Multiplexed analysis of polymorphic loci by concurrent interrogation and enzyme-mediated detection
3 Assignments
0 Petitions
Accused Products
Abstract
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.
-
Citations
131 Claims
-
1-69. -69. (canceled)
-
70. A method of identifying one or more nucleotides at each of two or more designated sites in one or more targets, the method comprising the following steps:
-
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 encoded probes wherein differently encoded probes have different nucleotide sequences, selected such that different probes are differently encoded and probes of a first type are complementary, in whole or in substantial part, to a subsequence of an amplicon sense strand, where said amplicon sense strand is in molar excess over its complementary antisense amplicon strand; and encoded probes of at least a second type are complementary, in whole or in substantial part, to a subsequence of an amplicon antisense strand, where the amplicon antisense strand is provided in molar excess over its complementary sense amplicon strand; d) associating said set of probes with a set of encoded carriers, such that the encoding indicates which types of probes are associated with which types of encoded carrier, and wherein more than one type of probe is associated with one type of encoded carrier; e) contacting the set of encoded probes with said amplicons under conditions permitting the formation of a probe elongation product, following annealing of encoded probes to amplicons, and wherein probes are capable of annealing to an amplicon such that an interrogation site within a probe is in alignment with a designated polymorphic site in said amplicon; and f) detecting probe elongation products. - View Dependent Claims (107, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123)
-
-
71-106. -106. (canceled)
-
108. (canceled)
-
124. A method for differentiating alleles which are differentiated by different nucleotides at a variable site of one or more nucleic acid sequences, comprising:
-
providing, for each designated variable site of a nucleic acid sequence, pairs of labeled primers, with members of a pair being differently labeled from each other and from other members of other pairs, one member having a subsequence complementary to a first subsequence which is identical to the 5′
terminal subsequence of one allele, and the other member of the pair being identical to said one member but for the nucleotide at its 3′
terminus, wherein, following annealing of said complementary subsequences on a primer with their respective complementary subsequences, said primers are capable, under appropriate conditions, of being extended to form elongation products by addition of dNTPs to their respective 3′
ends and wherein said labels are detectable in the elongation products;providing, for each anticipated elongation product, an oligonucleotide probe optically detectably-labeled having a subsequence complementary to a subsequence of a particular elongation-product; providing conditions for generation of elongation products; combining elongation products with oligonucleotide probes under conditions permitting annealing of elongation products and oligonucleotide probes having complementary subsequences; detecting elongation products by detecting the presence of the optically detectable labels; and identifying different elongation products by detecting and identifying the labels on the detected elongation products. - View Dependent Claims (125, 126, 127, 128, 129, 130, 131)
-
Specification