228 (SEQ ID NO: 46) 5' MG AAA CCA TGA TCT CTG TTA TAT intron JFL135 (SEQIDNO: 28) 5' GTG ATC AAG CAG GCT GAC TAT GTG exon Gs~ - 227 r263 b~) JFL229 (SEQ ID NO: 45) 5' CCC CAG TCC CTC TGG AAT AAC CAG intron JFL136 (SEQ ID NO: 29) 5' GCT GCT GGC CAC CAC GAA GAT GAT exon G~- 49 (111 bp) JFL226 (SEQ ID NO: 24) 5' AAC AGC AGA CCT CCC TGC CCA AAG intron JFL227 (SEQ ID NO: 25) 5' CCC CCC TGC ACA GAT TTG ACA CTT intron ~ - ~oln27 JFL223 (SEQ ID NO: 30) 5' TTG GAC AGA ATA GCT CAA CCA AAT exon JFL224 (SEQ ID NO: 32) 5' TAG AAC CAG GTC GTA GTC ACT exon Gia2 - 201n27 Q~P~imers ~L54 (SEQIDNO: 20) 5' CCC CCC ATC CCC AGC TAC CT exonrlntron JL57 (SEQlDNO:21) 5' TCT CAC CAT CTC CTC GTC CTC exonfimtron Gia2 - 201læ2~1nner Prin~rs f5~4 b~) JL55 (SEQIDNO:22) 5' ATT GCA CAG AGT GAC TAC ATC CCC exon ~56 (SEQ ID NO: 23) 5' GGC GCT CAA GGC TAC GCA GAA exon Gt~ - 201 Outer Primers JFLIQ9 (SEQ ID NO: 47) 5' TGT CTT TTA TTT AGT ATC AA e:~on~mtron JFL110 (SEQ ID NO: 1~) 5' GAT CTG GAT AGA ATA TCC CAG e~on~lntron a~/l~ .
~FLIIO (SEQIDNG: 16) 5' GAT CTG GAT AGA ATA TCC CAG exon JFL112 (SEQIDNO: 17) 5' GGT GAA ATG TGT TTC TAC AAT exon Gta3 - 227 Ollter Primers JFL113 (SEQIDNO: 18) 5' TTC CCC TTG CGC AGG ATG m exOnrmtrOn JFL114 (SEQIDNO:48) 5' ACA TAC CAT CTC CTC GTC CTC e:~onfimtron Gia3 - 2Q7 Inn~ Prime~rS (12Q b~) Jl:L115 (SEQIDNO: 19) 5' CAG AAC MG GTC ATA ATC ACT e~lon JFLI~3 (SEQIDNO: 18) 5' TTC CCC TTG CGC AGG ATG m .: . .
Goa 25LI (87 b~
SP9 (SEQIDNO: 38) 5' CTG GAC AGC CTG GAT CGG ATT GGG exon `
SP10 (SEQIDNO: 39) 5' GAG GTT CTT GAA TGT GAA GTG GGT e~
Goa - 2~1(129 bV) JFL139 (SEQIDNO:40) 5' AAC CTC CAC l-rC AGG CTG m e~con SPII (SEQIDNO:41) 5' GTG GAG CAC CTG GTC ATA GCC GCT e~on Qz~. 201n2? (-200bD~
JFL201 (SEQIDNO:43) 5' TGT GAC GCC CTC GAA GCA GT e~on SP15 (SEQIDNO:42) 5' AAC GAC Cl`G GAG CGC ATC GCC e~on :: .: . ~. ., . , , , , : - ., ,., . .. ",. . . -. ... ...... . . .. .. . .

.. . ,, : .,. . . ~ . - :

G~ e~bes for ~h~ Det~Qn of Point MUta~,Qn~Wi1d Gsa - 49 S ' TA GGT GCT GG~ GAA TCT GGT 3 ' Gly (SEQID NO:49) AGA Arg (SEQID NO: 50) CGA Arg (SEQID NO: 51) GAA Glu (SEQID NO:52) GCA Ala (SEQ ID NO- 53) Gscc :~QlGTA Val (SEQII)NO 54) 5' TT CGC TGC ~ GTC CTG ACI 3' ~ (SEQID NO:55) TGT Cys (SEQnDNO:56) GGT Gly (SEQID NO: 57) AGT Ser (SEQID NO:58) CAT His (SEQlI)NO-59) CCI' Pro (SEQID NO.60) GsQ~ - 227CTT Leu (SEOID NO: 61) 5' GTG GGT GGCCA~;i CGC GAT GA 3' ~ (SEQID NO: 62) TAG Thr (SEQlD NO: 63) GAG Glu (SEQID NO: 64) AAG Lys (SEQID NO: 65) CGT Arg (SEQID NO:66) CCG Leu (SEQID NO: 67) CTG Pro (SEQ 11) NO: 68) '~CAC His (SEQlD NO:69) CAT His (SEQID NO: 70) Gi~l - 201 5' TC AGA ACr ,~ GTG AAA ACT 3 ~e (SEQII)NO: 71) AGC Ser (SEQ ~ NO: 72) AGT Ser (SEQI[)NO: 73) GGA Gly (SEQ ~ NO: 74) ACA Tar (SEQID NO: 75) AAA Lys (SEQID NO: 76) Gil - 227 ATA Ile (SEQ ID NO: 77) S' TG GGA GGT ~ AGA TCr GAG 3' ~ (SEQID NO:78) GAG Glu (SEQlD NO: 79) AAG Lys (SEQID NO: 80) CTG Leu (SEQII)NO: 81) CCG Pro (SEQlD NO: 82) CGG ~ (SEQ~DNO:83) CAT His (SEQIl)NO:84) CAC His (SEQnDNO:85) PCTiUS ? 1/ ao 8 58 . 13 p~ y 1?? ~

Gia2 - 201 S' TA CGG ACC ~ GTA AAG ACC 3' ~ (SEQID NO:86) AGC Ser(SEQ ID NO: 87) GGC Gly (SEQ ~ NO: 88) TGC Cys (SEQ ~ NO:89) CAC His (SEQ ~ NO:90) CCC Pro (SEQ ~) NO: 91) CTC Leu (SEQ n~ NO: 92) Gin2 - 2~
10 5' GTG GGT GGT CA~ CG& TCT GA 3' ~ (SEQ ~ NO:93) GTG L~ (SEQ ~ NO.94) CCG ~o (SEQ ~ NO-95) -CGG Arg (SEQ ~ NO:96) AAG Lys (SEQ n~ NO: 97) . GAG Glu (SEQ ~ NO:98) CAT His (SEQ ~ NO:99) Gi~3 - 2Q1 CAC His (SEQ ~ NO:100) S' TT CGG ACG e,~e. GTG AAG ACC 3' ~ (SEQ lO NO: 101) AGC Ser ~SEQ ~ NO-102) AGT Ser (SEQ ID NO 103) GGA Gly (SEQ ~ NO:104) ATA ne (SEQ ~ NO:105) ACA Thr (SEQ ~ NO:l~) ,AAA Lys (SEQ ~ NO: 107) Gic~3 - ~.
5' GTA GGT GGC~9~ AGA TCA GA 3' ~ (SEQ ~ NO:108) --.-.--CAT His (SEQ ~ NO:l~) CAC His ~SEQ ~ NO:llO) CTA Leu (SFQII)NO. 111) :
CCA ~O (SEQ ~ NO,112) CGA ~g (SEQ ~ NO 113) AAA Lys (SEQ ~ NO.114) GAA Glu (SEQ ~ NO-115) Goa ~u S' CTC ~GA ACC AGG &TC AAA AC 3' ~ (SEQ ~ NO:116) GGG Gly (SEQ ~ NO-117) TGG Trp (SEQIDNO- 118) AAG Lys (SEQ ~ NO- 119) ACG Thr (SEQ ~ NO-120) ATG M~ (SEQ ~ NO-121) AGC Ser (SEQ ~ NO.122) AGT Ser (SEQ ~ NO:123) - . .: , . , . . - ,. . , . , . . . . .. : ~
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-;` PcTilJs9l/oo858 13 MAY lcgl Goc~ - 222 5 ' GTC GGA GGC CA~ CGA TCT GA 3 ' ~ (SEQIDNO: 124) AAG Lys (SEQIDNO:125) GA& Glu (SEQIDNO: 126) CCG Pro (SEQIDNO: 127) CGG Arg (SEQIDNO: 128) CTG Leu (SEQII)NO- 129) CAT His (SEQIDNO: 130) Gza - 201 CAC His (SEQIDNO:131) 5' TG CGC TCC ~ GAC ATG ACC 3 "~ (SEQIDNO: 132) GGG Gly (SEQIDNO: 133) TGG Trp (SEQIDNO: 134) CAG Gln (SEQIDNO: 135) CCG Pro (SEQlDNO: 136) CTG Leu (SEQ IDNO: 137) S ' TG GGG GGG CAG AGG TCA GAG 3 ' ~ (SEQ ID NO: 138) AAG Lys (SEQIDNO: 139) GAG Glu (SEQIDNO: 140) CCG Pro (SEQIDNO: 141) CTG Leu (SEQIDNO: 142) CAC His (SEQlI) NO- 143) CAT His (SEQIDNO 144) CGG Arg (SEQIDNO: 145) For each G-pr~tein codon to be characterized, oligonucleotide probes are shown in Table 4 as follows. For each positisn, i.e., Gsa 201, the full probe sequence is shown for the wild type allele. The wild type codon at the potentially oncogenic site is underlined, and the translated amino acid is shown to the right A set of prsbes is 30 provided where the sequence is identical to the wild type except at the codon to be characterized. Thus, for non-wild type probes only that codon and the transla~ed amino acid product is shown in the table. Only those point mutations encoding amino acids different from the wild type amino acid are shown.
Table 5 shows stret hes of C~s sequence surrounding the arginine 201 and 35 glu~inc-227 codons which are highly conserved in G-protein a chains of vertebtates, yeast, and slime mold. Published sequences include rat Gsa, Gia2, Gia3, and Goa,human Gza (Katada and Vi, 1982, oc. ~. ~. ~. ~2:3129~, Gta of bovine retinal rod cells ~Chambard ~ ~., 1987, Natu~ ~:800), the a cha~n of the G-protdn (called GPA 1/SCGI ) that mediates phcromone signalling in s~hammyG~
40 CkT~YiSia~ (C`olven ~ al-. 1989, ~. ~2, and Itoh ~; ~., 1988, 1. ~jQ1- ~m.
~:6656), and an a chain (Gal) from ~j~tyoste~ Çs~m (Zarbl et ~., 1985, --Naturç ;~:382). Thc number is parcnthesis following each scquence is the aclual - , ~ ,- , : , : -P~TIus 9tl3/M~ 8~5 1 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=~e .:

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Exama~ 2 Identifi~a~Qn of G~ ~n Point ~utatiQn~ in Gs Gen~
To deterrnine thc frequency of mutations in codons 201 and 227 of Gs genes in pituitary and other tumors, the polyrncrase chain reaction (PCR) was used tO alnplify 5 a specific region of genotnic DNA and high-stringency hybridization of sequence-specific oligonucleotides to dctect point mutations in the arnplified produc~. -To detect mutations in the Gs gene, a single region including both codons 201 and 227 and an intervening intron was amplificd from turnor genomic DNA prepared from fresh frozen or paraffin ernbedded sarnples as describcd in Example 1. The amplification prirners shown in Table 3, designated "Gs 201n27 Outer Prirners" and "Gscc 201/227 Inner Primers," were used according to the method described above.Oligonucleotides specific for wild type or single-base mutations at codon 201 (6possible missense mutations) or codon 227 (7 possible missense mutations,1 nonsense mutation) were hybridized to the amplified product (Table 4). Genomic DNA from 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 1tumors had low basal adenylyl cyclase activity that responded normally to stimulatory agents, group 2 tumofs had marked elevation of basal adenylyl cyclase activity that responded poorly to stimulatory agents.
The hybridization results are shown in Figure lA. The hybridization probes shown in Table 4 were used as follows: R201 indicates the wild type probe for Gsc~
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 Gln 227 containing a point mutation (CAG to CGG) encoding arginine.
Of the many turnor types analyzed, mutations were detected only in GH-secreting tumors of the pituitary gland. Among 42 GH-secrcting pitui~y tumors, 18 (43%) contained Gsa mutations. Of these, 16 mutations were in codon 201 (14 30 arginine to cysteine, 2 arginine to histidine) and two were in codon 227 (both glutamine to arginine). These results are summarized in Table 6. For two patients in whosetumors Gsa point mutadons were detected, samples of white blood cells were available; neither sample containcd a mutant Gs gene, whether assessed by sequencing cDNA clones (2) or by allele-specific oligonucleotide analysis. This result 35 indicates that the mu~ations are soma~c and thus likely to have played a direct causal role in the development of the turnors. Furtherm~rc, a norrnal Gsa allele was present :. : , ~ ' ':
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~` PCTIUS91/0085 13 I'~AY la9' in all turnors where a mutant allele was detected, suggcsting that mutations that activate Gs are dominant.
C)f 42 GH-secreting pituitary tumors, 16 were biochemically characterized in telms of adenylyl cyclase activity. Eight turnors showing elevated adenylyl cyclase 5 werc predicted to harbor an activated Gsa; each of the~e tumors contained a mutation in codon 201 or codon 227 (Figure lA). No mutations wc~ detected in eight tumors tha~
showed normal adenylyl cyclase activity. Although Gsa mutations in other codons may also inhibit GlPase, the strong concordance between elevated adenylyl cyclase activity and a muution in codon 201 or codon 227 indicates that activating muutions at 10 other sites are relatively infrequent.
Table 6 provides a summary of human turnors screened for mutations in codons 201 and 227 for Gs and codons 179 and 205 for Gia2. DNA for PCR arnplifica~ion Mullis ~ ~1., supra;, was isolated from either fresh tissue, Verlaan-de Vries et al-.
1986, Ge~ 50:313, or paraffin-embedded tissue Kozasa et al . 1988, proc. atl.
15 ~. ~. ~ ~:2081. Eighteen growth honnone (GH) secreting pituitary adenornas contained a mutation in eilher Gsa codons 201 or 227. One ovarian granulosa cell turnor and three adrenal cor~ical tumors (two adenomas, one carcinoma) contained a mutation~n Gia2 codon 179.

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PCTiUS 9 1 / 00 8 5 ~v~ IAY 1991 Table 7 provides a list of wi~d type codons for the conseIved arginine and conserved glutamine in Gsa and Gia2 genes. The table also shows single-nucleotide base changes (in bold3 and the resulting arn~no acid changes. Oligonucleotides specific for wild type and each missense or nonsense single-base change listed were used to screen human tumors, with the exception of base changes that would be silent (marked with asterisks). Mutations detected in the tumors listed in Table 6 are underlined.
"Term" indicates termination or stop signal.

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prr IIS 9 1/ 00 8 5 8 Example 3 Identification of G-Protein Point Mutations in Gia2 Genes In order ~o investigate the possibility that mutational activation of signaling pathways mediated by other G-proteins might lead ~o abnormal proliferation and tumor 5 formation, a large panel of human tumors was screened for mutaions in a Gio! gene.
In Giæ the coding sequence and intron between the two codons to be tested, arginine-179 (corresponding to Gsa Arg 201) and glutamine-205 (corresponding to Gsa Gln 227), is short enough to allow PCR amplification of a single genornic DNA
fragment containing both codons (Itoh ~ ~., 1988, l. ~Ql- Chem. ~:6656).
The prirners and probes used for detection and characterization of Gio~2 are shown in Tables 3 and 4. Samples werc 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 Gia2/201 probe of Table 2 15 containing a point mutation (CGT or TGT) encoding cysteine (R179C). The last two rows of each panel were hybridized to the Gia2/227 probe shown in Table 4 comprising a point mutation (CGT or CAT) encoding histidine (R179H). The amplification method~ are described in Example 1.
Table 6 surnrnarizes the hybridization results. Mutations in codon 179 of Gio~2 20 were detec~ed in two different endocrine tumor types 3 of 11 tumors of the adrenal cortex and one of 6 ova~ian granulosa cell tumors. The adrenal turnor lacking a wild type allele was an adenocarcinoma; the other 2 adrenal turnors were adenornas. No muta~ions were found in codon 205. The high frequency of codon 179 mutations in tumors of two related cdl types suggests that these mutations converted the Gic~2 gene 25 into an oncogene, referred to herein as gip2 (for Gi protein-2).
StriKngly, the amino acids that replaced arginine-179 in Gi~2, cysteine and hisddine, were the same as those that replaced the cognate arginine at position 201 in Gsa oncogene products found in pituitary tumors (Landis ~ ~1.). It is possible that, of she six possible missense mutations that can result from single-base changes in these 30 codons of Gsa and Gia2,`only these mutant proteins are biologically active. Also, all mutations found so far, in either Gs or Gia2, are transition mutations (Table 7);
consequently, these mutations rnay rcflect a common mutagenic mechanism.
In one turnor of the adrenal cortex, a no~nal a~lele of Gia2 was not detected --(Figurc lB). Scquence analysis of PCR products revealed a single scquence, corr~sponding to the codon 179 mutation. This result makes it likely that the norrnal allele was missing, although the possibility that both alleles contain the same mutation cannot be excluded. Loss of the ncmnal allele suggests that its protein product .

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`-```. PCTIUS91/00858 ay 1ga1 interferes with the oncogenic e~fect of the mutant protein, so that failure to express normal Gi2 confers an additional sclective advantage on cells carrying an activating Gic~2 mutation in the other allele.

Example 4 Detection of ~s,~ Mutations in Paraffin-Embedded Sarnples bv Microdisseçtion Changes in the t vo Gsa codons affected by ~ mutations were detected in formalin-fLxed, paraffin-embedded tissue blocks of human tumors by isolating genomic DNA, arnplifying appropriate Gsa sequences with the polyrnerase chain reaction (PCR), and screening the arnplified products for their ability to hybridize with allele-specific oligonucleotides. In Examples 1-3 DNA from all cells in a S mrn tissue section was analyzed. DNA from one thyroid turnor had two different ~2 mutations, in addition to the wild type allele. To ask whether the two mutations were located in different regions of the 5 m tissue slice, an adjacent 5 mm section from the same tumor was divided into several smaller fragrnents; genornic DNA was isolated from eachfragrnent separately and subjected ~o PCR amplification and screening with allele-specific oliy;onucleotides. The two ~ mutations were detected in different fragments;
in addition, several fr'agrnents contained only the wild type codons at positions 201 and 227 of Gsa.
The latter observation suggested that the heterogeneous distribution of mutations in 5 mm sections may go undetected due to diludon by wild type DNA
sequences from cells in parts of the sections that contained no mutations. Accordingly, this modified microdissection approach was applied to additional tumors. Identification of a high prcvalencc of g~ mutations some in turnors carlier tested as nega~ive indicated that this approach is more sensitive at detecting .e5~ mutations.

A. Mali~nantThvroidTumors To explore the roles of ~ mutations in pathogenesis of malignant thyroid :
turnors, the micTodissection technique to tissue blocks of p~imary tumor or Iymph node metastases from 37 patients with differentia~ed thyToid carcinoma. These tissue fragments were also tested for mutations in spccific codons of the three human ras genes. _ Stained scctions from these surgical specimens were examined and each microdissec~d fragment was classified as malignant or benign thyroid tissue; in every case, the pathologic diagnosis applied to at least 90% of the cells in the fragment. Both ;
the pathologic cxasnination and the DNA analysis wesc performed in a blinded fashion:
The pa~hologist diagnosed histology of tissue fragments without knowledge of the lr~ P~T IIS 9 1 / 00 8 5 8 JlAYl~9~, presence or absence of mutaions, and the DNA analyses were performed on coded samples.

B, ~le Pre~aratioll Muliple 5 mm sections were cut from each tissue block and one was stained 5 with hematoxylin and eosin. Regions of the stained slide, 30- 100 mm2 in area, were demarcated with a pen and designated by number, when possible, demarcations separated histologically distinct regions. Using the stained slide as a template, an adjacent unstained S mrn section was divided into corresponding fragments with a razor blade. These fragments were transferred into separa~e tubes and each was processed 10 for PCR and hybridizaion screening, exactly as described in Example 1.

C. PCRAmplification Using 20-25 base pair (bp) oligonucleotide prirners upstream and downstream of codons 201 and 227 in the human Gsa gene, templates ranging from 165 to I ,200 bp in length. Primers chosen for PCR arnplification of regions around codons 12, 13 15 and 61 of the human H-ra~, Ki-~a~ and N-ra~ genes yielded products of 112-117 bp.
Amplification of fonnalin-preserved, paraffin-embedded tissues was most effective with relatively short PCR products. Amplification with nested primers was re~uired when the first PCR of larger DNA fragments produced an unsatisfactory amount of arnplifled DNA, as judged by agarose gel electrophoresis and ethidium bromide 20 staining.
PCR conditions were as generally descrilxd in Example 1, however, the oligonucleoddes and cycling temperatures used were as were as follows.
For nested primer arnplification of Gsa codons 201-227: Outer sense, ~JFL69) (SEQ lD NO: 26) 5'GCG CTG TGA ACA CCC CAC GTG TCT; outer andsense 25 (JFL70)(SEQID NO 27), 5'CGC AGG GGG TGG GCG GTC ACT CCA; product 1,200 bp; opdmal PCR condition, 30 cycles of 1, 2, and 2 m~n at 95'/50-n2; innersense (JFL135)(SEQID NO:28), 5'GTG ATC MG CAG GCT GAC TAT GTG;
inner annsense (JFL136)(SEQll) NO:29),5'GCT GCT GGC CAC CAC GAA GAT
GAT; product 526 bp; op~mal PCR condi~on, 30cycles 30, 40, and 45 sec at 30 95-/57-m-.
For Gsa codon 201: scnse (JFL135) (SEQID NO:28), 5'GTG ATC AAG
CAG GCI'GAC TAT GTG; andsense (JFL286)(SEQID NO:44),5'TA ACA Gl~
GGC TTA CTG GAA;pqoduct 222bp;optimal PCR conditions: 40 cyclesof30,30, and 30 sec at 95-/55-172- For Gsct codon 227: sense~JFL229)(SEQ D~ NO:45),5' 35 CCC CAG TCC CTC TGG AAT AAC CAG; antdsense (JFL136) (SEQ lO NO: 29), .. . .. .
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PCTiUS 9 1 / O ~ 8 5 8 5 'GCT GCT GGC CAC CAC GAA GAT GAT; product 165 bp; optirnal PCR
condition,50 cycles of 60, 30, and 30 sec a~ 9s'155-n2-.
For ~5 gene amplications the following standard PCR conditions of 50 cycles of 60, 30, and 30 sec 95 /55 n2 were used. ~a~ primcrs were as follows.
Ha-ras codons 12 and 13: sense (JFL243) (SEQ ID NO: 146), S'AGA CCC
TGT AGG A&G ACC CCG GGC C; antisense (JFL244) (SEQ ID NO: 147), S'ATA
GTG GGG TCG TAT TCG TCC ACA A; product 150 bp.
For Ha-ras codon 61: sensc (JFL252) (SEQ ID NO: 148), 5'GTC ATT GAT
GGG GAG ACG TG; antisense (JFL253) (SEQ ID NO: 149), 5'ACA CAC ACA
GGA AGC CCT CC; product 112 bp;
for Ki-ras codons 12 and 13: sense (EK371) (SEQ ID NO: 150), 5'CCT GCT
GAA AAT GAC TGA ATA TAA A; antisense (EK372) (SEQ ID NO: 151),5'T ATT
GTT GGA TCA TAT TCG TCC ACA; product 118 bp;
for Ki-ras codon 61: sense (~FL248) (SEQ Il~ NO: 152), 5'GTA ATT GAT
GGA GAA ACC TG; antisense ~JFL249) (SEQ ID NO: 153), 5'ATA CAC AAA GAA
AGC CCl' CC; product 112 bp.
For N-~ codons 12 andl3: sense (JFL216) (SEQ ID NO: 154),5'CTT GCT
GGT GTG AAA TGA CT; antisense (lFL257) (SEQ ID NO: 155),5'GGT GGG ATC
ATA TTC ATC TA; product 150 bp.
ForN-rascodon 61: sense (JFL218) ~SEQII) NO: 156), S'GTTATA GAT
GGT GAA ACC TG; antisense (JFL242) (SEQ ID NO: 157),5'GGC AAA TAC ACA :
GAG GAA GCC TTC; product l l2 bp.
Hybridization dot blots like that shown were scored by counting in an AMBIS
radioanalytic imaging system as described above. Replicate PCR amplifications and 25 analyses of adjacent 5 tmn sections produced sirnilar results, indicating that the procedure was accurate and reproducible as well as sensitive.
To avoid false positives by nonspecific hybridization of mutant probes to the PCR product in the absence of mutations, a level of hybridizadon was set, below which a sample would be considcred negative for a particular mutation. This level was set at 30 20% of the signal detec;ed by hybridization of the wild type probe to unmutated DNA
in the sample. Hybridization signals were determined to be reproducible at this level, but not below it. Consequently, a positive result (20% or more of the arnplified DNA
contains a Lnutation) indicates that at least 40~o of the cells in the assayed tissue fragrnent contain the mutation, if--as expected for dominant somatic mutations--35 each cell has one u ild type and one mutant allele.

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`"~ PCTiUS 9 1 / 00 8 5 8 D. Hvbri~iz~Qn and Detectio~of Poilll Mut~tiQn~
For mutation-specific olinucleotide hybridization, the PCR product was spotted (dot-blot apparatus, Bio-Rad) and covalently bound to a nylon filter (Pall Biodyne-B, 0.45 um) using W light as the auto crosslink semng (Stratalinker, Stratagene).
5 Hybridization with [32P]-radiolabeled mutation-specific oligonucleotides 20 bp long were perfo~rned exactly as described in Exarnple l.

E. Cn~fQI presence ~f a Muta~n To verify the presence of point mutations we scanned all aS hybridization reachons with an AMBIS radioanalytic imaging system (Ambis Co., San Diego, CA), 10 which measures tbe emission of radioactivity from each mutant (CPMm) or wild type (CPMwt) dot on the filter. The [32p] b radioactivity of mutant oligonucleotides nonspecifically bound to the same filters after high stringency washing was termed background activity (CPMb), and ranged from S to 10 % of the wild type hybridization signal. A dot was considered to represent a gsp mutation if (CPMm - CPMb) divided 15 by (CPMwt - CPMb) was greater than or equal to 0.2. This CTiteriOn thus required that amplified DNA samples judged as positive for a mutation must exhibit a mutant signal 20% of that obserYed with wild type. Applying this criterion also minirnized the chance that a spunously positive result could result from contamination of a ~-negativesample by DNA from a ~2-positive sarnple.

20 F Controls The microdissection procedure gave negative results for Gsa and ~a~ mutations in sections of normal connective tissue and human thyroid removed during parathyroidectomy. To confirm positive results, PCR products from six fragments for which hy~ridization results indicated R2OlC mutations at levels near the demonstrated 25 cutoff point (i.e., 22-35%) were sequenced. Genomic DNA was amplified using one biotinylated and one non-biotinylated pnmer, ~o generate unilateral biotinylated PCR
products. A~er binding thc biotinylated PCR product to streptavidin coated beads(Dynabeads, l}ynal) the complementary strand was denatured and aspirated, leaving single stranded DNA. Sequencing was performed ushg the Sequenase kit (Sequenase,30 USB). In all six cases the sequenchg gels showed both the wild typc and the mutant 201 codon, conflln~ing the rcsults of dot blot hybridization.

G. Numbel and Distribution of Mutations ~ mutations were found in surgical specimens from 24 of 37 patients (65 %)with differcn~iated thyroid cancer. ~ mutations in these patients were .
- , , --.

F-~ prT us 91/OG85&
y 199 -heterogeneously distributed among rnicrodissected fragments; overall, 81 of 266 thyroid tissue fragrnents (30.5 %) contained detectable~Lmutations. Arnong the 24 surgical specimens with at least onc ~S32 positive fragrnent, a ~2 mutation was found in 60 of 120 histologically malignant fragmcnts (S0 %). The mutations were not confined 5 to fragrnents with obviously malignant tissue, however, indeecL in the same tumors 21 of 60 histologically benign fragrnents (36.2 %) also contained 3~ mutations.
Of the 24 patients in whom thc mic~disscction techni~ue revealed ~s32 mutations, only five were originally suspected of harboring ~ on the basis of the previous testing procedure, which us~d the entire 5 m section as a single sample. In 10 each of thesc five previously positive cases, more than half of the fragments tested with the new procedure were positive for~ mutations, a frequency found in only one other case. Thesc observations indicate that without microdissection the dilution of samples by DNA from regicns without mutations can cause a substantial proportion--perhaps more than 75%--of thyroid ~ mutations to be missed.
Specimens from six of the 37 patients harbored two different ~ mutations, -and the turnor of one patient (number æ, Table 8) contained three different ~
mutations. These multiple mutations were found in separate fragments of these specimens: Of the 31 ~positive fragments tested in patients with more than one mutation, only one fragment contained two mutations. Multiple ~ mutations in a 20 single tumor did not correlate with clinical aggressiveness or other characte~istics of the tumor (Table 8).
Of the 37 specimens exarmned, lS were obtained from tumor metastases to areas physically separate both from the original primary tumor and from nom~al thyroid tissue--i.e., spread beyond the strap muscles to lymph nodes in 13 cases and to lung 25 in two case:s. Of these l5 specimens, 11 harbored ~ mutadons. In three cases (patients 1:2, 19 and 24), these metastases revealed two different ~2 mutations.Occasional reports have documented more than one ras mutation in a tumor (e.g., in human cutaneous melanoma, see Van't Veer ~S ~., 19~9, ~ol. ~ell Biol.,9:3114-3116). The present study is the first to find a high incidence of muliple30 mutations of a single oncogene in individual tumors. The microdissection technique will likely find a similar multiplicity of oncogenes in other tumors as well.

~as M
Mutaional substitutions of an~Lno acid rcsidues in threc key positions of p2 1~
--glycine-12, glycinc-13, and glutarnine-61--inhibit GTP hydrolysis and promote its 35 ability to t~i~gger ncoplastic Iransfonnation. Other im~estiga;ors haw reported prevalences of ~ muudons in thyroid cancer ranging from 17 to 60 % (LeMoine ..... .. .

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'Tllls9l!oc858 1.~, ~/IAY 19qt ~1.. 1989, Q~Q~n~. 4:159-164 and Wright ~1-, 1989, Brit. J. Cancer, ~Q:576-577).Consequently, the 37 surgical specimens from patients with thyroid cancer were tested for oncogenic mutations affecting the three key codons of the three hurnan ra~ genes.
Microdissected fragrnents from 12 of 37 patients (32 %) conta~ned N-~ mutations.5 These included 12 fragments with codon-61 mutations (Q61R), 18 codon-13 mutations (nine G13C, nine G13D), and a single fragrnent with a codon-12 mutation (G12C).
N-ras mutations resembled g5p mutations in several respects: Both were heterogeneously distnbuted, sometimes multiple in a single patient, and present in benign as well as malignant thyroid dssue (Table 8). Of 117 microdissected fragrnents 10 tested in the 12 ras-positive patients, 31 (26 %) contained an N-r~s mutation. Two pa~ients had more dhan one different ras mutation.
Detection of ~L and ~ mutations in the same microdissected fragment does not mean that both mutations are present in the same cell; indeed, further microdissection would probably segregate different cell populations containing each 15 mutation.

The thyroid tumors exhibited four previously unreported mutations, including substdtutions of proline or serine for arginine-201 and subsdtutions of histidine or proline for glutamine-227.

20 J ConclusiQ~
The microdissection technique for finding point mutations gready extends the practical resolution for detecting certain oncogenes, to the point that it can detect a mutadon present in 40% or more of the few thousand cells in a 5 m x 30-lûO rnm2 fragment of tissue. Microdissection may serve to uncover heterogeneously distributed 25 oneogenes in non-thyroid tumors also. The increased sensidvity of this teehnique also shows that a eonelusion that a partieular oneogene mutadon is not present in a tumor can be wTong. If based upon PCR amplification of large fragrnents of tumor, such a negative eonclusion must be qualified; in fact, a negative result only indicates dhat the mutation is not present in a substandal proportion of the cells in the turnor fragrnent.
30 The results of this analysis are presentcd in Table 8.

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Patients numbered 1-27 and 28-37 (left colurnn) underwent sllrgery in Dusseldorf and San Francisco, respectively. Gap and ra mutations are listed as present or absent in microdissected fragrnents of thyroid tissue; fragrnents containing no thyroid tissue are not include~ Each fragment tested was classified as "carcinoma"
or "benign"; for each, 90% or more of the cells seen in the corresponding part of the stained scction were histologically malignant 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 present in the material available for analysis. Although Harvey- and Kirsten-rasmutations were also sought, only N-ras were found.
Clinical characteristics of patients: The '~isto.~rNM" column (third from the left) indicates whether the turnor was diagnosed as a papillary (P) or follicular (F) thyroid carcinoma, and provides the nurnerical clinical classification (TNM) of thyroid cancer devised by the World Health Organization2l, in which T, N, and M are numbers referring to different characteristics: T (varying from I to 4) indicates increasing size and extent of the tumor mass; N (varying from 1 to 3) indicates increasing numbers of Iymph node metastascs; M (0 or 1) indicates the absence or presents of distant metastases; "x" indicates that the information was not known to us. Fifteen patients (nos. 11, 12, 13, 15, 16, 17, l9, 21, 24, 25, 26, 27, 32, 34, and 37) had Iymph node metastases outside thc 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 (l3lI or external) before the operation.
Table 9 provides a summary of pituitary gsp mutations identified in Examples 1-4.

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p~ s a 1 / O O ~ 5 8 1 r~ p/!ll;V 1~1 Example Mate~ials and Method~
A. ~uman.~l~
Human tissues were obtained from surgical biopsy and either RNA immediately extracted or rapidly frozen to -70-C until use. All human tissues were obtained by informed consent in approval with the University of Califomia, Davis Human Subjecls ReYiew Committee. Tissues were also supplied by the Tissue Bank of the Department of Pathology University of Califomia, Davis) under direction of Dr. Robert D. Cardiff.
Microsc~pe slides with bone marrow smears on them were analyze~ One was hematoxylin and eosin (H+E) sta~ned slide, while the other was merely air dried bone marrow. Both slides 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.

B. CellLines Human tumor~cell lines previously characterized fo have known activating point mutations in various ~a~ alleles were used. Cell lines EJT24 (bladder transitional cell -carcinoma) (Tabin ~ a~-, 1982, ~ ~ 143-149, and Reddy ç~ ~., 19~2, ~QQ:149-152) and SK-N-SH (neuroblastoma) (Taparowsky ~ ~1.. 1983, ~:581-586) were a gift of Dr. R. Cardiff. Calu- 1 (lung carcinoma), SW 480 (colon carcinotna, and PA-1 (teratocarcinoma) were obtained from the Arnerican Type Culture ~ -Collection (ATCC) (Capoh et ~., 1983, Nature ~Q~:507-513, and Tainsky et ~., 1984, Sciençe ~:643-645). HL-60 (promyelocytic leukemia) was a gift of Dr. J.
Lawrence (Mu~ray ~ ~., 1983, ~ ;~;749-757).
Other cell lines were used as negative controls for ~ mutations because they are not known to contain activated alleles. K562 (erythroleukemia) was supplied by the Cetus rlssue Culturc Collection (CTCC) (Lozzio and Lozzio, 1975, ~:321-334). Cell line G-2101 (renal clear cell carcinorna) was originated in our lab (Gumerlock ~ ~., 1988, In ~Q ~ ~. ~QI. ~4:429-434). The cell strain T-3891 (fetal lung) is a normal, nonimrnortaLized fibroblastic culture (Rossitto ~t al-, 1988, 1 Yi~ Q:431 435). All of ~he above cell lines were maintained according tothe instrucdons of the supplier.

C. RNA Ex~acdons f~om Freshor Fr~zen Tissu~and Cell Lines Total cellular RNA was extracted f~m dssues and cell lines using modifications 35 of the prcviously descTibed guanidinium-isothiocyanate-phenol-chlorofonn methods - - - , . .

.

PCT!US91/0085 (Maniatis ~ , 1982, In. Molecular Cl~ing, New York, Cold Spring Harbor Page 190, and Chirgwin ~L~-. 1979, Bioehem. ~:5294-5299). Guanidinium isothiocyanate solution (5 M guanidinium isothiocyanate, 25 mM sodium citrate, 0.5%
sarcosyl, pH 7.0) (GITC) was prepared to 5% ~-mercaptoethanol (GITC-ME) just 5 prior to use. Tissue bits were powdered in liquid nitrogen in a mortar, further ground in the mortar upon additional of GITC-ME and 1.5 ml of the slurry was layered onto a cesiurn chloride (CSCL) density gradient in 13 x 51 mrn polyallomer tubes (Becl~nan Labora~ories). The CsC1 density gradient was prepared by layering 1.~ nl of a 40%
CsCI density gradient was prepared by layering 1.5 ml of a 40% CsCl solution in 20 10 rnM Tris-HCI, 2 rnM EDTA, pH 7.5 (IE) onto 2.0 nl of 5.7 M CsC1 in TE. RNA
was pelleted through this density gradient by ultracentrifugadon at 40,000 rpm in an SW-50~ 1 rotor at room temperature for 16 to 19 hours.
The RNA pellet was suspended in 50 ~I TE-SDS (10 mM Tris-HCl, I mM
EDTA, pH 7.4 wit 0.5% SDS) in a microcentrifuge tube for phenol-chloroforrn 15 extraction. TE saturated phenol was mixed 1:1 (v/v) with a chloroform:isoamyl alcohol (24:1) soludon. 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 precipitation. The RNA was 20 precipitated by addition of 5 M NaCI to create a final concentration of 0.3 M NaCI
followed by addition of two volurnes of ice-cold lOO'Yo ethanol. This solution was placed at -70-C for a minimum of one hour. The tube was then warmed to room temperature to melt the ice and spun in a rnie.~fuge at 4 C for 15 minutes to pellet the RNA precipitate. The supernant was decanted and residual liquid was rernoved by 25 vacuum desiecation. When nearly d y, the RNA pellet was redissolved in TE (without SDS) and precipitated a second time. This time the RNA pellet was redissolved in 50-100 ,ul of 0.2X TE. RNA coneentrations were deterrnined by reading opdeal density at 260 mm in a spectrophotometer and calculated by setting 1.0 O.D. equivalent to 40 lag per ml RNA.

30 D. RNA ExtraetjQn from Air-d~ied ~one Ma~Qw Slides Mi~,~roscopç slidcs of human 'oone marrow weN extracted for RNA. One was stained with H+E while the other was rnerely air~ried and left unstained. The cells on these slides were scraped with razor blades into rnicrocentrifuge tubes. To the tubes was added 1 ml of GITC-ME buffçr, and the tubes wçre shaken vigorously on a rotary 35 shaker for 60 n~inutes to dissolve the eells. The solution was then put in a 2 ml microcentrifuge tube. To preeipitate DNA away from the RNA in solution, 0.1 rnl of 2 , .

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P~TlllS 9 1 / 00 8 5 8 M sodium acetate (pH 4.8) was added to each tube. The DNA precipitation and an extraction were performed by adding 1 ml of phenol:chloroforrn to the tube, inverting the tubcs multiple times, and placing the tubes on wet ice for 15 minutes. 'rhis method of quick RNA extraction including the precipitation of DNA away from the RNA is 5 modification of that described by Chomczynski and Sacchi, 1987, ~n~l- Bioçhem.~:1 5~ 1 S9. Following the incubation of icc, DNA rema~ns at the inte~face between the organic and aqueous phases. The tubcs were spun in a rnicrofuge at 4 C for 20 minutes, and the aqueous phasc containing the RNA was removed and transf~Ted to a new 2 rnl tube for precipitation of the RNA. to each tube was added 750 ~1 of 10 isopropanol, and the tubes were inverted several times before placing at -20 C for one hour. Precipitated RNA was pelle~ed by spinning in a microfuge at C for 20 minutes.
The RNA was redissolved in 300 tul of Gll C-ME and precipitated a second tirne by addition of 300 ~1 of isopropanol and placing at -20-C for one hour. RNA was pelleted again as above, supernatant was discarded, and the RNA pellets washed with I ml of 15 70% ethano!. Once again, the RNA was pelleted, supernatant discarded, and the pellet dried under vacuum desiccation. The RNA was finally redissolved in 50 ~l of 0.2 X
TE and quanitated. Both slides each yielded over 15 ~g of RNA.

E. RNA Extraction from Alcohol-fixed Para~-Embedded Tissues Fifty rnicron scctions of paraffin blocks werc cut and deparaffir~zed in 1 ml of20 xylenes by vigorous shaking in a microfuge tube for 30 minutes. Tissue bits were pelleted by microfuging for five rninutes and the xylenes decanted. Residual xylenes were rernoved by washing with 100% ethanol and repelleting the tissue bits. To the tissue 1 ml of the (3ITC-Me so!ution described above was added. Tubes were vigorously shaken on a rotary shaker for one hour tO dissolve the tissues. All 25 subsequent steps in thc RNA isolation were the sarne as those described above for the bone rnarrow slides where DNA was precipitated away from RNA. Each fifty micron section yielded approxirnately 25 ,ug of RNA.

F. RNA/P~R Procedure The strategy for amplification of mRNA se~guences in the polymerase chain 30 rcaction (RNAIPCR) is based on that previously described (Kawasaki ~ al-. 1988, P~C. ~. ~d Sci. ~ 85:5698-5702j. Total cellular RNA was converted to a pool of cDNAs by reverse transcription. This cDNA was then subjectcd to PCR using gcne-specific pritner pairs. The primer pairs were designed to be homologous to exon sequcnces separated by one or more introns. Primer A, the upstream primer of the35 pair, comprised same scquence (5'--->3') as that detcrmined for the non-template -, - . , , - , : -, . , .. . , ., - -., ..... .. -, ..... - .. . . . . .. . .

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`~ PCTIUS 91/00858 13 MAY la91 so strand used in RNA transcription of the gene (the same sequence as the RNA itself with thymine substituted for uracil). Primer B, the downstrearn primer, was designed to be complementary to and ani-parallel to the non-template strand. Primers were generally 21 to 24 bases in length (21- to 2~mers) with a GC content of 40-60%.
With the primer pair spanning an intron, PCR arnplification of spliced mRNA
results in arnplified products of a predicted length containing only the exon sequences of the gene. Amplification of unspliced RNA or any contarninating genomic DNA inthe RNA preparation yields products of a larger size including the intron sequences.
The smaller product predicted fIom thc spliced nR NA sequcncc will only be prvduced if spliced mRNA transcribed from the gene of interest is present. Therefore, thepresent 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 Visualization Assay (GVA) for gene expression.
The predicted amplified products were confirmed by use of internal probe hybridization to the PCR products. For this reason, oligonucleotides were prepared in sets of three: two as the arnplimer pair and a third internal to both amplimers 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 expr~ssion in a extremely rapid and sensitive fashion.

G. Ras Primer5 and PT~bes Seven sets of ~-specific RNA/PCR primers were designed and are listed in Table 6. An upstream primer specific for exon 1 in each of the three human ra~ genes, c-N-~ (p;imer EK 221) (SEQ ID NO: 158), c-Ha-, a~- 1 (EK 222) (SEQ lD NO: 159), and c-Ki-~a -2 (EK 223) (SEQ ID NO: 160) was prepared. these were each used separatcly in com'oination with a generic exon 2 downstream primer (EK 225) (SEQ 11) NO: 162) targeted ~o a consNved region. In addition, a generic upstream p~imer (EK
224) (SEQ DD NO: 161) was used, paired w~th EK 225 (SEQ ID NO: 162) to detect "pan"-~ expression. The products of each of these pTimer scts were predicted to be a size of 200 bp.
Primer pairs wcre also dcsigned for each of the three genes to yield different-sized RNA/PCR products. Using GVA to score gene expression, the production of different-sized products allowed all three gene expression assays to be mn sirnultaneously in the sarne reaction mixture and then vicwed in a single lane of a gel.
The rcsult saved in enzyme expcnses and allowed scale-up of the number of samples to 35 be screened by a factor of three.

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RNA/PCR products from c-N-~ (primers EK 365 (SEQ ID NO: 163) and EK
366 (SEQ ID NO: 164)), c-Ha-~-l (EK 367 (SEQ DD NO: 165) and EK 368 (SEQ ID
NO: 166)), and c-Ki-ra~-2 (EK 369 (SEQ ID NO: 167) and EK 370 (SEQ ID NO:
168)) mes~sages of 299 bp, 259 bp, and 234 bp, respcctively. Each upstream primer S was designed to have a greater than six base misrnatch with the other two upstream primers to prevent cross amplification of the other ~a~ messages. These three sets of primers were able to be used simultaneously in the same reaction mixture.
In these experiments, allele-specific oligonucleotide (ASO) probes hybridizationwas used to detect point mutations at the 12th and 61st codons to screen for activating 10 point mutaions in ra~ alleles. The 20-mer probes used are listed in Table 7. These probes were used according to the procedure describe,d in Farr ~ al., 1988, Pr~c. Narl.
Acad. ~i. Il~ 8~:9268-9272. --H. Svnthesis of cDNA from RNA
Complementary DNA (cDNA) was synthesized from ~he ex~racted total cellular 15 RNA essentially as previously described (Kawasaki ç~ ~1. 1988, ~. ~I~l. .~i.
~i. ~ ~:5698-5702). One micrograrn of total RNA was reverse transcribed with Moloney Murine Leukemia Virus (Mo-MuLV) reverse transcriptase (Bethesda ResearchLaboratories) in a 20 111 reaction in 1 X PCR buffer (50 mM KC1, 20 mM Tris-HCI pH
8.3, 2.5 rnM MgCI2, and 0.01% BSA) containing 20 U RNAsin ~Promega), 1 ~LI of a 20 10 mM each stock of nucleotide ~iphosphates (dATP, dCTP, dGTP, and dTTP), and100 pmoles of random hexamer prirners. The change to random hexamer primers rather than oligo~T prirners was based on a recent report showing better yield of RNA/PCR products (Noonan and Roninson, 1988, ~I1IÇ1. ~isl B~- 16: 10366).
Reverse transcnption reactions were incubated at room temperature for 15 minutes, 25 42-C for 30 minutes, and 95 C for five minutes. The 95-C incubation was done to heat denan~re the Mo-MuLV reverse transcriptase enzymc. These cDNAs were stored at 4-C until use in the PCR reaction.

1. PCR Reaction~ll~ cl2NA
The PCR method was used according to Saiki ~ ~., as described for DNA
30 using rccombinant thermostable DNA polyrnerase originating from Th~ aquaticus(rTaq) (Perlcin Elmer-Cetus). Slight modifications included reducing the amount of primers to 10 pmoles each, reducing the dNTPs to 1 ~1 of the 10 mM each stock described above, and using the rTaq enzyme at 2 U per reaction in a total 100 ~lrcaction. The substrate for RNA/PCR was 2 ~1 of thc 20 111 cDNA described above. --35 This amount corresponds to 100 ng of the initial 1 llg of total cellular RNA used in the 1 ~ ~flAY ~

reverse transcription reaction. Where three gene reactions were run sirnultaneously, this corresponds to a minimum of 30 gene expression studies from 1 llg of total cellular RNA.
PCR therrnal profiles of 95 C for one minute (denaturing of double strands), 5 55 C for 30 seconds (annealing of amplimers), and 72 C for 30 seconds (synthesis of DNA) were performed in a programmable hea~ block (Perkin Elmer-Cetus) for 30-50 cycles. A 30 second synthesis step at 72 C was suff~cient to produce RNA/PCR
products of at least 935 bp.

J. Gel Yisu~liz~on Assav (GVA) for mRNA Exyression Discre~e gene expression was scored by the gel visualization assay (GVA) following RNA/PCR. RNA/PCR products were screened by running 9 ,~LI of the reaction mLxture in 2% NuSieve (E:MC, Rockland, MD), 1% agarose gels in Tris-borate EDTA buffer (TBE). For size mar~cers, the 123 bp DNA ladder (Bethesda Research Laboratories) was used. Gels of 75 ml were run in wide rnini-sub cells (Bio-15 Rad Laboratories) in TBE at a constant lO0 volts for approximately 90 rninutes. Gels were stained in an ethidium brornide solution (0.5 llg per rnl) for 30 minutes, detained for 30 minutes, and p~otographed under ultraviolet light with a Polaroid Land camera.

K. ASO Probin~ of RNA/PCR Product~
RNA/ PCR products to be probed were run in 2% agarose gels in a Tris-bora~e 20 EDTA electrophoresis buffer (l~E) in a rnini-gel systenL alkaline transfer to Zeta-Probe nylon filters ~Bio-Rad Laboratories) in a wiclc-acion transfer was done with a 0.4 N NaOH solution in water. Transfers werc allowed to proceed for 90 rninutes.Following transfer, blots were neutralized in 2X SSC for S m~nutes. Blots were prehybridized in a solution of 3 M tetra-methyl arnrnonium chloride (I~AC), 50 mM
25 Tris-HCI pH 7.5, 2 mM EDTA, 5X Denhardt's solution, and 0.3% SDS at 55'C for one hour with circular agitation. HybTidization with ASO probcs that were kinase-labelcd with gamma ~2P-ATP was done in 5 ml of the TMAC buffer listed above with2 x 106 cpm per ml of probe added. Hybridization continued at 55 C for one hour.The hybridization buffer and the first wash of 2X SSC with 0.1% SDS (50 rnl) at room 30 ternperature were discarded as radioactive waste. A second wash was done at room tcmperature with 2X SCC with 0.1% SDS. Blots were quickly rinsed in the TMAC --buffer rninus the Denhardt's solution and extensively washed in the sarne buffer at 61'C for one hour. This wash was the critical wash for allowing ~he discr~minatory ability of the ASO probes to distinguish point mutations. Blots were then blotted dry - - .- , . - - - . - ..................... . .
.. . , - . . . . . . .

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.

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with Whatmann 3 MM paper and exposed to Kodak XAR filrn for at -70 C for one hour. Fikn was then developed in an automatic processor and data interpreted.

Tabl~
Oligonucleotide pr~rners for ~ RNA/PCR. All sequences are listed in a 5'-~
5 3' direction (in EK 224 (SEQ lD NO: 161), K=T or G In EK 225 (SEQ lI) NO: 162), R-A or G).
E~K 221: N~ on I (ups~eam) GACTGAGTACAAAGTGGTGGTGG
(SEQ ID NO: 158) . ~ -EK 222: Ha-~-l e~on I (ups~am) GACGGAATATAAGCIGGTGGTGG
10 (SEQ ID NO: 159) EK æ3: Ki-~-2 exon I (ups~eam) GACTGAATATAAACTTGTGGTAG
(SEQ ID NO: 160) -~
EK 224: generic ras e~on I (upslream) ATGACTGAATATAAACTGGTGGTGGTKGG
(SEQ ID NO: 161) 15 EK 225: generic Ia5 exon 2 (downstream) CATGTACTGGTCCCI'CATGGCRCTG
(SEQ ID NO: 162) EK 365: N-G~ e~on I (ups~wn) CGGGCTCTCCAACA'rlTl~CC
(SEQ ID NO: 163) EK 366: N-~s e~on 2 (downs~eam) GTATTGGTCTCTCATGGCACT
20 (SEQ ID NO: 164) EK 367: Ha-ra~-1 e~on I (upstream) AG~AGACCCTGTAGC;AGGACC
(SEQ ID NO: 165) EK 368~ I exon 2 (downslream) GTACIGGTCCC&CATGGCGCT
(SEQ ID NO: 166) 25 E~C 369: Ki-ra5 2 e~on I (upstream) CGGGAGAGAGGCCTGCTGAAA
(SEQ ID NO: 167) EK37~.~ Ds-2e~2(dow~m) GTACTGGTCCCTCATTCCACT :~
(SEQ ID NO: 168) Table l l Oligonuclcoddes Por ~ ASO probing. All sequences are listed in a 5'--->3' direction (R=A or G; H=C, A, or T; V- A, C, or G; S=G, A, or T).
JNOl:Ha-~-112(w.t.) GTGGGCGCCGGCGGTGTGGG (SEQ ~ NO:169) .' JN02:Ha-D~-112-1 GTGGGCGC~HGCGGTGTGGG (SEQ ~ NO:170) nN03:Ha-~-112-2 GTGGGCGCCGHCGGTG~GGG (SEQ ~ NO:171) JN ~:Ha-~-161(w.t.) TACTCCTCCTGGCCGGCGGT (SEQ ~ NO:172) ~ - - ~ , .. . . .~

PCTIIIS q 1 / O 0 8 5 8 JN 05: Ha-~-l 61-1 TACTCCTCCTHGCCGGCGGT (SEQ ID NO: 173) JN 06: Ha-ra~-l 61-2 TACTCCTCCVGGCCGGCGGT (SEQ ID NO: 174) JN 07: Ha-la~-1 61-3 TACTCCTCRTGGCCGGCGGT (SEQ ID NO: 175) JN 08: Ki-~-2 12 (w.t.) CCTACGCCACCAGCTCCAAC (SEQ ID NO: 176) JN09: Ki-~-2 12-1 CCTACGCCACSAGCTCCAAC (SEQIDNO: 177) JN 10: Ki-~a~-2 12-2 CCTACGCCASCAGCTCCAAC (SEQ ID NO: 178) JN 11: Ki-ras-2 61 (w.t.) TACTCCTCTTGACCTGCTGT (SEQID NO: 179) JN 12: Ki-~-2 61-1 TACTCCTCTTHACCTGCTGT (SEQ ID NO: 180) JN 13: Ki-~-2 61-2 TACTCCTCTVGACCIGCTGT (SEQ ~) NO: 181) JN 14: Ki-~-2 61-3 TACTCCTCRTGACCTGCTGT (SEQ ID NO: 182) JN 1~: N-~ 12/13 (w t.) GGAGCAGGTGGTGTTGGGAA (SEQ ID NO: 183) JN 16: N-~ 12-1 GGAGCAHGTGGTGTTGGGAA (SEQ ID NO: 184) JN 17: N-~a~ 12-2 GGAGCAGHTGGTGTTGGGAA (SEQIDNO: 185) JN 18: N-~ 13- 1 GGAGCAGGTHGTGTTGGGAA (SEQ ID NO: 186) JN 19: N-ras 13-2 G~iAGCAGGTGHTGTTGGGAA (SEQ ID NO: 187~
JN 20: N-ras 61 (w.t.) TACTCTTCTTGTCCAGCTGT (SEQ ID NO: 188) JN 21: N-ras 61-1 TACICTTCrrHTCCAGCTGT (SEQ ID NO: 189) JN 22: N-~ 61-2 TACTCTTCI VGTCCAGCI`GT (SEQ ID NO: 190) JN 23: N-ras 61-3 TACTCTTCRTGTCCAGCTGT (SEQ ID NO: 191) Ex~rnple 6 The results of GVA of RNA/PCR amplification of human ras family mRNAs are shown in Figure 2. The samples used were a normal spleen and the cell line K562.
Lanes 1 and 14 contain ~he 123 bp DNA ladder. Negadve controls (no RNA) for each25 reaction are shown in lanes 4, 7, 10, and 13. Lanes 2-4 display the RNA/PCR
products utilizing the "pan" ~ primers EK 224 (SEQ ID NO: 161) and EK 225 (SEQ
ID NO: 162) on the RNA fTom the normal human spleen, the K562 cell line, and thenegative control of no RNA, respectively. As prcdicted, the 200 bp amplified product is present. Lanes 5-7 display the results using the c-N-r~-specific primers EK 365 30 (SEQ ~ NO: 163) and EK 366 (SEQ ~ NO: 164). The samples are displayed in the same sequencc and as predicted, a 299 bp product is prcsent indicating expression a the c-N-~ gene is both sarnples of human cells. c-Ha-~-1 cxpression is shown in lanes 8-10. The primers ER 367 (SEQ ID NO: 165) and EK 368 (SEQ ID NO: 166) produce a 2S9 bp product and that is clearly seen in lane 9 (K562 cells). No product is seen 35 ~om RNA isolated from the normal human spleen (lane 8). The lack of a product is inteIpreted to reflect lack of c-Ha-E~-2 mRNA or levels of the message below that .. - . . . . . .............. . . . .

- : . . . . , . .: . ~ .
. . . , ., .

P~'T~ 91 /~0858 .

ss de~ectable after 30 cycles of RNAIPCR in the normal human spleen. This result shows the utility of the GVA for the detcction of mRNA after RNA/PCR.
Lanes I I - 13 contain the RNA/PCR products using the c-Ki-~-2 primers EK
369 (SEQ ID NO: 167) and EK 370 (SEQ ID NO: 168). The predicted product of 234 S bp is presen~ in both lanes 11 (no{mal human spleen RNA) and 12 (K-562 RNA) indicating expression of the gene in both sarnples; however, the abundance of message is less in the no~mal human spleen f~om that in the K562 cell line.

~am~l~
Utiliz~n of the ras RNA/PCR ProdlLct~ in th~ Screenio~ for Ac~ating Point 10 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 cell line sarnples on each blot are thc same: lane 1, EJ/T24 ~NA amplified with prirners EK 222 (SEQ ~ NO: 159) and EK 225 (SEQ lD NO: 162) (c-Ha-ra~
15 lane 2, EJ/T24 RNA amplified with primers EK 224 (SEQ ID NO: 161) and EK 225 ~SEQ ID NO: 162) ("pan" ra~); lane 3, PA-l RNA amplified wi~h primers EK 221 (SEQ ID NO: 158) and EK 225 (SEQ ID NO: 162) (C-N-~); lane 4, SW-48- RNA
amplified with primers EK 223 ~SEQ ID NO: 160) and EK 225 (SEQ ID NO: 162) (c-Ki-~-2); lane 5, SW-480 RNA amplified with primers EK 224 ($EQ ID NO: 161) - .
20 and EK 225 (SEQ ID NO: 162) ("pan" ra~); lane 6, HI.-60 RNA amplified with pnmers EK 221 (SEQ ID NO: 158) and EK 22~ (SEQ ID NO: 162) (c-N-r~); lane 7, Calu-l RNA amplified with primers EK 223 (SEQ ID NO: 160) and EK 225 (SEQ ID
NO: 162) (c-Ki-~-2); lane 8, Calu-l RNA arnplified with primers EK 224 (SEQ ID
NO: 161) and EK 225 (SEQ ID NO: 162) ("pan" ~); lane 9, G2101 RNA amplified 25 with primers EK 224 (SEQ ID NO: 161) and EK 225 (SEQ ID NO: 162) ("pan" ~);
lane 20, 123 bp DNA ladder. The blot in Panel A has been probed with a pool of oligonueleotides speeific for aetivating point mutations at the second nucleotide of the 12th codon of c-Ha-~aS-l (JN 03) (SEQ ID NO: 171).
As predicted, lanes 1 and 2 containing the EJm4 RNA amplified both the c-Ha-30 ~-1 primers and the "pan" ~ primers are positive for the characterized EJ~r24mutation. Panel B blot has been probed with a pool of oligonucleotides speeific for aetivating point mutations at the seeond nueleotide of the 12th codon of c-N-~ (~N 17) (SEQ ID NO: 185). The PA-2 cell line is known to cont~n a mutad~n at this positdon, and lane 3 is posidve as expee~ed. Panel C blol has bcen probed with oligonueleotide 35 pool JN 09 (SEQ lD NO: 177) ~argeted to mutadons at the first nucleotide of codon 12 in e-Ki-~-2.

. - . . . . . ............. .

;' :' ~ PCT!US 91/008 58 The SW 480 cell line contains one of those mutations and lanes 4 and 5 containing RNAIPCR products for that cell line are positive. Because the signal in lane 5 is quite weak, it may indicate tha~ the mutant allele's message is in low abundance with respect to all other ~ messages in the cell as the "pan" ~a~ pnmers were used for 5 that lane or that the "pan" ~ primers are less efficient at amplifying c-Ki-~-2 messages with respect to the other two ~ genes.
Panel D blot has been probed with pool JN 22 (SEQ ID NO: 190) specific for mutations at the second position of the 61st codon of c-N-~. Cell line ~-60 has a mutation at the position and is positive in lane 6. This panel, in combination with Panel 10 B, illustrates that the RNA/PCR products amplified by the primers EK 221 (SEQ ID
NO: 158) and EK 225 (SEQ ID NO: 162) (c-N-~a~) contain sequences of both the 12th, 13th, and 61st codons of that gene. This is a significant advance for ~a~ point mutation screening in that a single PCR reaction allows one to sereen both activating hotspots in a single product as opposed to the need for two reactions when screening genomic15 DNA due to those two spots being present in exons 1 and 2 separated by a large intron.
There is also the additional value of s,,reening for expressed mutations in mRNA as opposed to the possibility of detecting mutations in non-expressed alleles.

Exam~le 8 RNAfPCR products from alcohol-fixed pa~affin-ernbedded samples were 20 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 arnplified with primers EK 36S (SEQ ID NO:
163) and 366 (SEQ ID NO: 164) (c-N-~: 299 bp), those in lanes 3, 7, and 10 with primers EK 367 (SEQ ID NO: 165) and EK 368 (SEO ID NO: 166) (c-Ha-~-1: 259 bp~, and those in lanes 4, 8, and 11 with primers EK 369 (SEQ 11) NO: 167) and EK
25 370 (SEQ lI) NO: 168) (e-Ki-~-2: 234 bp). Lanes 2-4 are the negative controls with no RNA added to the reverse transcTiptase reaction of RNA/PCR. No produets are seen in those lanes other than dle primer dimers mentioned above. Lanes ~8 eontain RNA/PCR products from the Calu- 1 eell line and the products co~responding to ~
messages from all three genes are present. Lanes 9-11 eontain the XNA/PCR produets 30 from the eell line G-2101. In this ease, thex is a laek of any signal from c-Ha-~s-1 messages indieating laek of expression. _ These reaetions were run for 50 eyeles of RNA/PCR whieh inereases the presenee of baekground bands, but also the teehnique for preeipitating DNA away from RNA which was used in the prcparation of these RNAs is not totally efficient and ean 35 result in eontaminating genomie DNA in the RNA preparations. The eon~aminating genornie DNA ean result in speeific amplification of the B~ genes eontaining the intron .

P~TIUS,l/CC85 between exons 1 and 2 but it will be a much larger size than that predicted for the spliced mRNA.

E2~am~le 9 The "pan" ~ prirners wcrc us~d to amplify reverse transcribed RNA products.
5 Sample preparation and the arnplification procedure were as described in Exarnple 4.
The results of "pan" ~a~ arnplification of RNA isolated from the air dried stained microscope slide preparations of hurnan bone rnarrow are shown in Figure 5. The predicted 200 bp RNA/PCR products using primers EK 224 (SEQ ID NO: 161) and EK 225 (SEQ ID NO: 162) arc shown for thc unstained slide (lane 3) and the stained slide (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.
~am~ '. . ~
etection of ras Mutadons bv Forrnat II
The synthesis of biotinylated primers, poly T tailing of the probes and preparation of the forrnat II filters are as described in con~nonly assigned, copending - ~ -U.S. Serial No. 197,bO0 incorporated herein by reference (also see Chiang et ~.,1989, ~i~h~i~ i~(4):360)- - , Three sets of filters representing 21 mutation spccific oligo probes for N-, H-,and K-~, respectively, are shown in Figure 6. The sequences of the ~ probes are 20 listed in Table 12. All probes were designed to have approxirnately the sarne melting temperature (~50 -52-C) so that hybridization conditions could be standardized for all the oligonucleotides. Five pmoles of each tailed probe were spott~d onto BiodyneNylon membranes (Pall Biosupport, NY) and W irnrnobilized.
l~e ~ oligonucleo~de bound filters were hybridized in 5X SSPE, 0.5% SDS
25 with alkali denatured PCR products for 60 minutes at 42-C. Washing was done in 3M
tctramethylammoniurn chloride to minimize the influence of base composition among the various nucleoddcs. The filters were briefly rinsed with 2X SSPE, 0.1% SDS, then incubated in the same buffer with 2 llg/ml streptavidin-horse radish paoxidase conjugate ("Sequencc," Cetus Co~poration) for 30 minutes at room temperanlre. The filters were then washed for five minutes with the same buffer without the conjugate. -Reagents of the ECL gene detection system (Amesrsham) werc addcd and incubated for one minute at room temperature. Filters were then wrapped in Saran wrap and the light signal produced was detected by exposing Kodak XRP film tO the filters for 20 seconds to onc minutc.

: ; : . ~ - . . , : ... .. , . ~ . . . . . .... ..
. , . : -, .. ~ , - . . -~ PCTIUS 5~ 0~0 8~591 ~abl~l~
~du~P~f~Fonmat~Detec~on N-~ codon12 YZl ~JAGCAGGTGGTGTTGG (SEQ ~ NO:192) YZ21 GAGCAAGTGGTGTTGG (SEQIDNO:193) YZ22 GAGCATGTGGTGTTGG (SEQIDNO:194~
YZ23 GAGCACGTGGTGTTGG (SEQnD NO:19S) YZ2 GAGCAGATGGTGTTGG (SEQDD NO:196) YZ24 GAGCAGCTGGTGTTGG (SEQ~)NO:197) YZ25 GAGCAGTTGGTGTTGG (SEQDD NO:198) N-~codon13 YZ26 GCAGGTAGTGTTGGGA (SEQrD NO:199) YZ50 GCAGGTTGTGTTGGGA (SEQIDNO:200) YZ27 GCAGGTCGTGTTGGGA (SEQIDNO:201) YZ28 GCAGGTGATGTTGGGA (SEQIDNO:202) YZ29 GCAGGTGCTGTTGGGA (SEQ~DNO:203) YZ3 GCAGGTGGTGTTGGGA (SEQID NO: 204) N-~ codon16 YZq AGCTGGACAAGAAGAGT (SEQIDNO:205) Yz30 AGCTGGAGAAGAAGAGT (SEQIDNO:206) YZ5 CAGCTGGAAAAGAAGAG (SEQIDNO:207) YZ31 AGCTGGACGAGAAGAG (SEQID NO: 2Q8) YZ 6 AGCTGGACTAGAAGAGT (SEQnD NO:209) YZ32 AGCTGGACCAGAAGAG (SEQIDNO:210) YZ51 AGCAGGACTGAAGAGT (SEQIDNO:211) Yz33 AGCAGGACACGAAGAG (SEQIDNO:212) H-~ codon12 YZ7 GGAGCCGGCGGTG (SEQIDNO:213) YZ34 GGCGCCAGCGGTGT (SEQIDNO:214) YZ35 GGCGCCTGCGGTGT (SEQlDNO:215) YZ~6 GGCGCCCGCGGTG (SEQ ~ NO:216) YZ37 GGCGCCGACGGTGT (SEQIDNO:217) YZ8 GGCGCCGTCGGTGT (SEQII~NO:218) YZ38 GGCGCCGCCGGTG (SEQIDNO:219) H~ codonl3 YZ39 GCCGGCAGTGTGGG (SEQII)NO:220) YZ9 GCCGGCTGTGTGGG (SEQIDNO:221) YZ40 GCCGGCCGTGTGGG (SEQIDNO:222) YZ41 GCCGG5GATGTGGG (SEQIDNO:223) YZ42 GCCGGCGCTGTGGG (SEQIDNO:224) YZ43 GCCGGCGTTGTGGG (SEQlDNO:225) H-~ codon61 YZ10 GCCGGCCAGGAGGA (SEQIDNO:226) YZ11 GCCGGCGAGGAGGA (SEQIDNO:22.7) YZ44 CGCCGGCAAGGAGG (SEQIDNO:228) YZ45 GCCGGCCGGGAGG (SEQIDNO:229) YZ46 GCCGGCCTGGAGGA (SEQIDNO:230) YZ47 GCCGGCCCGGAGG (SEQIDNO:231) YZ48 CGCCGGCCATGAGG (SEQIDNO:232) YZ49 GCCGGCCACGAGGA (SEQIl)NO:233) , . . . , . . . . . ~ . ~ . ......

.... .: . . . - . .. , .. . - : .

PcT~ o ~ 5 8 1~ M4Y la91 59 . -K-~ascodon 12 YZ52 GGAGCTGGTGGCGTA (SEQD:)NO:234) YZ53 GGAGCTAGTGGCGTAG (SEQ ID NO: 235) YZ54 GGAGCTTGTGGCGTAG (SEQ II) NO: 236) YZ55 GGAGCTCGTGGCGTA (SEQ ~) NO: 237) YZ56 GGAGCTGATGGCGTAG (SEQ ID NO: 238) - .
YZ57 GGAGCTGCTGGCGTA (SEQ ~D NO: 239) YZ58 GGAGCTGTTGGCGTAG (SEQIDNO:240) K-~co~ion 13 YZ59 GCTGGTAGCGTAGGC (SEQIDNO:241) . .
YZ60 GCTGGTTGCGTAGGC (SEQ ~) NO: 242) YZ61 GCTGGTCGCGTAGGC (SEQ ID NO: 243) YZ62 GCTGGTGACGTAGGC (SEQ ID NO: 244) YZ63 GCTGGTGTCGTAGGC (SEQ ID NO: 245) YZ64 GCTGGTGCCGTAGGC (SEQ ID NO: 246) K-~ codon 61 YZ 65 AGCAGGTCAAGAGGAG (SEQ ID NO: 247) YZ66 AGCAGGTGAAGAGGAG (SEQ ~:) NO: 248) YZ67 AGCAGGTAAAGAGGAGT (SE~2 ID NO: 249) YZ68 GCAGGTCGAGAGGAG (SEQIDNO:2S0) YZ69 AGCAGGTCTAGAGGAG (SEQIDNO:2Sl) YZ70 GCAGGTCCAGAGGAG (SEQ ~) NO: 252) YZ71 AGCAGGTCATGAGGAG (SEQ ID NO: 2S3) - .
YZ72 GCAGGTCACGAGGAG (SEQIDNO:254) . . ., .. , : . :

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~ p~ / o n Q s s 1 .~ M lY ~.~. `^ I

SEQUENCE LISTING

(1) GENERAL INFOR~ATION:
(i) APPLICANT: McCormick, Fra~cis P.
Lyo~s, John F.
(ii) TITLE OF INVENTION: Detection of Poin~ Mutations in Genes Encoding GTP Binding Proteins ~iii) NUM3ER OF SEQUENCES: 254 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Cetus Corporation (B) STREET: 1400 Fifty-Third Street (C) CITY: Emeryville (D) STATE: California (E) COUNTRY: USA
(F) ZIP: 94608 (v) COMæUTER READABLE FO~M:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) aPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.25 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION: ~ :
~A) NAME: Sias Ph.D., Stacey R.
(B) REGISTRATION NUMBER: 32,630 (C) REFERENCE/DOCKET NUMBER: 2537.1 (ix~ TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (415) 420-3197 (B) TELEFAX: (415) 658-5239 (C) TELEX: 4992659 - :, ',IAY lr~l (2) INFORMATION FOR SEQ ID NO:l: -(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: l9 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
TCGCTGCCGT GTCCTGGAC l9 (2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: l1near (ii) MOLECULE TYPE: DNA (genomic) (xi~ SEQUENCE DESCRIPTION: SEQ ID NO:2:

(2) INFORMATION FOR SEQ ID NO:3: ~
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE 3ESCRIPTION: SEQ ID NO:3:

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:
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~ PCTIUS91,/00858 (2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUE~CE DESCRIPTION: SEQ ID NO:5:

(2) INFORMATION FOR SEQ ID NO:6:
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(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:

(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE C~ARACT~RISTICS:
(A) LENGT~: 20 base pairs -(B) TYPE: nucleic acid (C) STRANDEDNESS: single _ : -(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: :

`~' 1 3 M~ 9~1 63 : -(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs (~) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:

(2) INFORMATION FOR SEQ ID NO:9: -(i) SEQUENCE CHARACTERISTICS: -(A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUE~CE DESCRIPTION: SEQ ID NO:9:
TGGGTGGCCC GCGCGATG 1 a (2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:

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-PCTIUS 9 1/ 0~,815q~

(2) INFORMATION FOR SEQ ID NO~
~i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:

(2) INFORMATION FOR SEQ ID NO:12: ~`
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUEN~E DESCRIPTION: SEQ ID NO:12:

(2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS: '~
(A) LENGTH: 19 base pairs .
(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:

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P~Tlus 9 1 / 0~

(2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: l9 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:

(2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
TGGGTGGCCA CCGCGATG l8 ~ -(2) INFORMATION FOR SEQ ID NO:l6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:

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(2) INFORMATION FOR SEQ ID NO:17: -(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:

(2) INFORMATION FOR SEQ ID NO:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D~ TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
TTCCCCTTGC 5CAGGATGTT T 21 : -: ~ .
(2) INFORMATION FOR SEQ ID NO:l9: .
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single ~ ~-(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) -(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l9:
CAGAACAAGG TCATAATCAC T 21 ;~

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.

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, ~ PC~IUS S 1/00858 1,ny l~ t (2) INFORMATION FOR SEQ ID NO:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:

(2) INFORMATION FOR SEQ ID NO:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear .
(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:

(2) INFORMATION FOR SEQ ID No:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA ~genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:

~ PCTIUS 9~ 81598 (2) INFORMATION FOR SEQ ID NO:23: .
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULr TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:

.
(2) INFORMATION FOR SEQ ID NO:24:
, ;
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear -(ii) MOLECULE TYPE: DNA (ge~omic) (xi) SEQUEN~E DESCRIPTION: SEQ ID NO:24:

(2) INFORMATION FOR SEQ ID NO:25:
(i) SEQUENCE CHARACTERISTICS: .
(A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) : :
(xi) SEQUENCE DESCRIPTION: SEQ ID NO 25:

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P~TilJS 91/00858 1 3 ~

(2) INFORMATION EOR SEQ ID NO:26:
~i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) T020LOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:
GCGCTGTGAA CACCCCACGT GTCT 24 : - :

(2) INFORMATION FOR SEQ ID NO:27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: sinqle - .
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:
CGCAGGGGGT GGGCGGTCAC TCCA . 24 ' (2) INFORMATION FOR SEQ ID NO:28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs ~B) TYPE: nucleic acid (C) STR~NDEDNESS: single (D) TOPOLOGY: linear ~ .
(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: :.

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PCT!US 9~ 0?n0y8 5 8 (2) INFORMATION FOR SEQ ID NO:29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs (B~ TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:

(2) INFORMATION FOR SEQ ID NO:30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single ~ :
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUEN~CE DESCRIPTION: SEQ ID NO:30:

(2) INFORMATION FOR SEQ ID NO:31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESC~IPTION: SEQ ID NO:31:

~ PcTllis9l!oo858 (2) INFORMATION FOR SEQ ID NO:32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single ~ . .
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:
TAGAACCAGG TCGTAGTCAC T 21.
(2) INFORMATION FOR SEQ ID NO:33:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid : :
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUE~CE DESCRIPTION: SEQ ID NO:33:

(2) INFORMATION FOR SEQ ID NO:34:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs (P) TYPE: nucleic acid ~-(C) STRANDEDNESS: single :
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:

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prTIl1s 91 / 00 8 5 1 3 ~ .Y lq~l (2) INFORMATION FOR SEQ ID NO:35:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:

.:
(2) INFORMATION FOR SEQ ID NO:36:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:
GCTCACTTGA AGTGTAGG 18 -.

(2) INFORMATION FOR SEQ ID NO:37:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:

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c~ 3 ~,?AY l~Sl (2) INFORMATION FOR SEQ ID NO:38:
ti) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear .
(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:

(2) INFORMATION FOR SEQ ID NO:39:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - -(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:
GAGGTTCTTG AATGTGAAGT GGGT 24 ;.~

(2) INFORMATION FOR SEQ ID NO:40: ~ . .
(i) SEQUENCE C~ARACTERISTICS:
(A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:

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p~T~ qllnn~58 !"Y 19~?

(2) INFORMATION FOR SEQ ID NO:41:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGT~: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:

(2) INFORMATION FOR SEQ ID NO:42:
(i) SEQ~ENCE CHARACTERISTICS:
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(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear --(ii) MOLECULE TYPE: DNA (genomic) ~xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:

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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:

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(2) INFORMATION FOR SEQ ID NO:46:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) ~xi) SEQUENCE DESCRIPTION: SEQ ID NO:46: -AAGAAACCAT GATCTCTGTT ATAT 24 ~ ~

~ P~Tllls 9~ ,/ oy8~5c81 (2) INFORMATION FOR SEQ ID NO:47:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:

(2) INFORMATION FOR SEQ ID NO:48:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:

(2) INFORM~TION FOR SEQ ID NO:49:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:

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~ PCT~ s9,1/~,C~40815;8 ~2) INFORMATION FOR SEQ ID NO:50:
(i) SEQUENCE C~ARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid - -(C) STRANDEDNESS: single (D) TOPOLOGY: linear -~
(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:

(2) INFORMATION FOR SEQ ID NO:51:
(i) SEQUENCE CHARACTERISTICS:
- (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) :
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:
TAGGTGCTCG AGAATCTGGT 20 . . .

(2) INFORMATION FOR SEQ ID NO:52: : -(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs :i .
(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear , (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:

'' ' ~ : ,. ' `T ~ 9 1 / 0 0 8 5 8 13 MAY ~^~9t (2) INFORMATION FOR SEQ ID NO:53:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECVLE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:

(2) INFORMATION FOR SEQ ID NO:54:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single :
(D) TOPOLOGY: linear ~ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:

.

(2) INFORMATION FOR SEQ ID NO:55:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:

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PCT!US a 1 /008 58 i 3 I~?"Y ^~-(2) INFORMATION FOR SEQ ID NO:56: -(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (3) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:

, ;,, . , (2) INFORMATION FOR SEQ ID NO:57:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:

(2) INFORMATION FOR SEQ ID NO:58:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (8) TYPE: nucleic acid `
(C) STRANDEDNESS: single .
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) ;~
~xi) SEQUENCE DESCRIPTION: SEQ ID NO:58:

P~TIIJS ~/M(~895~8 (2) INFORMATION FOR SEQ ID NO:59:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single -(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQ~ENCE DESCRIPTION: SEQ ID NO:59:

(2) INFORMATION FOR SEQ ID NO:60:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUE~CE DESCRIPTION: SEQ ID NO:60:

(2) INFORMATION FOR SEQ ID NO:61:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear ~ii) MOLECULE TYPE: DNA (genomic) ~xi) SEQUENCE DESCRIPTION: SEQ ID NO:61:

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PCT~US 9113/ OA 81o?1 (2) INFORMATION FOR SEQ ID NO:62:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs tB) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear :-(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:62:

(2) INFORM~TION EOR SEQ ID NO:63:
(i) SEQUENCE CHARACTERISTICS:
(A~ LENGTH: 20 base pairs ' - .
(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:63:
GTGGGTGGCT AGCGCGATGA 20 ~

(2) INFORMATION FOR SEQ ID NO:64: ;
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid .
(C) STRANDEDNESS: single (D) TOPOLOGY: linear -(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:64:

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PCTIUS 9 1 / O O ~ 5 8 13~.Y l9~l (2) INFORMATION FOR SEQ ID NO:65:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:65:

(2) INFORMATION FOR SEQ ID NO:66:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) -(xi) SEQUENCE DESCRIPTION: SEQ ID NO:66:

(2) INFORMATION FOR SEQ ID NO:67:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECUI,E TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:67:

PCTIUS9~/00858 13 MAY 1~1 (2) INFORMATION FOR SEQ ID NO:68:
(i) SEQUENCE CHARACTERISTICS:
(A) ~ENGTH: 20 base pairs (~) TYPE: nucleic acid -.
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:68:

(2) INFORMATION FOR SEQ ID NO:69:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - .
(ii) MOLECULE TYPE: DNA (genomic) .
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:69:

(2) INFORMATION FOR SEQ ID NO:70:
(i) SEQUENCE CHAXACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear .
~ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:70:

(2) INFORMATION FOR SEQ ID NO:71: : , (i) SEQUENCE C~ARACTERISTICS:
~A) LENGTH: 20 base pairs (B) TYPE: nucleic acid _ : -tC) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:71:
-TCAGAACTAG AGTGAAAACT 20 ;
' ~ -P~T/1lS 9 1 /008 5 13 M.9Y ~¢~l (2) INFORMATION FOR SEQ ID NO:72:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid ~C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:72:

(2) INFORMATION FOR SEQ ID NO:73:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:73: -(2) INFORMATION FOR SEQ ID NO:74:
(i) SEQUENCE C~ARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:74:

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P~TIUS 9113/~ 8~5~ ~

(2) INFORMATION FOR SEQ ID NO:75: ~ -(i) SEQ~ENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:75: .

(2) INFORMATION FOR SEQ ID NO:76:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA ~genomic) . .
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:76:
TCAGAACTAA AGTGAAAACT 20 ~
:, ~ .
(2) INFORMATION FO~ SEQ ID NO:77:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs : :
~B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi~ SEQUENCE DESCRIPTION: SEQ ID NO:77:

(2) INFORMATION FOR SEQ ID NO:78:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (8) TYPE: nucleic acid (C) STRANDEDNESS: single .
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) .
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:78:

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, . . - ;: :

:
--~ PCTIUS 9 1 / O 0 8 5 8 (2) INFORMATION FOR SEQ ID NO:79:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 Dase pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPGLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:79:

(2) INFORMATION FOR SEQ ID NO:80:
(i) Sr QUENCE CHARACTERISTICS: -(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:80: -(2) INFORMATION FOR SEQ ID NO:81: ~ .
(i) SEQUENCE C~ARACTERISTICS:
(A) LENGTH: 20 base pairs ~B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:81:

.:~ PCTIUS 91/00858 1~ MAY 1991 (2) INFORMATION FOR SEQ ID NO:82:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid ~C) STRANDEDNESS: single ~ -(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:82:

.
(2) INFORMATION FOR SEQ ID NO:83:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear -(ii) MOLECULE TYPE: DNA (genomic) . ~:.
(xi) SEQUE~CE DESCRIPTION: SEQ ID NO:83: ~ :

~ . . .
(2) INFORMATION FOR SEQ ID NO:84:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) ~xi) SEQUENCE DESCRIPTION: SEQ ID NO:84: ' : . . : : : ~

- . . , : . .

~, PCTiUS 51/0085 l~M~Y l^~

(2) INFORMATION FOR SEQ ID NO:85:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (3) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii~ MOLECULE TYPE: DNA (genomic) (xi) SEQUEN OE DESCRIPTION: SEQ ID NO:85:

(2) INFORMATION FOR SEQ ID NO:86:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRAN3EDNESS: single ~ -(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:86:

(2) INFORMATION FOR SEQ ID NO:87:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:87:

(2) INFORMATION FOR SEQ ID NO:88:
(i) SEQUENCE CHARACTERISTICS:
(A~ LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) ~OLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:88:

..

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Pr~Tllscl /oo858 ~ 3 MAY l~al (2) INFORMATION FOR SEQ ID NO:89:
~i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear ~ .:
(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:89:
TAC~GACCTG CGTAAAGACC 20 (2) INFORMATION FOR SEQ ID NO:90: -(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C~ STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:90:
TACGGACCCA CGT ~ GACC 20 (2) INFORMATION FOR SEQ ID NO:91: ::~
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (3) TYPE: nucleic acid ~-(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:91:

(2) INFORMATION FOR SEQ ID NO:92:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (~) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear ~ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:92:

' . '': ` . :' , 1 3 I~AY ~ - ' ?

(2) INFORMATION FOR SEQ ID NO:93:
~1) SrQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs tB) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: 1 inear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:93:

(2) INFORMATION FOR SEQ ID NO:94: .
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs ~B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) -(xi) SEQUENCE DESCRIPTION: SEQ ID NO:94:

(2) INFORMATION FOR SEQ ID NO:95:
~i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:95:

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PCT!US 9 1 / O 0 8 5 8 13~AYl~'' (2) INFORMATION FOR SEQ ID NO:96:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (3) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear :
(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:96:

(2) INFORMATION FOR SEQ ID NO:97:
(i) SEQUENCE CHARACTERISTICS-(A) LENGTH: 20 base pairs .
(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:97:

(2) INFORMATION FOR SEQ ID NO:98: - .
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs :
(~) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear ;
(ii) MOLECULE TYPE: DNA (genomic) ~xi) SEQUENCE DESCRIPTION: SEQ ID NO:98: , (2) INFORMATION FOR SEQ ID NO:99:
~i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 20 base pairs _ :
~B) TYPE: nucleic acid ~C) STRANDEDNESS: single ~D) TOPOLOGY: linear ~ii) MOLECULE TYPE: DNA ~genomic) ~xi) SEQUENCE DESCRIPTION: SEQ ID NO:99:
,~
GTGGGTGGTC ATCGGTCTGA 20 : ~

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PCT/~S 9 1 ~ 00 8 5 8 1 3 l~llAY 1- r 4 .

(2) INFORMATION FOR SEQ ID NO:100:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base ~airs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) -(xi) SEQUENCE DESCRIPTION: SEQ ID NO:100:
GTGGGTGGTC ACCGGTCTGA . 20 (2) INFORMATION FOR SEQ ID NO:101:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs ~B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLEC~LE TYPE: DNA (genomic) :
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:101:

(2) INFORMATION FOR SEQ ID NO:102:
(i) SEQUENCE CHARACTERISTICS: : -(A) LENGTH: 20 base pairs -(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear :.
(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:102:
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P~T~ Q ~/3(~Ag 5c31 (2) INFORMATION FOR SEQ ID NO:103:
(i) SEQUENCE C~ARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:103:

(2) INFORMATION FOR SEQ ID NO:104:
~i) SEQUENCE CHARACTERISTICS:
(A) LENG1'H: 20 base pairs ~B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:104:

(2) INFORMATION FOR SEQ ID NO:105:
(i) SEQUENCE C~ARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single ~D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SE~UENCE DESCRIPTION: SEQ ID NO:105:

(2) INFORMATION FOR SEQ ID NO:106:
(i) SEQUENCE C~ARACTERISTICS:
(A) LENGTH: 20 base pairs (~) TYPE: nucleic acid (C) ST~ANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:106:

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~i) SE~UENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear ~ii) MOLECULE TYPE: DNA (genomic) ~xi) SEQUENCE DESCRIPTION: SEQ ID NO:107:

~2) INFORMATION FOR SEQ ID NO:108:
~i) SEOUENCE CHARACTERISTICS:
~A) LENGTH: 20 base pairs ~B) TYPE: nucleic acid ~C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLEC~LE TYPE: DNA (genomic) -.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:108:

- (2) INFORMATION FOR SEQ ID NO:109:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid ~C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:109:
GTAGGTGGCC ATAGATCAGA 20 ~ -.... . . . . .

~ PCT!US 9 13 0aOy8l5q8 ss (2) INFORMATION FOR SEQ ID NO:110:
ti) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLrCULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:110:

(2) INFORMATION FOR SEQ ID NO:lll:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPO10GY: linear (ii) MOLECVLE TYPE: DNA (genomic) (xi) SEQUE~CE DESCRIPTION: SEQ ID NO:lll:

(2) INFORMATION FOR SEQ ID NO:112:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:112:

1 31~ 1ql (2) INFORMATION FOR SEQ ID NO:113: :~-(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:113:
GTAGGTGGCC GAAGATCAGA 20 . .
(2) INFORMATION FOR SEQ ID NO:114: -(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs --(B) TYPE: nucleic acid . -(C) STRANDEDNESS: single (D) TOPOLOGY: linear -(ii) MOLECULE TYPE: DNA ~genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:114:

(2) INFORMATION FOR SEQ ID NO:115: .
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs :
(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D~ TOPOLOGY: linear ~ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:115:

(2) INFORMATION FOR SEQ ID NO:116:
~i) SEQUENCE CHARACTERISTICS: . : (A) LENGTH: 20 base pairs ~B) TYPE: nucleic acid (C) STRANDEDNESS. single ~D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi~ SEQUENCE DESCRIPTION: SEQ ID NO:116: ~ -- , . - , . . . - .

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PCTIUS91/00858 .
13 MAY '~1 (2) INFORMATION FOR SEQ ID NO:117:
~i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:117:

(2) INFORMATION FOR SEQ ID NO:118:
(i) SEQUENCE CH~RACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) ~ .
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:118:

(2) INFORMATION FOR SEQ ID NO:119:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:ll9:

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P~TIUS 91/ 00 8 5 8 (2) INFORMATION FOR SEQ ID NO:120:
(i) SEQUENCE C~ARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:120:

(2) INFORMATION FOR SEQ ID NO:121:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear .
(ii) MOLECULE TYPE: DNA (genomic) ~xi) SEQUENCE DESCRIPTION: SEQ ID NO:121:

(2) INFORMATION FOR SEQ ID NO:122:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) ~xi) SEQUENCE DESCRIPTION: SEQ ID NO:122:
CTCCGAACCA GCGTCA~AAC 20 (2) INFORMATION FOR SEQ ID NO:123:
~i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 20 base pairs ~B) TYPE: nucleic acid ~C) STRANDEDNESS: single ~D) TOPOLOGY: linear ~ii) MOLECULE TYPE: DNA ~genomic) ~xi) SEQUENCE DESCRIPTION: SEQ ID NO:123:

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(2) INFORMATION FOR SEQ ID NO:124:
(i) SEQUENCE CHA~ACTERISTICS:
(A) LENGTH: 20 base Dairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:124:

(2) INFORMATION FOR SEQ ID NO:125:
(i) SEQVENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLEC~LE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:125:

(2) INFORMATION FOR SEQ ID NO:126: . ~ -~
(i) SEQVENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs~ .
(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D~ TOPOLOGY: linear : .
(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:126:

PCTjUS91/00858 (2) INFORMATION FOR SEQ ID NO:127:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (3) TYPE: nucleic acid (C) STRANDEDNESS: single (D~ TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:127:

(2) INFORMATION FOR SEQ ID NO:128:
(i) SEQUENCE C~ARACTERISTICS:
(A) LENGT~: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) -;
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:128: :

(2) INFORMATION FOR SEQ ID NO:129: .
(i) SEQUENCE CHARACTERISTICS: ~ -(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear ~ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:129: .
GTCGGAGGCC TGCGATCTGA 20 .
(2) INFORMATION FOR SEQ ID NO:130:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid .
(C) STRANDEDNESS: single (D) TOPOLOGY: linear ~i;
(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQ~ENCE DESCRIPTION:-SEQ ID NO:130:

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ti) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:131:

~2) INFORMATION FOR SEQ ID NO:132:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi~ SEQUENCE DESCRIPTION: SEQ ID NO:132:

(2) INFORMATION FOR SEQ ID NO:133:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (~) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:133:

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(2) INFORMATION FOR SEQ ID NO:134:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:134:
TGCGCTCCTG GGACATGACC 20 ~ .

(2) INFORMATION FOR SEQ ID NO:135:
(i) SEQUENCE CHARACTERISTICS-(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:135:
TGCGCTCCCA GGACATGACC 20 :

(2) INFORMATION FOR SEQ ID NO:136: .:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear ~ii) ~OLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:136:

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(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:137:

(2) INFORMATION FOR SEQ ID NO:138:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:138:

(2) INFORMATION FOR SEQ ID NO:139:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single -(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:139: . .

(2) INFORMATION FOR SEQ ID NO:140:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) .
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:140:

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(2) INFORMATION FOR SEQ ID NO:142:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:141: .
TGGGGGGGCC GAGGTCAGAG
(2) INFORMATION FOR SEQ ID NO:142:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear . - .
(ii) MOLECULE TYPE: DNA (genomic) ::
(xi) SEQUE~CE DESCRIPTION: SEQ ID NO:142:
TGGGGGGGCT GAGGTCAGAG 20 ~-(2) INFORMATION FOR SEQ ID NO:143:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C~ STRANDEDNESS: single (D) TOPOLOGY: linear .
(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:143:

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(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs ~B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:144:

(2) INFORMATION FOR SEQ ID NO: 145:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:145:

(2) INFORMATION FOR SEQ ID NO:146:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs (B) TYPE: nucleic acid -(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:146:

(2) INFORMATION FOR SEQ ID NO:147:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:147:

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l06 ''~: :'' ' (2) INFORMATION FOR SEQ ID NO:148:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (x_) SEQUENCE DESCRIPTION: SEQ ID NO:148: -(2) INFORMATION FOR SEQ ID NO:149:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear . -(ii) MOLEC~LE TYPE: DNA (genomic) .
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:149:

(2) INFORMATION FOR SEQ ID NO:150:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:150:
CCTGCTGAAA ATGACTGA~T ATAAA 25 ':
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PCTIUS91!00858 13 ~J~IAY 1~'`4 (2) INFORMATION FOR SEQ ID NO:151:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:151:

(2) INFORMATION FOR SEQ ID NO:152:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (~) TYPE: nucleic acid ~C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUE~CE DESCRIPTION: SEQ ID NO:152:

(2) INFORMATION FOR SEQ ID NO:153:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULB TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:153:

~2) INFORMATION FOR SEQ ID NO:154:
(i) SEQUENCE C~ARACTERISTICS:
(A) LENGTK: 20 base pairs (B) TYPE: nucleic acid (C) ST~ANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi~ SEQUENCE DESCRIPTION: SEQ ID NO:154:
CTTGCTGGTG TGAAATGACT . 20 - : . - ~ - - : .

`~ PCTIUS 9 1 / O 0 8 5 8 1~ MAY 1991 (2) INFORMATION FOR SEQ ID NO:155:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:155: . -GGTGGGATCA TATTCATCTA 20 ~ .
.
(2) INFORMATION FOR SEQ ID NO:156:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single - . . .
~D) TOPOLOGY: linear .
(ii) MOLEC~LE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:156:

(2) INFORMATION FOR SEQ ID NO:157:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs :
(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:157:

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- -PCT!US51/00858 13 MAY ~q~1 (2) INFORMATION FOR SEQ ID NO:158:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs (~) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:158: -(2) INFORMATION FOR SEQ ID NO:159:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs (3) TYP: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear . -(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUERCE DESCRIPTION: SEQ ID NO:159: ~ -(2) INFORMATION FOR SEQ ID NO:160:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGT~: 23 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear ~ii) MOLECULE TYPE: DNA ~genomic) ~xi) SEQUENCE DESCRIPTION: SEQ ID NO:160:

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~ PCT!US 5~ q8 (2) INFORMATION FOR SEQ ID NO:161:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA ~genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:161: , (2) INFORMATION FOR SEQ ID NO:162:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: sinsle (D) TOPOLOGY: linear .
(ii) MOLECULE TYPE: DNA (genomic) -(xi) SEQUENCE DESCRIPTION: SEQ ID NO:162:

(2) INFORMATION FOR SEQ ID NO:163:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs (B~ TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE ~YPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:163:

(2) INFORMATION FOR SEQ ID NO:164:
(i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 21 base pairs (B~ TYPE: nucleic acid ~C) STRANDEDNESS: single (D) .OPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:164:

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111 ~

(2) INFORMATION FOR SEQ ID NO:165:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs (E) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) .
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:165:

(2) INFORMATION FOR SEQ ID NO:166:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA ~genomic) :
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:166:
GTACTGGTCC CGCATGGCGC T 21 ~:-(2) INFORMATION FOR SEQ ID NO:167:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear ~ii) MOLECULE TYPE: DNA ~genomic) ~xi3 SEQUENCE DESCRIPTION: SEQ ID NO:167:
CGGGAGAGAG GCCTGCTGAA A 21 ~.
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(2) INFORMATION FOR SEQ ID NO:168:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) ~ .
(xi` SEQUENCE DESCRIPTION: SEQ ID NO:168:

(2) INFORMATION FOR SEQ ID NO:169: ~:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid ~C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUE~CF DESCRIPTION: SEQ ID NO:169:

(2) INFORMATION FOR SEQ ID NO:170:
(i) SEQUENCE CHARACTERISTICS:
(A) ~ENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single ~D) I'OPOLOGY: linear ~ii) MOLECULE TYPE: DNA ~genomic) ~xi) SEQUENCE DESCRIPTION: SEQ ID NO:170:

~2) INFORMATION FOR SEQ ID NO:171:
(i) SEQUÆNCE CHARACTERISTICS:
~A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear tii) MOLECULE TYPE: DNA ~genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:171:

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(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:172:

(2) INFORMATION FOR SEQ ID NO:173:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:173:

(2) INFORMATION FOR SEQ ID NO:174: -.
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:174:

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~CTIUS ?1/00858 lS91 (2) INFORMATION FOR SEQ ID NO:175:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:175:

(2) INFORMATION FOR SEQ ID NO:176:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B! TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUE~CE DESCRIPTION: SEQ ID NO:176:

(2) INFORMATION FOR SEQ ID NO:177:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear ~ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:177:

(2) INFORMATION FOR SEQ ID NO:178:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs :~
(B) TYPE: nucleic acid ~C) ST~ANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:178:

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(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:179:

(2) INFORMATION FOR SEQ ID NO:180:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY. linear (ii) MOLECULE TYPE: DNA (genomic) ~
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:180: ~.

(2) INFORMATION FOR SEQ ID NO:181:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) I'YPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:181:

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3 ~ I ~ Y , ~ 3 1 (2) INFORMATION FOR SEQ ID NO:182:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs tB) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:182:

(2) INFORMATION FOR SEQ ID NO:183:
(i) SEQUENCE CHARACTERISTICS:
(~) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:183:

(2) INFORMATION FOR SEQ ID NO:l84:
(i) SEQUENCE CHARACTERISTICS: :~
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid --(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA lgenomic) ~xi) SEQUENCE DESCRIPTION: SEQ ID NO:184:

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(2) INFORMATION FOR SEQ ID NO:185:
~i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:135:

(2) INFORMATION FOR SEQ ID NO:186:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) -(xi) SEQUENCE DESCRIPTION: SEQ ID NO:186:
GGAGCAGGTH GTG~TGGGAA 20 :
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(2) INFORMATION FOR SEQ ID NO:187:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) txi) SEQUENCE DESCRIPTION: SEQ ID NO:187:
GiGAGCAGGTG HTGTTGGGAA 20 (2) INFORMATION FOR SEQ ID NO:188:
(i) SEQUENCE CHARACTERISTICS:
tA) LENGTH: 20 base pairs tB) TYPE: nucleic acid _ :
tC) STRANDEDNESS: single tD) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA tgenomic) txi) SEQUENCE DESCRIPTION: SEQ ID NO:188:
TACTCTTCTT GTCCAGCTGT -- 20 - ~:

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(2) INFORMATION FOR SEQ ID NO:l89:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid ~C) STRANDEDNESS: single (D) TOPOLOGY: linear tii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:189:

(2) INFORMATION FOR SEQ ID NO:l90:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUE~CE DESCRIPTION: SEQ ID NO:l90:

(2) INFORMATION FOR SEQ ID NO:l9l:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single ~ :
(D) TOPOLOGY: linear . -(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l9l:

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.--. PCTIUS 9 1 / O 0 8 5 8 (2) INFORMATION FOR SEQ ID NO:192:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 base pairs (~) TYPE: nucleic acid ~C) STRANDEDNESS: single (D) TOPOLOGY: linear ~ii) MOLECULE TYPE: DNA (genomic) : --(xi) SEQUENCE DESCRIPTION: SEQ ID NO:192:

(2) INFORMATION FOR SEQ ID NO:193:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 base pairs (8) TYPE: nucleic acid (C) S~RANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE; DNA (genomic) (xi) SEQUE~CE DESCRIPTION: SEQ ID NO:193:

(2) INFORMATION FOR SEQ ID NO:194: ~ -(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 base pairs (B) TYPE: nucleic acid .
(C) STRANDEDNESS: single (D) TOPOLOGY: linear ~ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:194:

(2) INFORMATION FOR SEQ ID NO:195: -(i) SEQUENCE CHARACTERISTICS: .
(A) LENGTH: 16 base pairs (B) TYPE: nucleic acid _ .
(C) STRANDEDNESS: single : .
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:195: -`

~ .

- . . - . . . : .- : .: ~ :.: : . . . : : ~:
.. ., . , .. . .. .. .- ..

~;' 1 3 MAY 1991 ;

(2) INFORMATION FOR SEQ ID NO:196:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:196:

(2) INFORMATION FOR SEQ ID NO:197:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLEC~LE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:197: .
GAGCAGCTGG TGTTGG 16 ::
(2) INFORMATION FOR SEQ ID NO:198:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single . (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:198:

.; . . -- . , PCT!US 91/0085 1 3 MAY l99l (2) INFORMATION FOR SEQ ID NO:199:
(i~ SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l99:

(2) INFORMATION FOR SEQ ID NO:200: ~ ~ .
(i) SEQUENCE CHARACTERISTICS:
(A) LENGT~: 16 base pairs (B) TYPE: nucleic acid ~-(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:200: ~
GCAGGTTGTG TTGGGA 16 ::

(2) INFORMATION FOR SEQ ID NO:201:
(i) SEQUENCE CHARACTERISTICS:
(A) EENGTH:. 16 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear :
(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:201:

(2) INFO~MATION FO~ SEQ ID NO:202: .
(i) SEQUENCE CHA~ACTERISTICS: .
(A~ LENGTH: 16 base pairs (B) TYPE: nucleic acid . (C~ STRANDEDNESS: single (D~ TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESC~IPTION: SEQ ID NO:202:

,~ PCT1US 91/00858 flAY ~99 (2) INFORMATION FOR SEQ ID NO:203:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 base pairs (B) TYPE: nucleic acid (C) ST~ANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:203:

(2) INFORMATION FOR SEQ ID NO:204:
(i) SEQUENCE CHARACTERISTICS: : .
(A) LENGTH: 17 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single ~D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUEhCE DESCRIPTION: SEQ ID NO:204:

(2) INFORMATION FOR SEQ ID NO:205:
(i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 17 base pairs ~B) TYPE: nucleic acid ~C) STRANDEDNESS: single ~D) TOPOLOGY: linear ~ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:205:

IY!AY 'qql (2) INFORMATION FOR SEQ ID NO:206:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:206:

(2) INFORMATION FOR SEQ ID NO:207:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear :
(ii) MOLECULE TYP~: DNA (genomic) (xi) SEQUE~CE DESCRIPTION: SEQ ID NO:207: -CAGCTGGAAA AGAAGAG l?

(2) INFORMATION FOR SEQ ID NO:208:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 base pairs ~B) TYPE: nucleic acid (C) STRANDEDNESS: single ~D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA ~genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:208: -: ~ . . - , . , . ~ . . . , . - .. : .
- ~ - - . . , -: .......... . :

PrT'II~ 9113/MOA~8 ~

(2) INFORMATION FOR SEQ ID NO:209:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single -(D) TOPOLOGY: linear -(ii) MOLECULE TYPE: DNA (genomic) ::
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:209:

(2) INFORMATION FOR SE2 ID NO:210:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 base pairs (B) TYPE: nucleic acid (C) STRANDFDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:210:

(2) INFORMATION FOR SEQ ID NO:211:
(i) SEQUENCE CHARACTERISTICS: -(A) LENGTH: 16 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:211:

(2) INFORMATION FOR SEQ ID NO:212:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 base pairs ~B) TYPE: nucleic acid (C) STRANDEDNESS: single _ ~D) TOPOLOGY: linear : -~ii) MOLECULE TYPE: DNA ~genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:212:

,, , . , - , . .

PCrlUS 9114`~

(2) INFORMATION FOR SEQ ID NO:213:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:213:

(2) INFORMATION FOR SEQ ID NO:214: ~ :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single ......
(D) TOPOLOGY: linear -(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUE~CE DESCRIPTION: SEQ ID NO:214:

:., -.
(2) INFORMATION FOR SEQ ID NO:215:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 base pairs (B) TYPE: nucleic acid (C~ STRANDEDNESS: single ~ :
(D) TOPOLOGY: linear :
(ii) MOLECULE TYPE: DNA tgenomic) txi) SEQUENCE DESCRIPTION: SEQ ID NO:215:
GGCGCCTGCG GTGT 14 ..

- .. , - . : . , . - ~ . -, : : .

,. ~ :. : . - ~ , . . .

' ' : ' ' - : -, . . ., - . . - . :' .

13 MAY l99l l26 (2) INFORMATION FOR SEQ ID NO:216:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:216:

(2) INFORMATION FOR SEQ ID NO:217:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUE~CE DESCRIPTION: SEQ ID NO:217:

(2) INFORMATION FOR SEQ ID NO:218:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear --(ii) MOLECULE TYPE: DNA (genomic) -(xi) SEQUENCE DESCRIPTION: SEQ ID NO:218:

(2~ INFORMATION FOR SEQ ID NO:219:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:219:

.. : , , . ,- .... -. ~ ,. . ... : . . - ., - - . - . . ::, - . . ..

: : : --1 ~ MAY 1991 ~2) INFORMATION FOR SEQ ID NO:220:
(i) SEQUENCE CHARACTERISTICS: ~.
(A) LENGTH: 14 base pairs (B) TYPE: nucleic ac1d (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA tgenomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:220: ~
GCCGGCAGTG TGGG 14 ~ -(2) INFORMATION FOR SEQ ID NO:221:
(i) SEQUENCE CHARACTE RISTICS:
(A) LENGTH: 14 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLEC~LE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:221: :

(2) INFORMATION FOR SEQ ID NO:222:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 base pairs .
(B) TYPE: nucleic acid -(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) .
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:222: ; .

,: . . ;' '' . `. ':;' ' , ~ ' PCTIUS 9ll3/MoA~

(2) INFORM~TION FOR SEQ ID NO:223:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 base pairs (B) TYPE: nucleic acid (C3 STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:223:
GCCGGCGATG TGGG l4 (2) INFORMATION FOR SEQ ID NO:224:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 base pairs (3) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:224:
GCCGGCGCTG TGGG l4 : ' ..
(2) INFORMATION FOR SEQ ID NO:225:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 base pairs (B) TYPE: nucleic acid : .
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:225: .
GCCGGCGTTG TGGG l4 (2) INFORMATION FOR SEQ ID NO:226:
(i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: l~ base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:226:
GCCGGCCAGG AGGA -- ~ l4 . . ......... ~. ,: ~ . . . -. .: , : - :; . ~
.. . .- . , ~ ; , , . - :

13 I~AY l991 l29 (2) INFORMATION FOR SEQ ID NO:227:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 base pairs (B) TYPE: nucleic acid ~C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQQENCE DESCRIPTION: SEQ ID NO:22~:

(2) INFORMATION FOR SEQ ID NO:228:
(i) SEQUENCE CHARACTERISTICS: :
(A) LENGTH: 14 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear .;
(ii) MOLECULE TYPE: DNA (genomic) :
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:228: -CGCCGGCAAG GAGG 14 ' (2) INFORMATION FOR SEQ ID NO:229:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear ~ .
(ii) MOLECVLE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:229:

.
.
- :. : , ~ , . ... . . . . .

~ PCTIUS 9 1 / 00 8 5 ~

(2) INFORMATION FOR SEQ ID NO:230:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA ~genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:230:

.
(2) INFORMATION FOR SEQ ID NO:231:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear ~ -(ii) MOLECULE TYPE. DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:231:

(2) INFORMATION FOR SEQ ID NO:232:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 base pairs (~) TYPE: nucleic acid (C) STRANDEDNESS: single : `
(D) TOPOLOGY: linear ~ii) MOLECULE TYPE: DNA ~genomic) ~xi) SEQUENCE DESCRIPTION: SEQ ID NO:232:
CGCCGGCCAT GAGG 14 .: .

. .. ~ ., . . ,. , . ~ . .. ~ . - - . . ..

~ PCTIUS 9~ 9~

(2) INFORMATION FOR SEQ ID NO:233:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single :
(D) TOPOEOGY: linear (ii) MOLEC~LE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:233:
GCCGGCCACG AGGA 14 -.
(2) INFORMATION FOR SEQ ID NO:234:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear ~:~
(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:234: :
GGAGCTGGTG GCG~A 15 -(2) INFORMATION FOR SEQ ID NO:235:
(i) SEQUENCE CHARACTERISTICS: :
(A) LENGTH: 16 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single .
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:235:

(2) INFORMATION FOR SEQ ID No:236:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:236:

prTIlls 91 /0085 1 :~ MAY la9 l32 (2) INFORMATION FOR SEQ ID NO:237:
~i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 base pai~s (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xl) SEQUENCE DESCRIPTION: SEQ ID NO:237:

(2) INFORMATION FOR SEQ ID NO:238:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 base pairs (3~ TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUE~CE DESCRIPTION: SEQ ID NO:238:

(2) INFORMATION FOR SEQ ID NO:239:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) .
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:239:

-: . . : : -, .- - , : . :

PCT~ US 91~ Op~ 8q5 8 (2) INFORMATION FOR SEQ ID NO:240:
ti) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear ~ .--(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:240:

(2) INFORMATION FOR SEQ ID NO:241:
(i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 15 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA tgenomic) (xi) SEQUE~CE DESCRIPTION: SEQ ID NO:241:

(2) INFORMATION FOR SEQ ID NO:242:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:242:

(2) INFORMATION FOR SEQ ID NO:243:
(i) SEQUENCE C~ARACTERISTICS:
(A) LENGTH: 15 bas~ pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii~ MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:243:
~ GCTGGTCGCG TAGGC 15 ~~

- .. ., -- ~

:: , : : . ~

., : . , - . - .

~ PCTIUS 9 1/ 0Oy8 5 &

l34 (2) INFORMATION FOR SEQ ID NO:244:
(i) SEQUENCE C~ARACTERISTICS:
~A) LENGTH: 15 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:244: .

(2) INFORMATION FOR SEQ ID NO:245:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 base pairs (B) TYPE: nucleic acid -(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECyLE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:245: :-(2) INFORMATION FOR SEQ ID NO:246:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:246:

- .- :, . - -.. :- . : . - : - . .. . . -PC ~ i ~S 1 1 / OyO 8 5 8 (2) INFORMATION FOR SEQ ID NO:247:
~i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear . -(ii) MOLECULE TYPE: DNA (gen~mic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:247:

(2) INFORMATION FOR SEQ ID NO:248: -(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 base pairs (B) TYPE: nucleic acid .
(C) STRANDEDNESS: single -(D) TOPOLOGY: linear -(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:248:

(2) INFORMATION FOR SEQ ID NO:249:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) ~xi) SEQUENCE DESCRIPTION: SEQ ID NO:249:

~2) INFORMATION FOR SEQ ID NO:250:
~i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 15 base pairs ~B) TYPE: nucleic acid ~C) STRANDEDNESS: single ~D) TOPOLOGY: linear ~ii) MOLECULE TYPE: DNA tgenomic) ~xi) SEQUENCE DESCRIPTION: SEQ ID NO:250:

- . . ~ . ., . .- , . . . ~ -- . .~ . . ~ , . , - - , ': : ' :'. ~. ' . ~ . , :.............. - ~ . - . . .
.:
- , pt`T 115 9 (2) INFORMATION FOR SEQ ID NO:251:
(i) SEQUENCE CHARACTERISTICS:
(A~ LENGTH: 16 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:251:

(2) INFORMATION FOR SEQ ID NO:252:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: lS base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:252:

(2) INFORMATION FOR SEQ ID NO:253:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 base pairs .. ~.-(B) TYPE: nucleic acid (C) STRANDEDNESS: single ~D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:253:

P~T I I~S ~ 1 / 0 0 8 ~; 8 (2) INFORMATION FOR SEQ ID NO:254:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA ~genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:254:

. . ~
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Claims (20)

In the Claims

1. A method for detecting a G-protein a subunit point mutation in a nucleic acid segment present in a biological sample, said method comprising:
(a) treating the sample with a G-protein a 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 a 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 a 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 a mutant nucleic acid sequence within said amplified DNA; and (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.

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 will amplify a nucleic acid segment encoding a subsequence of a G-protein a subunit selected from the group consisting of Gi.alpha.1, Gi.alpha.2, Gi.alpha.3, Gz.alpha., Go.alpha., and Gs.alpha..

7. The method of Claim 6, wherein said nucleic acid segment encodes at least one amino acid selected from the group consisting of the amino acids corresponding to Gs.alpha. 49, 201, and 227.

8. The method of Claim 7, wherein said primer pair is selected from the group consisting of: JFL69 (SEQ ID NO: 26) and JFL70 (SEQ ID NO: 27); JFL135 (SEQ ID NO: 28) and JFL136 (SEQ ID NO:29); JFL228 (SEQ ID NO: 46) and JFL135 (SEQ ID NO: 28); JFL229 (SEQ ID NO: 45) and JFL136 (SEQ ID NO: 29);
JFL226 (SEQ ID NO: 24) and JFL227 (SEQ ID NO: 25); JFL223 (SEQ ID NO: 30) and JFL224 (SEQ ID NO: 32); JL54 (SEQ ID NO: 20) and JL57 (SEQ ID NO: 21);
JFL109(SEQ ID NO:47) and JFL110 (SEQ ID NO: 16); JFL110 (SEQ ID NO: 16) and JFL112 (SEQ ID NO:17);JFL113 (SEQ ID NO: 18) and JFL114 (SEQ ID NO:
48); JFL115 (SEQ ID NO:19) and JFL113 (SEQ ID NO: 18); SP9 (SEQ ID NO: 38) and SP10 (SEQ ID NO: 39); JFL139 (SEQ ID NO: 40) and SP11 (SEQ ID NO: 41);
JFL201 (SEQ ID NO: 43) and SP15 (SEQ ID NO: 42); JL55 (SEQ ID NO: 22) and JL56 (SEQ ID NO: 23); JFL223 (SEQ ID NO: 30) and SP33 (SEQ ID NO: 31); and JFL224 (SEQ ID NO: 32) and SP34 (SEQ ID NO: 33); JFL235 (SEQ ID NO: 34) and JFL237 (SEQ ID NO: 35); JL55 (SEQ ID NO: 22) and JFL212 (SEQ ID NO: 36);
JFL215 (SEQ ID NO: 37) and JL56 (SEQ ID NO: 23); and JFL135 (SEQ ID NO: 28) and JFL286 (SEQ ID NO: 44).

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 Gi.alpha.1, Gi.alpha.2, Gi.alpha., Go.alpha., Gs.alpha., and Gz.alpha..

10. The method according to Claim 1, wherein said probe hybridizes to a G-protein subsequence encoding the amino acid corresponding to the Gs.alpha. amino acid at position 49, 201, or 227.

11. The method of Claim 10, wherein said probe is selected from the group consisting of ;
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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 oligonucleotide primer pair for the amplification of a subsequence of a nucleic acid encoding a G-protein .alpha. subunit wherein said subsequence comprises a nucleic acid sequence encoding an amino acid corresponding to the Gs.alpha. amino acid at a position selected from the group consisting of 49, 201, and 227.

15. An oligonucleotide primer pair according to Claim 14, wherein said G-protein .alpha. subunit is selected from the group consisting of Gs.alpha., Go.alpha., Gi.alpha.1, Gi.alpha.2, Gi.alpha.3, and Gz.alpha..

16. An oligonucleotide probe for distinguishing between oncogenes and proto-oncogenes encoding G-protein .alpha. subunits, wherein said probe hybridizes to a region of nucleic acid encoding an amino acid selected the the group consisting amino acids corresponding to the Gs.alpha. amino acid at positions 49, 201, and 227.

17. An oligonucleotide primer pair for the amplification of a subsequence of a nucleic acid encoding a G-protein a subunit wherein the primer pair is selected from the group consisting of: JFL69 (SEQ ID NO: 26) and JFL70 (SEQ ID NO: 27); JFL135 (SEQ ID NO: 28) and JFL136 (SEQ ID NO: 29); JFL228 (SEQ ID NO: 46) and JFL135 (SEQ ID NO: 28); JFL229 (SEQ ID NO: 45) and JFL136 (SEQ ID NO: 9);
JFL226 (SEQ ID NO: 24) and JFL227 (SEQ ID NO: 25); JFL223 (SEQ ID NO: 30) and JFL224 (SEQ ID NO: 32); JL54 (SEQ ID NO: 20) and JL57 (SEQ ID NO: 21);
JFL109 (SEQ ID NO: 47) and JFL110 (SEQ ID NO: 16); JFL110 (SEQ ID NO: 16) and JFL112 (SEQ ID NO:17); JFL113 (SEQ ID NO: 18) and JFL114 (SEQ ID NO:
48); JFL115 (SEQ ID NO: 19) and JFL113 (SEQ ID NO: 18); SP9 (SEQ ID NO: 38) and SP10 (SEQ ID NO: 39); JFL139 (SEQ ID NO: 40) and SP11 (SEQ ID NO: 41);
JFL201(SEQ ID NO: 43) and SP15 (SEQ ID NO: 42); JL55(SEQ ID NO: 22) and JL56 (SEQ ID NO: 23); JFL223 (SEQ ID NO: 30) and SP33 (SEQ ID NO: 31); and JFL224 (SEQ ID NO: 32) and SP34 (SEQ ID NO: 33); JFL235 (SEQ ID NO: 34) and JFL237 (SEQ ID NO: 35); JL55 (SEQ ID NO: 22) and JFL212 (SEQ ID NO: 36);
JFL215 (SEQ ID NO: 37) and JL56 (SEQ ID NO: 23); and JFL135 (SEQ ID NO: 28) and JFL286 (SEQ ID NO: 44).

18. A probe for the detection of a point mutation in a nucleic acid encoding a segment of a G-protein a subunit wherein the probe is selected from the group consisting of:
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19. A method for detecting a point mutation, if present, in a nucleic acid encoding a G-protein .alpha. subunit in a sample comprising:
(a) hybridizing a G-protein .alpha. subunit probc 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 .alpha. subunit, comprising, in a separate container:
(a) a G-protein primer pair suitable for providing an amplified G-protein DNA segment in a PCR reaction;
(b) a G-protein probe comprising a wild type sequence which will hybridized to said DNA segment if it is a wild type; and (c) a G-protein probe comprising a sequence containing a point mutation for detecting a point mutation if present in said DNA segment.