Genotyping of mutations by combination of in-tube hybridization and universal tag-microarray

An assay for detecting single-nucleotide polymorphisms (SNPs) in gene fragments, the assay comprising:

• amplifying gene fragments to form an initial amplification product, wherein the amplifying comprises using reverse primers configured to hybridize with fluorescent oligonucleotides;
• isolating single strand deoxyribose nucleic acid (ssDNA) from the initial amplification product, the ssDNA including the reverse primers;
• forming a solution comprising the ssDNA and reporting molecules, each reporting molecule comprising a first domain configured to hybridize with a wild-type or mutated sequence that may potentially be included in the ssDNA, the mutated sequences each comprising a SNP, and a second oligonucleotide domain configured to hybridize with a complementary capture probe;
• hybridizing, in the solution, the ssDNA with the reporter molecules that have first domains that arm complementary to the ssDNA;
• applying the solution to the surface of a microarray comprising an array capture probes fixed to a microarray slide, each capture probe being configured to hybridize with a complementary second domain of a corresponding reporter molecule;
• capturing the hybridized ssDNA on the microarray by hybridizing the second oligonucleotide domains of the reporter molecules with complementary capture probes of the microarray;
• and
• detecting the hybridized ssDNA captured on the array by detecting fluorescent oligonucleotides hybridized with the primers.

An assay for detecting single-nucleotide polymorphisms (SNPs) in gene fragments, the assay comprising:

• amplifying gene fragments to form an initial amplification product;
• isolating single strand deoxyribose nucleic acid (ssDNA) from the initial amplification product;
• forming a solution comprising the ssDNA and different reporting molecules, each reporting molecule comprising a first domain configured to hybridize with a wild-type or mutated sequence that may potentially be included in the ssDNA, the mutated sequences each comprising a SNP, and a corresponding second oligonucleotide domain configured to hybridize with a complementary capture probe;
• hybridizing, in the solution, the ssDNA with first reporter molecules that have first domains that have a first nucleotide sequence, by maintaining the solution at a first hybridization temperature corresponding to the first sequence for a first time period, and hybridizing, in the solution, the ssDNA with a second type of the reporter molecules that have first domains that have a second nucleotide sequence, by maintaining the solution at a different second hybridization temperature corresponding to the second nucleotide sequence for a second time period;
• applying the solution to the surface of a microarray comprising an array capture probes fixed to a microarray slide, each capture probe being configured to hybridize with a complementary second domain of a corresponding reporter molecule;
• capturing the hybridized ssDNA on the microarray by hybridizing the second oligonucleotide domains of the reporter molecules with complementary capture probes of the microarray;
• and
• detecting the hybridized ssDNA captured on the array.