Methods and Kits for Multiplex Hybridization Assays

Abstract

The invention provides a method for genotyping interfering polymorphic loci in a target polynucleotide, such as a strand of genomic DNA, in a multiplex hybridization-based assay. The invention also provides nucleic acid standards for validating the performance of such hybridization-based assays. In one aspect, the method of the invention is carried out by providing for each interfering polymorphic locus one or more probes so that at least one probe is capable of forming a perfectly match duplex at the locus regardless of the characteristic sequence of an adjacent polymorphism.

1 . A methods of determining a genotype of interfering polymorphic laid in a hybridization-based assay, the method comprising the steps of:
 providing for substantially every allele of each locus of interfering polymorphic loci a probe for substantially every allele of each adjacent loci of the interfering polymorphic loci, each allele of the adjacent loci having a characteristic sequence, and each such probe being capable of forming a stable duplex with the characteristic sequence of a different allele of each such adjacentloci;   hydridizing the probes to a target polynucleotide containing the interfering polymorphic loci under conditions that allow stable duplexes to form whenever a probe has a sequence complementary to a locus of the interfering polymorphic loci and a characteristic sequence of an allele of an adjacent locus of the interfering polymorphic loci; and   detecting the presence of probes forming stable duplexes with the target polynucleotide to determine the genotype of the interfering polymorphic loci.   
     
     
         2 . The method of  claim 1  wherein said interfering polymorphic loci contain from two to three loci. 
     
     
         3 . The method of  claim 2  wherein said interfering polymorphic loci are in genes selected from HLA genes, tumor suppressor genes, cell cycle control genes, oncogenes, or genes encoding xenobiotic metabolizing enzymes. 
     
     
         4 . The method of  claim 3  wherein said probes are molecular inversion probes. 
     
     
         5 . The method of  claim 3  wherein said interfering polymorphic loci are in genes encoding said xenobiotic metabolizing enzymes that are selected from the group consisting of ABCB1, ABCC2, GYP1A2, CYP2A6, CYP2B6, CYP2C19, CYP2C8, CYP2C9, CYP2D6, CYP3A4, CYP3A5, DPYD, FMO3, GSTM1, NAT1, NAT2, SLC21A6, SLC22A1, SLC22A2, TPMT, and UGT1A1. 
     
     
         6 . The method of  claim 3  wherein said interfering polymorphic loci are in genes encoding said xenobiotic metabolizing enzymes that are selected from the group consisting of CYP1A1, CYP1B1, CYP2C18, CYP3A7, GSTT1, GSTM3, GSTA1, UGTIA6, UGTIA7, UGT2B4, UGT2B7, UGT2B15, ADH1B, ALDH2, APE1, CDKN2A, COMT, DRD2, DRD4, EPHX1, ERCC1, ERCC2, ERCC4, ERCC5, GRPR, GSTA4, LIG3, MDM2, MGMT, MPO, NQO1, OGG1, PCNA, POLB, SLC6A3, SOD2, TP53, XRCC1, XRCC2, XRCC3, XRCC9, ABCB1, CYP2E1, GSTP1, SLC22A2, ABCC2, CYP2J2, NAT1, TPMT, CDA, CYP3A4, NAT2, UGT1A1, CYP1A2, CYP3A5, CYP2C8, CYP2A6, DPYD, CYP2C9, CYP2B6, FMO2, SLC15A2, CYP2C19, FMO3, SLC21A6, CYP2D6, GSTM1, and SLC22A1. 
     
     
         7 . The method of  claim 6  wherein said probes are molecular inversion probes. 
     
     
         8 . A kit of nucleic acid standards for determining the presence in a probe mixture of selected probes specific for interfering polymorphic loci, the kit comprising: a plurality of nucleic acid standards, wherein each nucleic acid standard comprises a double stranded nucleic acid containing a nucleic acid sequence for at least one haplotype of the interfering polymorphic loci and wherein there is at least one nucleic acid standard having a nucleic acid sequence complementary with each probe of the probe mixture. 
     
     
         9 . The kit of  claim 8  wherein each of said nucleic acid standards is capable of replication. 
     
     
         10 . The kit of  claim 9  wherein each of said nucleic acid standards comprises a first primer binding site, a second primer binding site, and disposed therebetween at least one DNA segment containing a sequence of each haplotype of said interfering polymorphic loci. 
     
     
         11 . The kit of  claim 9  wherein each of said nucleic acid standards comprises a plasmid. 
     
     
         12 . The kit of  claim 11  wherein said nucleic acid standards are provided in pairwise mixtures corresponding to different homozygous and heterozygous combinations of non-interfering polymorphic loci and different diploid combinations of haplotypes of interfering polymorphic loci. 
     
     
         13 - 15 . (canceled) 
     
     
         16 . The method of claim  14  wherein said interfering polymorphic loci are in genes encoding said xenobiotic metabolizing enzymes that are selected from the group consisting of CYP1A1, CYP1B1, CYP2C18, CYP3A7, GSTT1, GSTM3, GSTA1, UGT1A6, UGT1A7, UGT2B4, UGT2B7, UGT2B15, ADH1B, ALDH2, APE1, CDKN2A, COMT, DRD2, DRD4, EPHX1, ERCC1, ERCC2, ERCC4, ERCC5, GRPR, GSTA4, LIG3, MDM2, MGMT, MPO, NQO1, OGG1, PCNA, POLB, SLC6A3, SOD2, TP53, XRCC1, XRCC2, XRCC3, XRCC9, ABCB1, CYP2E1, GSTP1, SLC22A2, ABCC2, CYP2J2, NAT1, TPMT, CDA, CYP3A4, NAT2, UGT1A1, CYP1A2, CYP3A5, CYP2C8, CYP2A6, DPYD, CYP2C9, CYP2B6, FMO2, SLC15A2, CYP2C 19 , FMO3, SLC21A6, CYP2D6, GSTM1, and SLC22A1.