WO1998038338A1 - Procedes d'analyse d'un acide nucleique - Google Patents

Procedes d'analyse d'un acide nucleique Download PDF

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Publication number
WO1998038338A1
WO1998038338A1 PCT/US1998/004003 US9804003W WO9838338A1 WO 1998038338 A1 WO1998038338 A1 WO 1998038338A1 US 9804003 W US9804003 W US 9804003W WO 9838338 A1 WO9838338 A1 WO 9838338A1
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probe
nucleic acid
sample
nucleotide
probes
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PCT/US1998/004003
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English (en)
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Anthony P. Shuber
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Exact Laboratories, Inc.
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Priority to AU66768/98A priority Critical patent/AU6676898A/en
Priority to EP98908830A priority patent/EP0975797A1/fr
Priority to CA002282705A priority patent/CA2282705A1/fr
Publication of WO1998038338A1 publication Critical patent/WO1998038338A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6832—Enhancement of hybridisation reaction

Definitions

  • This invention relates generally to methods for using segmented oligonucleotides. Methods of the invention are especially useful for disease diagnosis by detecting the presence of genetic mutations in cellular samples.
  • Cancer has been associated with genetic mutations in a number of oncogenes and tumor suppressor genes. Duffy, Clin. Chem., 41: 1410-1413 (1993). For example, point mutations in the ras genes have been shown to convert those genes into transforming oncogenes. Bos et ai, Nature, 315: 726-730. Mutations and the loss of heterozygosity at the p53 tumor suppressor locus have been correlated with various types of cancer. Ridanpaa et al., Path. Res. Pract, 191: 399-402 (1995); Hollstein et ai, Science, 253: 49-53 (1991 ).
  • Colorectal cancer is an example of a disease that is highly curable if detected early. With early detection, colon cancer may be effectively treated by, for example, surgical removal of the cancerous tissue. Surgical removal of early-stage colon cancer is usually successful because colon cancer begins in cells of the colonic epithelium and is isolated from the general circulation during its early stages. Thus, detection of early mutations in colorectal cells would greatly increase survival rate.
  • Current methods for detection of colorectal cancer focus on extracellular indicia of the presence of cancer, such as the presence of fecal occult blood or carcinoembryonic antigen circulating in serum. Such extracellular indicia typically occurs only after the cancer has become invasive. At that point, colorectal cancer is very difficult to treat.
  • a variety of detection methods have been developed which exploit sequence variation in DNA using enzymatic and chemical cleavage techniques.
  • a commonly- used screen for DNA polymorphisms consists of digesting DNA with restriction endonucleases and analyzing the resulting fragments by means of southern blots, as reported by Botstein et ai, Am. J. Hum. Genet, 32: 314-331 (1980) and White et ai, Sci. Am., 258: 40-48 (1988). Mutations that affect the recognition sequence of the endonuclease will preclude enzymatic cleavage at that site, thereby altering the cleavage pattern of the DNA. Sequences are compared by looking for differences in restriction fragment lengths.
  • restriction fragment length polymorphism mapping A problem with this method (known as restriction fragment length polymorphism mapping or RFLP mapping) is its inability to detect mutations that do not affect cleavage with a restriction endonuclease.
  • RFLP mapping restriction fragment length polymorphism mapping
  • a mutation is interrogated by two oligonucleotides capable of annealing immediately adjacent to each other on a target DNA or RNA molecule, one oligonucleotide having the 3' end complementary to the point mutation. Adjacent oligonucleotide sequences are only covalently attached when both oligonucleotides are correctly base-paired. Thus, the presence of a point mutation is indicated by the ligation of the two adjacent oligonucleotides. Grossman et ai, Nucleic Acid Research, 22: 4527-4534 (1994). However, the usefulness of this method for detection is compromised by high backgrounds which arise from tolerance of certain nucleotide mismatches or from non-template directed ligation reactions. Barringer et ai, Gene, 89: 117-122 (1990).
  • a number of detection methods have been developed which are based on a template-dependent, primer extension reaction. These methods fall essentially into two categories: (1 ) methods using primers which span the region to be interrogated for the mutation, and (2) methods using primers which hybridizes proximally and upstream of the region to be interrogated for the mutation.
  • primer-dependent DNA polymerases have, in general, a low replication error rate. This feature is essential for the prevention of genetic mistakes which would have detrimental effects on progeny. Methods in a second category exploit the high fidelity inherent in this enzymological reaction.
  • Detection of mutations is based on primer extension and incorporation of detectable, chain-terminating nucleoside triphosphates.
  • the high fidelity of DNA polymerases ensures specific incorporation of the correct base labeled with a reporter molecule.
  • Such single nucleotide primer-guided extension assays have been used to detect aspartylglucosaminuria, hemophilia B, and cystic fibrosis; and for quantifying point mutations associated with Leber Hereditary Optic Neuropathy (LHON). See. e.g., Kuppuswamy et al., Proc. NatI. Acad. Sci.
  • the selectivity and stability of the oligonucleotide primer extension assay is determined by the length of the oligonucleotide primer.
  • short primers i.e., less than about a 15-mer
  • short primers exhibit transient, unstable hybridization and, consequently, do not readily prime the extension reaction.
  • short primers exhibit nonspecific binding to a wide variety of perfectly-matched complementary sequences.
  • detection methods based on primer extension assays use oligonucleotide primers ranging in length from 15-mer to 25-mer.
  • genomic DNA is isolated from the biological sample and/or amplified with PCR using primers which flank the region to be interrogated.
  • the primer extension analysis is then conducted on the purified PCR products. See PCT Patent Publications WO 91/13075; WO 92/15712; and WO 96/30545.
  • WO 91/13075 a primer for the extension reaction
  • WO 92/15712 a primer for the extension reaction
  • WO 96/30545 since considerable optimization is required to ensure that only the perfectly annealed oligonucleotide functions as a primer for the extension reaction, only limited multiplexing of the primer extension assays is possible.
  • Krook et ai, supra report that multiplexing can be achieved by using primers of different lengths and by monitoring the wild-type and mutant nucleotide at each mutation site in two separate single nucleotide incorporation reactions.
  • the present invention provides methods for increasing the selectivity of nucleic acid hybridization reactions.
  • Methods of the invention comprises using segmented oligonucleotides in order to simultaneously achieve the hybridization stability characteristic of relatively long probes and the nucleic acid selectivity (i.e., intolerance of mismatches) characteristic of shorter probes.
  • Use of segmented oligonucleotides of the invention allows stable, selective (i.e., perfectly matched) hybridization of oligonucleotide probes for the identification of target nucleic acids, for template- dependent extension of such probes, for cleavage of the nucleic acids or the probes hybridized thereto, and for other nucleic acid reactions that benefit from stable, selective hybridization.
  • methods of the present invention permit the use of multiple segmented oligonucleotides under one set of reaction conditions.
  • methods of the. invention comprise exposing a sample suspected to contain a target nucleic acid to a short first probe and a longer second probe capable of hybridizing to substantially contiguous portions of the target nucleic acid, thereby to detect the target nucleic acid.
  • the two probes are exposed to sample under conditions that do not favor the hybridization of short first probe in the absence of longer second probe.
  • Factors affecting hybridization are well known in the art and include temperature, ion concentration, pH, probe length, and probe GC content.
  • a first probe because of its small size, hybridizes numerous places in an average genome.
  • any given 8-mer occurs about 65,000 times in the human genome.
  • an 8-mer has a low melting temperature (T m ) and a single base mismatch greatly exaggerates this instability.
  • T m melting temperature
  • a second probe is larger than the first probe and will have a higher T m .
  • a 20-mer second probe typically hybridizes with more stability than an 8-mer.
  • a longer probe will form a stable hybrid but will have a lower selectivity because it will tolerate nucleotide mismatches.
  • the first probe hybridizes with high selectivity (i.e., hybridizes poorly to sequence with even a single mismatch), but forms unstable hybrids when it hybridizes alone (i.e., not in the presence of a second probe).
  • the second probe will form a stable hybrid but will have a lower selectivity because of its tolerance of mismatches.
  • Methods of the invention comprise conducting a reaction that would not occur absent contiguous hybridization of the first and second probes.
  • the first and second probes hybridize to substantially contiguous portions of the target.
  • substantially contiguous portions are those that are close enough together to allow hybridized first and second probes to function as a single probe (e.g., as a primer of nucleic acid extension).
  • Substantially contiguous portions are preferably between zero (i.e., exactly contiguous so there is no space between the portions) nucleotides and about one nucleotide apart.
  • a linker is preferably used where the first and second probes are separated by two or more nucleotides, provided the linker does not interfere with the assay (e.g., nucleic acid extension reaction).
  • linkers are known in the art and include, for example, peptide nucleic acids, DNA binding proteins, and ligation. It has now been realized that the adjacent probes bind cooperatively so that the longer, second probe imparts stability on the shorter, first probe. However, the stability imparted by the second probe does not overcome the selectivity (i.e., intolerance of mismatches) of the first probe. Therefore, methods of the invention take advantage of the high selectivity of the short first probe and the hybridization stability imparted by the longer second probe.
  • segmented oligonucleotides eliminates the need for careful optimization of hybridization conditions for individual probes, as presently required in the art, and permits extensive multiplexing. Several segmented oligonucleotides can be used to probe several target sequences assayed in the same reaction, as long as the hybridization conditions do not permit stable hybridization of short first probes in the absence of the corresponding longer second probes.
  • methods of the invention comprise improvements in the detection of mutant nucleic acids.
  • a feature of the invention is the recognition that the selectivity of an oligonucleotide primer extension assays is significantly improved with the use of segmented oligonucleotides as primers for template-based extension.
  • a first (proximal) probe hybridizes adjacent to a nucleic acid suspected to be mutated.
  • the first probe comprises between about 5 and about 10 nucleotides.
  • the first probe alone is not a primer for template-based nucleic acid extension because it will not form a stable hybrid under the reaction conditions used in the assay.
  • a second (distal) probe in mutation detection methods of the invention hybridizes upstream of the first probe and to a substantially contiguous region of the target (template).
  • the second probe alone is not a primer of template-based nucleic acid extension because it comprises a 3' non-extendible nucleotide.
  • the second probe is larger than the first probe, and is preferably between about 15 and about 100 nucleotides in length.
  • template-dependent extension takes place only when a first probe hybridizes next to a second probe. When this happens, the short first probe hybridizes immediately adjacent to the site of the suspected single base mutation. The second probe hybridizes in close proximity to the 5' end of the first probe. The presence of the two probes together increases stability due to cooperative binding effects.
  • the two probes are recognized by polymerase as a primer.
  • This system takes advantage of the high selectivity of a short probe and the hybridization stability imparted by a longer probe in order to generate a primer that hybridizes with the selectivity of a short probe and the stability of a long probe. Accordingly, there is essentially no false priming with segmented primers. Since the tolerance of mismatches by the longer second probe will not generate false signals, several segmented primers can be assayed in the same reaction, as long as the hybridization conditions do not permit the extension of short first probes in the absence of the corresponding longer second probes.
  • first and second probes are hybridized to substantially contiguous regions of target, wherein the first probe is immediately adjacent and upstream of a site of suspected mutation, for example, a single base mutation.
  • the sample is then exposed to dideoxy nucleic acids that are complements of possible mutations at the suspected site.
  • dideoxy nucleic acids are labeled.
  • Deoxynucleotides may alternatively be used if the reaction is stopped after the addition of a single nucleotide.
  • Polymerase either endogenously or exogenously supplied, catalyzes incorporation of a dideoxy base on the first probe. Detection of label indicates that a non-wild-type (i.e., mutant) base has been incorporated, and there is a mutation at the site adjacent the first probe. Alternatively, methods of the invention may be practiced when the wild-type sequence is unknown. In that case, the four common dideoxy nucleotides are differentially labeled. Appearance of more than one label in the assay described above indicates a mutation may exist.
  • segmented oligonucleotides comprising first and second probes, are used to increase the selectivity (i.e., reduce the possibility of false positives) of target nucleic acid detection methods.
  • a sample suspected to contain a target is exposed to first and second probes, wherein the first probe is a shorter, less stable, but selective probe; and the second probe is a longer, more stable, less selective probe.
  • a target is detected by hybridization of the first and second probes to substantially contiguous portions of the target.
  • the first and second probes are detectably labeled.
  • a segmented oligonucleotide comprises a series of first probes, wherein sufficient stability is only obtained when all members of the segmented oligonucleotide simultaneously hybridize to substantially contiguous portions of a nucleic acid.
  • segmented primer comprising a series of three first probes (i.e., three short probes with no terminal nucleotide capable of hybridizing to a substantially contiguous portion of a nucleic acid upstream of the target nucleic acid)
  • the concurrent hybridization of the three probes will generate sufficient cooperative stability for the three probes to prime nucleic acid extension and the short probe immediately adjacent to a suspected mutation will be extended.
  • segmented probes comprising a series of short first probes offer the high selectivity (i.e., intolerance of mismatches) of short probes and the stability of longer probes.
  • first and second probes are constructed such that the first probe hybridizes immediately adjacent to the single base being investigated.
  • the second probe hybridizes to a region that is substantially contiguous with the region to which the first probe hybridizes.
  • the segmented nucleotide can comprise a series of short first probes that can hybridize to a contiguous region immediately adjacent to and upstream of the single base being investigated.
  • the sample is then exposed to the four common dideoxy nucleotides, or to other 3' terminal (i.e., unextendible) nucleotides.
  • a single-base extension reaction is conducted, and the 3' terminal nucleotides incorporated into the first probe are detected.
  • the incorporation of two different, but complementary nucleotides is evidence of heterozygosity at the single base locus.
  • deoxynucleotides may be used if extension is terminated (e.g., by enzyme inactivation) after addition of one nucleotide or if only one labeled nucleotide is present for incorporation during extension.
  • methods according to the invention also are useful to detect a loss of heterozygosity at an allele by determination of the amounts of maternal and paternal alleles comprising a genetic locus that includes at least one single-base polymorphism.
  • a statistically- significant difference in the amounts of each allele is indicative of a mutation in an allelic region encompassing the single-base polymorphism.
  • a region of an allele comprising a single-base polymorphism is identified.
  • Segmented oligonucleotides are designed to hybridize to corresponding regions on both paternal and maternal alleles immediately 3' to the single base polymorphism. After hybridization, a mixture of at least two of the four common dideoxy nucleotides (or deoxynucleotides if the reaction is stopped after addition of one nucleotide) are added to the sample, each labeled with a different detectable label. A DNA polymerase is also added. Using allelic DNA adjacent the polymorphic nucleotide as a template, the hybridized segmented oligonucleotide is extended by the addition of a single dideoxynucleotide that is the binding partner for the polymorphic nucleotide.
  • the dideoxynucleotides that have been incorporated into the first probe extension are detected by determining the number of bound extended probes bearing each of the two dideoxynucleotides in, for example, a flow cytometer or impedance counter.
  • the presence of an almost equal number of two different labels mean that there is heterozygosity at the polymorphic nucleotide.
  • the presence of a statistically-significant difference between the detected numbers of the two labels means that a deletion of the region encompassing the polymorphic nucleotide has occurred in one of the alleles.
  • Deoxynucleotides may be used as the detectable single extended base in any of the reactions described above that require single base extension. However, in such methods, the extension reaction must be stopped after addition of the single deoxynucleotide. Such methods may be employed regardless of whether a specific mutation is known (i.e., C- G). Moreover, the extension reaction need not be terminated after the addition of only one deoxynucleotide if only one labeled species of deoxynucleotide is made available in the sample for detection of the single base mutation. This method may actually enhance signal if there is a nucleotide repeat including the interrogated single base position.
  • methods of the invention may be used to detect target nucleic acid sites for subsequent cleavage of the hybridized (probe/target) nucleic acid. Cleavage is preferably accomplished with an enzyme, for example, a restriction endonuclease. Further aspects and advantages of the invention are apparent upon consideration of the following detailed description thereof.
  • Figure 1 is a diagram depicting the use of a segmented primer in a single base extension reaction for the detection of single base polymorphisms.
  • the white bar represents the template
  • the dark gray bar represents second probe which hybridizes to a region on the template that is substantially contiguous with the first probe (light gray).
  • the site suspected to be a single base mutation is labeled A.
  • the detectable label is marked B.
  • Methods of the invention are useful for a variety of reactions in which substantially contiguous hybridization of two probes is desired.
  • methods of the invention may be used to detect a target nucleic acid sequence using first and second probes as described above. Due to their increased selectivity for target, methods of the invention may be used to detect target nucleic acid that is available in small proportion in a sample and that would normally have to be amplified by, for example, PCR in order to be detected.
  • a particularly-preferred use of methods of the invention is to detect single-base mutations, especially in a heterogeneous sample, such as stool. Accordingly, methods of the invention are exemplified below by reference to detection of a single-base mutation in an oncogene. It is recognized that the single-base mutation may be part of a larger mutation. Often, however, as with the ras oncogenes, a single point mutation may be responsible for activation of the mutation.
  • Preferred methods of the invention comprise using segmented primers to enhance template-dependent nucleic acid polymerization. Such methods are especially useful for " detection of mutations, especially point mutations.
  • Methods of the invention comprise hybridizing two probes adjacent to a site of suspected mutation, wherein neither probe alone is capable of being a primer for template-dependent extension, but wherein adjacent probes are capable of priming extension.
  • methods of the invention comprise hybridizing to a target nucleic acid a probe having a length from about 5 bases to about 10 bases, wherein the probe hybridizes immediately upstream of a suspected mutation.
  • Methods of the invention further comprise hybridizing a second probe upstream of the first probe, the second probe having a length from about 15 to about 100 nucleotides and having a 3' non-extendible nucleotide. The second probe is substantially contiguous with the first probe.
  • substantially contiguous probes are between 0 and about 1 nucleotide apart.
  • a linker is preferably used where the first and second probes are separated by two or more nucleotides, provided the linker does not interfere with the nucleic acid extension reaction.
  • linkers are known in the art and include, for example, peptide nucleic acids, DNA binding proteins, and ligation.
  • methods of the invention comprise conducting an extension reaction to add nucleotides to the segmented primer resulting from co-hybridization of the above-described probes in a template-dependent manner.
  • methods of the invention are useful to detect mutations in a subpopulation of a polynucleotides in any biological sample.
  • methods disclosed herein may be used to detect mutations associated with diseases such as cancer.
  • methods of the invention may be used to detect a deletion or a base substitution mutation causative of a metabolic error, such as complete or partial loss of enzyme activity.
  • the following provides details of the use of methods according to the present invention in colon cancer detection. Inventive methods are especially useful in the early detection of a mutation. Accordingly, while exemplified in the following manner, the invention is not so limited and the skilled artisan will appreciate its wide range of applicability upon consideration thereof. Exemplary Methods for Detection of Colon Cancer or Precancer
  • a sample for analysis according to the invention is selected from stool, urine, sputum, blood, lymphatic fluid, semen, biopsy tissue, cerebrospinal fluid, and pus.
  • the sample is a cross-section of stool.
  • a preferred method for preparing a cross-section of stool is provided in co-owned, co-pending patent application Serial No. 08/699,678 (Attorney docket No. EXT-002), incorporated by reference herein.
  • As stool passes through the colon it adheres cells and cellular debris sloughed from colonic epithelial cells.
  • cells and cellular debris are sloughed by a colonic polyp (comprising mutated DNA).
  • sample is homogenized in an appropriate buffer, such as phosphate buffered saline comprising a salt, such as 20-100 mM NaCI or KCI, and a detergent, such as 1 -10% SDS or TritonTM, and/or a proteinase, such as proteinase K.
  • the buffer may also contain inhibitors of DNA and RNA degrading enzymes. Double-stranded DNA in the sample is melted (denatured to form single-stranded DNA) by well-known methods See, e.g., Gyllensten et ai, in Recombinant DNA Methodology
  • DNA or RNA may optionally be isolated from the sample according to methods known in the art. See, Smith-Ravin et al., Gut, 36: 81 -86 (1995), incorporated by reference herein. Once sample is prepared, it is exposed to one or more set of segmented primers according to the invention.
  • Genomic regions suspected to contain one or more mutations are identified by reference to a nucleotide database, such as GenBank, EMBL, or any other appropriate database or publication, or by sequencing.
  • genetic mutations in a number of oncogenes and tumor suppressor genes are known. Duffy, Clin. Chem., 41: 1410-1413 (1993).
  • Preferred genes for use in mutation detection methods of the invention include one " or more oncogenes and/or one or more tumor suppressor genes.
  • Specifically preferred genes include the ras oncogenes, p53, dec, ape, mcc, and other genes suspected to be involved in the development of an oncogenic phenotype.
  • methods of the invention permit the detection of a mutation at a locus in which there is more than one nucleotide to be interrogated. Moreover, methods of the invention may be used to interrogate a locus in which more than one single base mutation is possible.
  • at least one segmented primer is prepared to detect the presence of a suspected mutation.
  • a segmented primer comprises at least two oligonucleotide probes, a first probe and a second probe, which are capable hybridizing to substantially contiguous portions of a nucleic acid.
  • a first probe of the invention preferably has a length of from about 5 to about 10 nucleotides, more preferably between about 6 and about 8 nucleotides, and most preferable about 8 nucleotides.
  • a second probe of the invention has a preferable length of between about 15 and 100 nucleotides, more preferably between about 15 and 30 nucleotides, and most preferably about 20 nucleotides. Further, a second probe is incapable of being a primer for template-dependent nucleic acid synthesis absent a first probe because it has a 3' terminal nucleotide that is non-extendible.
  • Preferred non-extendible 3' terminal nucleotides include dideoxy nucleotides, C3 spacers, a 3' inverted base, biotin, or a modified nucleotide. Although, longer probes have a lower selectivity because of their tolerance of nucleotide mismatches, second probes are non-extendible and will not produce false priming in the absence of the proximal probe.
  • a segmented primer comprises a series of first probes, wherein each member of the series has a length of from about 5 to about 10 nucleotides, and most preferable about 6 to about 8 nucleotides. Although the first probes do not have a terminal nucleotide, nucleic acid extension will not occur unless all members of the series are hybridized to substantially contiguous portions of a nucleic acid.
  • the oligonucleotide probes of the segmented primer may be natural or synthetic, and may be synthesized enzymatically in vivo, enzymatically in vitro, or non- enzymatically in vitro.
  • Probes for use in methods of the invention are preferably selected from oligodeoxyribonucleotides, oligoribonucleotides, copolymers of deoxyribonucleotides and ribonucleotides, peptide nucleic acids (PNAs), and other functional analogues.
  • PNAs peptide nucleic acids
  • oligoamides comprising repeating amino acid units to which adenine, cytosine, guanine, thymine or uracil are attached. See Egholm, et ai, Nature, 365: 566-568 (1993); Oerum, et al. Nuci Acids Res., 23: 5332-36 (1993); Practical PNA: Identifying Point Mutations by PNA Directed PCR Clamping, PerSeptive Biosystems Vol. 1 , Issue 1 (1995). Peptide nucleic acid synthons and oligomers are commercially available form PerSeptive Biosystems, Inc., Framingham, MA. See, e.g., PCT publications EP 92/01219, EP 92/01220,
  • PNA probes are preferred to nucleic acid probes because, unlike nucleic acid/nucleic acid duplexes, which are destabilized under conditions of low salt, PNA/nucleic acid duplexes are formed and remain stable under conditions of very low salt. Additionally, because PNA/DNA complexes have a higher thermal melting point than the analogous nucleic acid/nucleic acid complexes, use of PNA probes can improve the reproducibility of blotting assays.
  • probes designed to detect mutations in the K-ras gene are provided below.
  • probes complementary to either portions of the coding strand or to portions of the non-coding strand may be used.
  • probes useful for detection of mutations in the coding strand are provided below. Mutations in K-ras frequently occur in the codon for amino acid 12 of the expressed protein. Several of the possible probes for detection of mutations at each of the three positions in codon 12 are shown below.
  • the wild-type codon 12 of the K-ras gene and its upstream nucleotides are: wild-type template 3 ' - TATTTGA ⁇ CACCATCAACCTCGACCA- 5 ' (SEQ ID NO: 1 )
  • the three nucleotides encoding amino acid 12 are underlined.
  • First probes and second probes capable of interrogating the three nucleotides coding for amino acid 12 of the K-ras gene are provided below.
  • First probe A is a first probe as described generally above, and has a sequence complementary to the nucleotides immediately upstream of the first base in codon 12 (i.e., immediately adjacent to the cytosine at codon position 1 ).
  • Second probe A is a second probe as generally described above.
  • Hybridization of first and second probes suitable for detection of a mutation in the first base of K-ras codon 12 are shown below: second probe A 5 ' -ATAAACTTGTGGTAG (SEQ ID NO: 2) first probe A TTGGAGCT (SEQ ID NO: 3) wild-type template 3 ' -TATTTGAACACCATCAACCTCGACCA-5 ' (SEQ ID NO: 1) Detection of a mutation in the second base in codon 12 may be performed by using the same second probe as above (second probe A), and a first probe, identified as first probe B below, that is complementary to a sequence terminating immediately adjacent (3') to the second base of codon 12.
  • second probe A 5 ' -ATAAACTTGTGGTAG SEQ ID NO: 2
  • first probe B TGGAGCTG SEQ ID NO: 4
  • wild-type template 3' -TATTTGA ⁇ CACCATCAACCTCGACCA-5 ' (SEQ ID NO: 1)
  • Detection of a mutation at the third position in codon 12 is accomplished using the same second probe as above, and first probe C, which abuts the third base of codon 12.
  • Hybridization of probes suitable for detection of a mutation in the third base of codon 12 are shown below second probe A 5 ' -ATAAACTTGTGGTAG (SEQ ID NO: 2)
  • the second probe is 1 and 2 nucleotides, respectively, upstream of the region to which the first probe hybridizes.
  • second probes for detection of the second and third nucleotides of codon 12 may directly abut (i.e., be exactly contiguous with) their respective first probes.
  • an alternative second probe for defection of a mutation in the third base of codon 12 in K-ras is:
  • the detection of mutations can also be accomplished with a segmented primer comprising a series of at least three first probes.
  • a series of first probes suitable for detection of a mutation in the third base of codon 12 is shown below: first probe X 5 ' -ATAAACTT (SEQ ID NO: 7) first probe Y TGGTAGTT (SEQ ID NO: 8) first probe Z GGAGCTGG (SEQ ID NO: 6) wild-type template 3 ' -TATTTGAACACCATCAACCTCGACCA-5 ' (SEQ ID NO: 1)
  • First and second probes are exposed to sample under hybridization conditions that do not favor the hybridization of the short first probe in the absence of the longer second probe.
  • Factors affecting hybridization are well known in the art and include raising the temperature, lowering the salt concentration, or raising the pH of the hybridization solution.
  • unfavorable hybridization conditions e.g., at a temperature 30-40 °C above first probe T m
  • first probe forms an unstable hybrid when hybridized alone (i.e., not in the presence of a second probe) and will not prime the extension reaction.
  • the longer, second probe having a higher T m , will form a stable hybrid with the template and, when hybridized to substantially contiguous portions of the nucleic acid, the second probe will impart stability to the shorter first probe, thereby forming a contiguous primer.
  • the sample may optionally be washed to remove unhybridized probes.
  • a modification of the dideoxy chain termination method as reported in Sanger, Proc. Nat'l Acad. Sci. (USA), 74: 5463-5467 (1977), incorporated by reference herein, is then used to detect the presence of a mutation.
  • the method involves using at least one of the four common 2', 3'-dideoxy nucleoside triphosphates (ddATP, ddCTP, ddGTP, and ddTTP).
  • ddNTPs dideoxy nucleoside triphosphates
  • a DNA polymerase such as SequenaseTM (Perkin-Elmer) is also added to the sample mixture.
  • the polymerase uses the substantially contiguous first and second probes as a primer, the polymerase adds one ddNTP to the 3' end of the first probe, the incorporated ddNTP " being complementary to the nucleotide that exists at the single- base polymorphic site. Because the ddNTPs have no 3' hydroxyl, further elongation of the hybridized probe will not occur. Chain termination will also result where there is no available complementary ddNTP (or deoxynucleoside triphosphates) in the extension mixture. After completion of the single base extension reaction, extension products are isolated and detected.
  • labeled deoxynucleotides may be used for detection if either the extension reaction is stopped after addition of only one nucleotide or if only one labeled nucleotide, corresponding to the complement of the expected mutation, is exposed to the sample.
  • the nucleoside triphosphate mixture contains just the labeled ddNTP or dNTP complementary to the known mutation.
  • second probe A and first probe A are exposed to an extension reaction mixture containing labeled ddTTP or dTTP.
  • the incorporation of a labeled ddTTP or dTTP in first probe A indicates the presence of a C ⁇ A mutation in the first nucleotide of codon 12 of the K-ras gene in the sample tested.
  • First probe A co- hybridized with second probe A to a wild-type template will not be extended or, alternatively, will be extended with unlabeled ddGTP or dGTP if available in the reaction mixture.
  • a detection method for this disease preferably screens a sample for the presence of a large number of mutations simultaneously in the same reaction (e.g., ape, K-ras, p53, dec, MSH2, and DRA).
  • ape e.g., K-ras, p53, dec, MSH2, and DRA.
  • a detection method for this disease preferably screens a sample for the presence of a large number of mutations simultaneously in the same reaction (e.g., ape, K-ras, p53, dec, MSH2, and DRA).
  • the primer extension reactions are conducted in four separate reaction mixtures, each having an aliquot of the biological sample, a polymerase, and the three labeled complementary non-wild-type ddNTPs (or dNTPs).
  • the reaction mixtures may also contain the unlabeled complementary wild- type ddNTP (or dNTP).
  • the segmented primers are multiplexed according to the wild- type template.
  • the first two nucleotides coding for amino acid 12 of the K-ras gene are cysteines.
  • second probe A and first probes A and B are added to a reaction mixture containing labeled ddATP (or dATP), ddTTP (or dTTP), and ddCTP (or dCTP).
  • Second probe C and first probe C are added to a reaction mixture containing labeled ddATP (or dATP), ddCTP (or dCTP), and ddGTP (or dGTP).
  • Any incorporation of a labeled ddNTP in a first probe indicates the presence of a mutation in codon 12 of the K-ras gene in the sample. This embodiment is especially useful for the interrogation of loci that have several possible mutations, such as codon 12 of K-ras.
  • the primer extension reactions are conducted in four separate reaction mixtures, each containing only one labeled complementary non-wild-type ddNTP or dNTP and, optionally, the other three unlabeled ddNTPs or dNTPs. Segmented primers can be thus be exposed only to the labeled ddNTP or dNTP complementary to the known mutant nucleotide or, alternatively, to all three non-wild-type labeled ddNTPs or dNTPs.
  • first probe A and second probe A are added to only one reaction mixture, the reaction mixture containing labeled ddCTP (or dCTP).
  • methods of the invention may be practiced as described above using labeled deoxynucleotides.
  • second probe A and first probes A and B are added to the three reaction mixtures containing labeled ddATP (or dATP), ddTTP (or dTTP), or ddCTP (or dCTP).
  • Second probe C and first probe C are added to the three reaction mixtures containing one of labeled ddATP (or dATP), ddCTP (or dCTP), and ddGTP (or dGTP).
  • the extension of a first probe with a labeled terminal nucleotide indicates the presence of a mutation in codon 12 of the -ras gene in the biological sample tested.
  • Labeled ddNTPs or dNTPs preferably comprise a "detection moiety" which facilitates detection of the short probes that have been extended with a labeled terminal nucleotide.
  • Detection moieties are selected from the group consisting of fluorescent, luminescent or radioactive labels, enzymes, haptens, and other chemical tags such as biotin which allow for easy detection of labeled extension products.
  • Fluorescent labels such as the dansyl group, fluorescein and substituted fluorescein derivatives, acridine derivatives, coumarin derivatives, pthalocyanines, tetramethylrhodamine, Texas Red®, 9-(carboxyethyl)-3-hydroxy-6-oxo-6H-xanthenes, DABCYL® and BODIPY® (Molecular Probes, Eugene, OR), for example, are particularly advantageous for the methods described herein. Such labels are routinely used with automated instrumentation for simultaneous high throughput analysis of multiple samples.
  • first probes comprising a portion of a segmented primer
  • ddNTPs or dNTPs are separated from unincorporated labeled ddNTPs or dNTPs. Such separation is accomplished by methods known in the art.
  • first probes comprise a "separation moiety.”
  • separation moiety is, for example, hapten, biotin, or digoxigenin.
  • the separation moiety in first probes does not interfere with the first probe's ability to hybridize with template and be extended.
  • the labeled ddNTPs comprise a separation moiety.
  • both the first probes and the labeled ddNTPs comprise a separation moiety.
  • reaction components are then separated on the basis of molecular weight using techniques known in the art such as gel electrophoresis, chromatography, or mass spectroscopy. See, Ausubel et ai, Short Protocols in Molecular Biology, 3rd ed. (John Wiley & Sons, Inc., 1995); Wu Recombinant DNA Methodology II, (Academic Press, 1995).
  • first probes are immobilized to a solid support after extension as described above.
  • the solid support is selected from the group consisting of glass, plastic, and paper.
  • the support is fashioned as a column, bead, dipstick, test tube.
  • the support is a microtiter dish, having a multiplicity of wells.
  • the conventional 96-well microtiter dishes used in diagnostic laboratories and in tissue culture are a preferred support.
  • the use of such a support allows the simultaneous determination of a large number of samples and controls, and thus facilitates the analysis.
  • automated systems can be used to provide reagents to such microtiter dishes. Any of a variety of methods known in the art may be used to immobilize short probes to a solid support.
  • a commonly used method consists of the non-covalent coating of the solid support with streptavidin or avidin and the immobilization of biotinylated oligonucleotide probe.
  • first probes comprise at least one biotinylated nucleotide.
  • the support is washed to remove any unbound probe.
  • the support is then tested for the presence of short probes that have been extended with a labeled terminal nucleotide by, for example, spectrophotometric methods.
  • the first probes are immobilized on the solid support prior to the extension reaction.
  • the immobilized first probes are then exposed to the sample, the second probes, a polymerase, and labeled ddNTPs or dNTPs.
  • the support is washed to remove the unincorporated labeled ddNTPs and the other reaction mixture components and the support is for the presence of short probes that have been extended with a labeled terminal nucleotide.
  • ADDRESSEE Patent Administrator, TESTA, HURWITZ &

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Abstract

La présente invention concerne des procédés de détection de la présence, dans un échantillon biologique, de séquences mutantes dans une sous-population de séquences géniques. Lesdits procédés sont utiles, en particulier, pour identifier des individus porteurs d'un indicateur de mutation génique du cancer colectoral primaire.
PCT/US1998/004003 1997-02-28 1998-02-27 Procedes d'analyse d'un acide nucleique WO1998038338A1 (fr)

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000009751A1 (fr) * 1998-08-14 2000-02-24 Exact Laboratories, Inc. Methodes diagnostiques dans lesquelles l'essai en serie de loci polymorphes est utilise
WO2000031305A2 (fr) * 1998-11-23 2000-06-02 Exact Laboratories, Inc. Methodes d'extension d'amorces permettant de detecter des acides nucleiques au moyen de molecules donneuses et receveuses
WO2001036687A2 (fr) * 1999-11-15 2001-05-25 Qiagen Genomics, Inc. Hybridation-amorcage simultanes destines a l'analyse du polymorphisme d'un seul nucleotide (snp)
US6280947B1 (en) 1999-08-11 2001-08-28 Exact Sciences Corporation Methods for detecting nucleotide insertion or deletion using primer extension
US6428964B1 (en) 2001-03-15 2002-08-06 Exact Sciences Corporation Method for alteration detection
EP1903117A1 (fr) * 2006-09-22 2008-03-26 Veterinärmedizinische Universität Wien Méthodes pour la détection de mutations au moyen d' amorces qui hybrident de façon contigue
US9109256B2 (en) 2004-10-27 2015-08-18 Esoterix Genetic Laboratories, Llc Method for monitoring disease progression or recurrence
US9777314B2 (en) 2005-04-21 2017-10-03 Esoterix Genetic Laboratories, Llc Analysis of heterogeneous nucleic acid samples

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EP0185494A2 (fr) * 1984-12-13 1986-06-25 Applied Biosystems, Inc. Détection de la séquence spécifique des acides nucléiques
WO1992015712A1 (fr) * 1991-03-05 1992-09-17 Molecular Tool, Inc. Determination d'acides nucleiques par extension de la polymerase d'oligonucleotides a l'aide de melanges terminateurs
WO1993006240A1 (fr) * 1991-09-13 1993-04-01 Cytocell Limited Technique d'amplification d'acides nucleiques

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0185494A2 (fr) * 1984-12-13 1986-06-25 Applied Biosystems, Inc. Détection de la séquence spécifique des acides nucléiques
WO1992015712A1 (fr) * 1991-03-05 1992-09-17 Molecular Tool, Inc. Determination d'acides nucleiques par extension de la polymerase d'oligonucleotides a l'aide de melanges terminateurs
WO1993006240A1 (fr) * 1991-09-13 1993-04-01 Cytocell Limited Technique d'amplification d'acides nucleiques

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000009751A1 (fr) * 1998-08-14 2000-02-24 Exact Laboratories, Inc. Methodes diagnostiques dans lesquelles l'essai en serie de loci polymorphes est utilise
WO2000031305A2 (fr) * 1998-11-23 2000-06-02 Exact Laboratories, Inc. Methodes d'extension d'amorces permettant de detecter des acides nucleiques au moyen de molecules donneuses et receveuses
WO2000031305A3 (fr) * 1998-11-23 2000-11-16 Exact Lab Inc Methodes d'extension d'amorces permettant de detecter des acides nucleiques au moyen de molecules donneuses et receveuses
US6280947B1 (en) 1999-08-11 2001-08-28 Exact Sciences Corporation Methods for detecting nucleotide insertion or deletion using primer extension
WO2001036687A2 (fr) * 1999-11-15 2001-05-25 Qiagen Genomics, Inc. Hybridation-amorcage simultanes destines a l'analyse du polymorphisme d'un seul nucleotide (snp)
WO2001036687A3 (fr) * 1999-11-15 2002-06-06 Qiagen Genomics Inc Hybridation-amorcage simultanes destines a l'analyse du polymorphisme d'un seul nucleotide (snp)
US6428964B1 (en) 2001-03-15 2002-08-06 Exact Sciences Corporation Method for alteration detection
US9109256B2 (en) 2004-10-27 2015-08-18 Esoterix Genetic Laboratories, Llc Method for monitoring disease progression or recurrence
US9777314B2 (en) 2005-04-21 2017-10-03 Esoterix Genetic Laboratories, Llc Analysis of heterogeneous nucleic acid samples
EP1903117A1 (fr) * 2006-09-22 2008-03-26 Veterinärmedizinische Universität Wien Méthodes pour la détection de mutations au moyen d' amorces qui hybrident de façon contigue
WO2008034640A1 (fr) * 2006-09-22 2008-03-27 Veterinärmedizinische Universität Wien Procédé pour la détection des mutations au moyen d'amorces qui s'hybrident de manière contigüe

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