C - Chemistry – Metallurgy – 12 – Q
Patent
C - Chemistry, Metallurgy
12
Q
C12Q 1/68 (2006.01) C12N 15/09 (2006.01) G01N 33/573 (2006.01) G01N 33/68 (2006.01)
Patent
CA 2385144
Methods are provided for determining the identity of a polymorphic nucleotide in a complex mixture of nucleic acids where one or more distinct polymorphisms can be present in the mixture, and multiple polymorphisms can be screened in parallel. Target nucleic acids are amplified using bridge amplification techniques. The detection and identification of the specific polymorphic residue(s) is based on readout methods that utilize the specificity of specific enzymes for complementary DNA sequences. These approaches result in a labeled nucleotide covalently attached to the amplicon, where the identity of the nucleotide is informative of the polymorphic sequence. in one aspect, the readout process uses primer extension protocols, where the specific base incorporated by DNA polymerise is determined by the sequence at the polymorphic site. In another aspect, the identity of a specific base hybridized and ligated to the amplicon is determined by the sequence at the polymorphic site. The polynucleotide to which the label has been attached can be detected in situ, i.e. bound to the solid substrate used for amplification; or can be released and detected.
Jones Keith
Leuther Kerstin
Shapero Michael H.
Ogilvy Renault Llp/s.e.n.c.r.l.,s.r.l.
Smithkline Beecham Corporation
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