Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-05-03
2003-07-29
Housel, James (Department: 1648)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S005000, C435S007500, C435S007700, C435S091500, C435S091200, C435S069100, C204S450000
Reexamination Certificate
active
06599704
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a method for detecting genetic modifications, particularly a method for detecting a few mutated alleles in an excess of wild type alleles.
BACKGROUND OF THE INVENTION
The detection of mutated alleles in an excess of wild type alleles offers a significant diagnostic potential. Illustrative fields of application include, for example:
(i) detection of tumor cells in the stool of patients suspected of having colorectal carcinomas;
(ii) detection of tumor cells in the sputum and bronchial lavage of patients suspected of having bronchial carcinomas;
(iii) detection of tumor cells in the urine of patients suspected of having bladder carcinomas; and
(iv) detection of tumor cells in tissue biopsy samples.
The detection of mutant alleles of tumor genes in specimens such as urine, pancreatic juice, sputum, or stool holds great promise for an early diagnosis of cancer (Sidransky, D. (1997), “Nucleic acid-based methods for the detection of cancer,” Science 278:1054-1059; Nollau, P. et al. (1996), Int. J. Cancer 66:332-336). In addition, the detection of mutant tumor genes in tissue samples such as lymph nodes or resection margins may allow a sensitive diagnosis of residual malignant disease. When few tumor cells are present with an excess of nonmalignant cells, mutant tumor alleles constitute a minor fraction compared to wild-type alleles, so that detecting point mutated alleles presents a major analytical problem. So far, this problem has been solved only when the point mutations in the respective tumor genes are known a priori.
Since K-ras is among the tumor genes most frequently mutated in human tumors, most experience in the aforementioned diagnostic applications has been obtained using this oncogene as a target. Various methods have been described for detecting mutant K-ras alleles in the presence of an excess of wild-type alleles. These methods include detection of cloned PCR products by allele-specific oligonucleotide hybridization (Sidransky, D. et al. (1992). Science 256:102-105); digital PCR (Vogelstein, B. and Kinzler, K. W. (1999), Proc. Natl. Acad. Sci. U. S. A. 96:9236-9241); allele-specific PCR (Smith-Ravin, J., England, J., Talbot, I. C., and Bodmer, W. (1995), Gut 36:81-86); a modification of the oligonucleotide ligation assay, termed Point-EXACCT (Somers et al. (1994), Nucleic Acids Res. 22:4840-4841; Somers et al. (1998), Biochim. Biophys. Acta 1379:42-52), mutant-enriched PCR (Chen and Viola, (1991), Anal. Biochem. 195:51-56; Kahn et al. (1991), Oncogene 6:1079-1083); and a modification of the latter making use of a thermostable restriction enzyme that is included in the PCR reaction and cuts any amplification products derived from the wild-type sequence (Fuery et al. (2000), Clin. Chem. 46:620-624).
Using these methods, only the well-calculated amplification of defined point mutations has heretofore been possible, it being necessary for this purpose to know precisely the location and identity of the point mutation. The allele-specific oligonucleotide hybridization of the cloned PCR products has not been suited to detect a few point-mutated alleles in an excess of wild type alleles when the position of the mutation, e.g., a point mutation or deletion, is not known in advance.
Therefore, it is an object of the present invention to provide a method for detecting and separating a few mutated alleles in an excess of wild type alleles.
SUMMARY OF THE INVENTION
The invention provides methods to achieve an enrichment of mutant alleles, by removing wild-type alleles by differential hybridization to carrier-bound complementary oligonucleotides whose sequences span the region of the gene in which point mutations are expected. The mutant sequences bind less tightly to the carrier-bound complementary oligonucleotides than do wild type sequences, and are separated therefrom and reamplified by PCR. By iterating this process, mutant alleles can be detected in the presence of an excess of wild-type alleles with high sensitivity.
The invention in one aspect relates to a method for detecting mutated alleles in an excess of wild typed alleles, comprising the separation of the wild type alleles by means of a separation process using a carrier to which one or several oligonucleotides complementary to the wild type alleles are bonded.
Another aspect of the invention relates to a method for detecting mutated alleles in an excess of wild type alleles in an examination sample, comprising the steps of:
isolating DNA from the examination sample;
amplifying from the isolated DNA a DNA sequence region that contains a target DNA sequence suspected of containing one or more mutations;
converting the amplified DNA sequence region to single stranded DNA; and
separating mutated single stranded DNA sequences from unmutated single stranded DNA sequences by a separation step that employs preferential binding of the unmutated single stranded DNA sequences to one or more oligonucleotides; wherein
the oligonucleotides comprise DNA sequences of 12 to 25 bases;
the oligonucleotide DNA sequences are complementary to DNA sequences in the unmutated target DNA sequence and together include all of the unmutated target DNA sequence; and
the oligonucleotides are covalently bound to carrier materials.
The mutated target DNA sequence may contain one or more mutations such as point mutations, deletions, inversions, insertions, and substitutions. The separation step may be a chromatographic step, and the mutated single stranded DNA sequences may be selectively eluted from column-bound or carrier-bound oligonucleotides. The carrier or column material(s) to which the oligonucleotides are bound can be selected from the group consisting of glasses, gel materials, and polymer materials. The separation step may employ sense strands, antisense strands, or both. Sensitivity is enhanced when both sense and anti-sense strands are employed in the same separation vessel. In such a case, the oligonucleotide DNA sequences comprise at least one DNA sequence complementary to a sense strand and at least one DNA sequence complementary to an antisense strand of the amplified DNA sequence region.
The method is applicable to analysis of samples wherein the target DNA sequence is suspected of containing a point mutation. In that case, one oligonucleotide whose sequence is complementary to the unmutated target DNA sequence is employed.
For more complex analyses, the oligonucleotides may comprise a plurality of oligonucleotides having different sequences. Mutated single stranded DNA sequences may be separated from unmutated single stranded DNA sequences by preferential binding of the unmutated single stranded DNA sequences to the different sequence oligonucleotides by separation steps configured in series or in parallel flow arrangements.
In another aspect of the invention, referred to as subtractive iterative PCR (siPCR), mutant detection sensitivity is enhanced by employing one or more repetitions of the sequential steps of (a) amplifying the mutated single stranded DNA sequences obtained by separation from unmutated single stranded DNA sequences; and then (b) subjecting the amplified mutated single stranded DNA sequences to a separation step to remove residual unmutated single stranded DNA sequences, where the separation step employs preferential binding of the unmutated single stranded DNA sequences to one or more carrier-bound oligonucleotides. To obtain extremely high sensitivity, e.g., where the mutations are not known a priori, the one or more repeated separation step(s) employs a plurality of oligonucleotides having different sequences, and mutated single stranded DNA sequences are separated from unmutated single stranded DNA sequences by preferential binding of the unmutated single stranded DNA sequences to the different sequence oligonucleotides configured in a parallel flow arrangement.
In a further aspect, the invention relates to identifying a genetic modification in the mutated DNA sequences by methods such as size-sorting electrophoresis to show restriction fragment length
Fuierer Marianne
Housel James
Hultquist Steven J.
Li Bao Qun
Yang Yongzhi
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