Patent ID: 6638719
Filing Date: 2003-10-28
Classification: C12Q

Abstract:
A method to aid in determining a ratio of alleles at a polymorphic locus in a sample, comprising the steps of:(a) amplifying a region of double stranded DNA in a sample, wherein the region comprises a polymorphic locus to form amplified DNA products using a first and a second pair of primers, wherein the first pair of primers specifically amplifies a first allelic form of the polymorphic locus and the second pair of primers specifically amplifies a second allelic form of the polymorphic locus, wherein each pair of primers comprises an upstream and a downstream primer, wherein each upstream primer is complementary to a strand of the DNA which is opposite to a strand of the DNA to which the downstream primer is complementary, wherein each upstream primer is labeled with a color tag, wherein the first upstream primer is labeled with a first color tag and the second upstream primer is labeled with a second color tag, wherein the upstream primer of the first primer pair terminates in a 3â€² nucleotide which is complementary to the first allelic form but not complementary to the second allelic form, wherein the upstream primer of the second primer pair terminates in a 3â€² nucleotide which is complementary with the second allelic form but not complementary to the first allelic form; (b) hybridizing the amplified DNA products to at least two probes which are immobilized to known locations on a solid support, wherein a first probe is complementary to the first allelic form and a second probe is complementary to the second allelic form of the polymorphic locus, whereby a pattern of hybridization is formed on the solid support; and (c) determining a pattern of positive and intermediate-positive hybridizations to the first and second probes, wherein (i) cross-amplification is indicated if positive hybridization of the first color tag to the first probe is observed and intermediate-positive hybridization of the second color tag to the second probe is observed at a level of from 1-20% of the average of all positive hybridizations on the solid support; (ii) cross-amplification is indicated if positive hybridization of the second color tag to the second probe is observed and intermediate-positive hybridization of the first color tag to the first probe is observed at a level of from 1-20% of the average of all positive hybridizations on the solid support; (iii) cross-hybridization is indicated if positive hybridization of the first color tag to the first probe is observed and intermediate-positive hybridization of the first color tag to the second probe is observed at a level of from 1-20% of the average of all positive hybridizations on the solid support; (iv) cross-hybridization is indicated if positive hybridization of the second color tag to the second probe is observed and intermediate-positive hybridization of the second color tag to the first probe is observed at a level of from 1-20% of the average of all positive hybridizations on the solid support; and (v) cross hybridization is indicated if positive hybridization of the first color tag to the first probe and positive hybridization of the second color tag to the second probe, and either intermediate-positive hybridization of the second color tag to the first probe at a level of from 1-20% of the average of all positive hybridizations on the solid support, or intermediate-positive hybridization of the first color tag to the second probe at a level of from 1-20% of the average of all positive hybridizations on the solid support are observed, whereby hybridization patterns determined in step (c) aid in determining a ratio of alleles at the polymorphic locus.