P450 single nucleotide polymorphism biochip analysis
View Patent ↗This invention relates to methods and compositions for determining single nucleotide polymorphisms (SNPs) in P450 genes. In preferred embodiments, self extension of interrogation probes is prevented by using novel non self-extension probes and/or methods, thereby improving the specificity and efficiency of P450 SNP detection in target samples with minimal false positive results. The invention thus describes a variety of methods to decrease self-extension of interrogation probes. In addition, this invention provides a unique collection of P450 SNP probes on one assay, primer sequences for specific amplification of each of the seven P450 genes and amplicon control probes to evaluate whether the intended p450 gene targets were amplified successfully. The invention also describes a variety of array platforms for performing the assays of the invention; for example: CodeLink™, eSensor™, multiplex arrays with cartridges etc., all described herein.
1. A method of determining the identification of a nucleotide at a detection position in a target DNA sequence comprising:
a) amplifying the target DNA using random primers to generate DNA amplicons;
b) transcribing said DNA amplicons to generate RNA target sequences;
c) providing a solid support with a first surface comprising at least one extension probe wherein said extension probe includes an interrogation nucleotide;
d) hybridizing said RNA target sequence to said extension probe to form a hybridization complex;
e) contacting said surface with:
i) a modified reverse transcriptase; and
ii) at least one chain terminating nucleotide comprising a hapten; under conditions whereby if said chain terminating nucleotide is perfectly complementary to the base of the target sequence immediately adjacent to the 3′ end of extension probe in the hybridization complex, said chain terminating nucleotide is added to extension probe to form a modified extension probe;
f) contacting said modified extension probe with the binding partner of said hapten, wherein said binding partner is labeled; and
g) detecting the presence of said label to determine the nucleotide at said detection position.
2. The method of claim 1 , wherein said modified reverse transcriptase only extends extension probes bound to RNA.