Multiplexed analysis of polymorphic loci by concurrent interrogation and enzyme-mediated detection

• US 20070264641A1
• Filed: 05/22/2006
• Published: 11/15/2007
• Est. Priority Date: 10/15/2001
• Status: Abandoned Application

• Current Assignee: Alice Li, Ghazala Hashmi, Michael Seul
• Original Assignee: Alice Li, Ghazala Hashmi, Michael Seul
• Inventors: Seul, Michael, Hashmi, Ghazala, Li, Alice
• Application Number:
  US11/438,723
• Publication Number:
  US 20070264641A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism

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.

Claims

1-48. -48. (canceled)

49. A method of determining the nucleotides at two or more predetermined polymorphic 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 different types of encoded probes have different nucleotide sequences and different types of probes are differently encoded and are complementary, in whole or in substantial part, to different amplicon strands or different subsequences of amplicon strands, and wherein at least one type of probe is selected so that the nucleotides in said type which align, upon annealing, with non-designated or non-selected polymorphic sites in amplicon strands are all outside the terminal elongation initiation region;

d) contacting the set of encoded probes with said amplicons under conditions permitting annealing of encoded probes to amplicons and the formation of a probe elongation product by the probes having sites in the terminal elongation region complementary to nucleotides in the aligned region in the amplicons, upon annealing of probes and amplicons, but wherein probes having sites outside the terminal elongation region complementary to non-designated or non-selected polymorphic sites in the aligned region in the amplicons, upon annealing of probes and amplicons, are not elongated;

e) detecting probe elongation products and the probes which are not elongated; and

g) decoding the identities of the elongated and non-elongated probes.

50. The method of claim 49 wherein the interrogation site is at the 3′

terminus of the probe.

51. The method of claim 49 wherein the different probe types are associated with encoded carriers.

52. The method of claim 49 wherein the encoded probe set includes a subset of four different types of probes, each with a different nucleotide which aligns with a designated polymorphic site.

53. The method of claim 49 wherein said target is an mRNA, cDNA or a double-stranded polynucleotide including DNA.

54. The method of claim 51 wherein encoding of probes is by associating probes with different sequences to carriers, including beads, having different optical signatures.

55. The method of claim 54 wherein the encoding is with color.

56. The method of claim 49 wherein the elongation of the probes comprises adding one or more types of deoxyribonucleotide triphosphates or di-deoxyribonucleotide triphosphates for elongating the set of probes.

57. The method of claim 49 wherein only one type of deoxyribonucleotide triphosphates or di-deoxyribonucleotide triphosphate is involved in elongating the set of probes.

58. The method of claim 56 wherein a fraction of at least one type of deoxyribonucleotide triphosphate or di-deoxyribonucleotide triphosphate is labeled so as to generate an optically detectable signature associated with the elongation product following its incorporation into the probe.

59. The method of claim 56 wherein all types of deoxyribonucleotide triphosphate or di-deoxyribonucleotide triphosphate are labeled so as to generate an optically detectable signature associated with the elongation product following its incorporation into the probe.

60. The method of claim 59 wherein a polymerase is included for mediating the elongation of the probes.

61. The method of claims 60 wherein the polymerase lacks 3′

->

5′

exonuclease activity.

62. The method of claim 49 wherein one of the complementary strands of each amplicon pair is selectively removed by digesting it with an enzyme.

63. The method of claims 62 wherein an amplicon is preselected for digestion by phosphorylating the primer incorporated in it.

64. The method of claim 49 wherein the 3 base segment at the 3′

terminus of a probe is perfectly complementary to the subsequence including the designated polymorphism of the complementary amplicon strand.