Method for detecting mutated alleles
OwnerChristoph Wagenerinventor
Life of this patent
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Abstract
A method for detecting mutated alleles in an excess of wild type alleles in a sample by isolating sample DNA; amplifying a target DNA sequence; and separating mutated DNA sequences from wild type DNA sequences by virtue of the preferential binding of the wild type sequences to carrier-bound complementary oligonucleotides. The amplification and separation steps may be iterated through one or more additional cycles to enhance sensitivity.
Claims15
A method for detecting the presence of tumor cells in an examination sample from a patient suspected of having cancer, wherein the method comprises detecting mutated alleles in an excess of wild type alleles by 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;
- 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.
A method for detecting the presence of tumor cells in an examination sample from a patient suspected of having cancer, wherein the method comprises detecting mutated alleles in an excess of wild type alleles by the steps of:
- isolating from an examination sample a DNA sample comprising unmutated wild typed alleles and suspected mutated alleles, wherein the nucleotide sequence is known for the unmutated wild typed alleles;
- amplifying the DNA sample by PCR;
- introducing the amplified DNA sample into at least one chromatographic column wherein the at least one chromatographic column comprises at least one carrier-bound oligonucleotide sequence that either alone in a single column or in multiple columns is complementary to the entire region of interest of the unmutated wild type sequence suspected of including at least one point mutation, and wherein the wild type alleles bind to the carrier bound oligonucleotide DNA sequences and the mutated alleles do not bind under hybridizing conditions maintained at the melting temperature of the wild-type specific oligonucleotide and its complementary sequence or if the mutated alleles do bind they are eluted from the column before the wild type alleles when a buffer is added;
- and detecting the mutation in the mutated alleles eluted from the at least one chromatographic column.
Dependencies are inferred from the claim text.