Methods and apparatus for screening and detecting multiple genetic mutations

• US 8,772,467 B2
• Filed: 10/07/2009
• Issued: 07/08/2014
• Est. Priority Date: 07/26/2002
• Status: Active Grant

• Current Assignee: Gamida for Life B.V.
• Original Assignee: Gamida for Life B.V.
• Inventors: Radtkey, Ray R., Held, Lance C., Wallace, Robert B., Menge, Karen, Canter, David
• Primary Examiner(s): Lu, Frank
• Application Number:
  US12/574,875
• Publication Number:
  US 20100167960A1
• Time in Patent Office: 1,735 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 435/183, 435/287.1, 435/287.2, 436/94, 436/501, 536/23.1, 536/24.3, 536/24.33, 536/25.3
• US Class Current: 536/24.3
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2535/131 Allele specific probes
• CPC Class Codes: C12Q 2537/149 Sequential reactions
• CPC Class Codes: C12Q 2565/518 characterised by the immobilisation of the nucleic acid sample or target
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

An assay system and methods are described where patient samples containing genomic DNA are analyzed for the presence of known genetic polymorphisms using a universal reporter strategy. In a preferred embodiment, the amplified DNA is localized at test sites in an array of sites on a microchip followed by a series of hybridization reactions that screen for the presence of a single mutation from among a number of mutations, and allow the identification of specific mutations. In addition to universal reporters, the assay may use blockers and discriminators for screening and identification of known polymorphisms.

Claims

1. A kit for use in a process of detecting members of a set of polymorphisms that occur at identified loci in samples of a patient nucleic acid, said kit comprising:

a group of unlabeled blockers capable of hybridizing under stringent conditions with markers in at least one locus contained in the patient sample nucleic acid thereby blocking said markers from further hybridizing to a detectable discriminator, wherein a specific nucleotide sequence of at least one unlabeled blocker of said group of unlabeled blockers is fully complementary to a nucleotide sequence comprising a mutation or a polymorphism in said at least one locus, and wherein the at least one unlabeled blocker is capable of hybridizing under said stringent conditions to the nucleotide sequence comprising the mutation or the polymorphism;

more than one detectable discriminator, at least one of said more than one detectable discriminator capable of hybridizing under said stringent conditions with the nucleotide sequence comprising said mutation or polymorphism, said at least one unlabeled blocker being capable of blocking said at least one detectable discriminator from hybridizing with the nucleotide sequence comprising said mutation or polymorphism under said stringent conditions; and

at least one universal reporter capable of binding with said more than one detectable discriminator,

wherein said at least one of said more than one detectable discriminator is denatured from the nucleotide sequence comprising said mutation or said polymorphism in a hybridization product formed by said at least one detectable discriminator and the nucleotide sequence comprising said mutation or said polymorphism under denaturation condition while said at least one unlabeled blocker has a sufficient length so that said at least one unlabeled blocker remains to hybridize with the nucleotide sequence comprising said mutation or said polymorphism in a hybridization product formed by said at least one unlabeled blocker and the nucleotide sequence having said mutation or said polymorphism under said denaturation condition.

2. The kit of claim 1, further comprising at least one amplification control being capable of binding with the patient nucleic acid.

3. The kit of claim 2, further comprising an additional universal reporter capable of binding with the at least one amplification control.

4. The kit of claim 1, further comprising an actively addressable electronic microarray capable of being loaded with said patient sample nucleic acid at its individually addressable sites.

5. The kit of claim 1, wherein at least one of said unlabeled blockers and said at least one of said more than one detectable discriminator comprise oligonucleotides that are complementary to the at least one locus contained in the patient sample nucleic acid.

6. The kit of claim 5, wherein the universal reporter comprises a nucleotide sequence that is complementary to the nucleotides of said at least one of said more than one discriminator.

7. The kit of claim 1, wherein said at least one unlabeled blocker of said group of unlabeled blockers is capable of hybridizing to a locus contained in the patient sample nucleic acid that is different from a locus to which said at least one of said more than one detectable discriminator is capable of hybridizing.