Multiplexed analysis of polymorphic loci by concurrent interrogation and enzyme-mediated detection
The invention provides methods and processes for the identification of polymorphisms at one or more designated sites, without interference from non-designated sites located within proximity of such designated sites. Probes are provided capable of interrogation of such designated sites in order to determine the composition of each such designated site. By the methods of this invention, one or more mutations within the CFTR gene and the HLA gene complex can be can be identified.
1 - 84 . (canceled)
85 . A method of determining the nucleotides at two or more predetermined polymorphic sites in one or more targets, the method comprising:
a) providing a set of oligonucleotide primer pairs, each pair capable of annealing with complementary polynucleotide strands to delineate a region of the corresponding target which includes a designated polymorphic site;
b) contacting said set of oligonucleotide primers with said targets under conditions allowing formation of pairs of complementary amplicon strands including designated polymorphic sites corresponding to designated polymorphic sites in corresponding targets;
c) selecting a set of probes wherein different types of encoded probes have different nucleotide sequences, and different types of probes are differently encoded and are complementary, in part, to different amplicon strands or different subsequences of amplicon strands,
d) contacting the set of probes with said amplicons at first temperature and conditions which favor annealing of encoded probes to amplicons and then providing a second temperature and conditions which favor formation of probe elongation products, and wherein probes are capable of annealing to an amplicon such that an interrogation site within a probe is in direct alignment with a designated polymorphic site in said amplicon, whereby a probe elongation product can be formed; and
e) detecting one or more probe elongation products.
86 . The method of claim 85 wherein the second temperature is higher than the first temperature.
87 . The method of claim 86 wherein the first temperature is about 55° C. and the second temperature is about 72° C.
88 . The method of claim 86 further including, following formation of the elongation product, providing a third temperature so as to permit amplicons to separate from elongation products and repeating the temperature cycle favoring, in sequence, annealing, elongation and separation.
89 . The method of claim 86 wherein the elongation products are detected before and after each temperature cycle.
90 . The method of claim 86 wherein elongation product formation is continuously monitored over a given period of time.
91 - 127 . (canceled)