Method for analyzing a polynucleotide containing a variable sequence

• US 6,150,095 A
• Filed: 10/06/1997
• Issued: 11/21/2000
• Est. Priority Date: 04/07/1995
• Status: Expired due to Term

• Current Assignee: Oxford Gene Technology IP Limited (Sysmex Corporation)
• Original Assignee: Oxford Gene Technology Limited (Sysmex Corporation)
• Inventors: Southern, Edwin Mellor, Pritchard, Clare Elizabeth, Case-Green, Stephen Charles
• Primary Examiner(s): Horlick, Kenneth R.
• Application Number:
  US08/930,798
• Time in Patent Office: 1,142 Days
• Field of Search: 435/6, 435/91.1, 536/24.3, 536/25.3
• US Class Current: 435/6.11
• CPC Class Codes: B01J 19/0046 Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays synthesis methods per se C40B50/00
• CPC Class Codes: B01J 2219/00608 DNA chips
• CPC Class Codes: B01J 2219/0061 The surface being organic
• CPC Class Codes: B01J 2219/00626 Covalent
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/6862 Ligase chain reaction [LCR]
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2521/101 DNA polymerase
• CPC Class Codes: C12Q 2521/501 Ligase
• CPC Class Codes: C12Q 2525/151 repeat or repeated sequences, e.g. VNTR, microsatellite, concatemer
• CPC Class Codes: C12Q 2533/107 Probe or oligonucleotide ligation
• CPC Class Codes: C12Q 2535/125 Allele specific primer extension
• CPC Class Codes: C12Q 2537/125 Sandwich assay format
• CPC Class Codes: C12Q 2561/125 Ligase Detection Reaction [LDR]
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides
• CPC Class Codes: C12Q 2565/518 : characterised by the immobilisation of the nucleic acid sample or target
• CPC Class Codes: C12Q 2565/537 : characterised by the capture oligonucleotide acting as a primer
• CPC Class Codes: C12Q 2600/156 : Polymorphic or mutational markers

Abstract

A method of analyzing a polynucleotide target involves incubating the target with an oligonucleotide probe, generally an array of immobilised oligonucleotide probes, to form a duplex, and using ligase or polymerase to extend one chain of the duplex. A point mutation or variable number tandem repeat section may be analysed. Arrays of immobilised oligonucleotides are provided for use in the method.

Claims

1. A method of analysis which comprises:

providing a polynucleotoide target including a nucleotide at a specified position, and two or more different oligonucleotide probes tethered to different locations of a support in the form of an array, each said probe being complementary to the target including an expected variant of the target and terminating at or close to the said specified position; and

performing the steps;

a) incubating the target with the probes to form a duplex,

b) incubating the duplex under ligation conditions with a labelled oligonucleotid ementary to the target,

c) and monitoring ligation in step b) as an indication of a point mutation at the specified position in the target.

2. A method of analysis which comprises:

providing a polynucleotide target having a variable number tandem repeat section and a flanking section, and two or more different oligonucleotide probes tethered to different locations of a support in the form of an array, each said probe having a section complementary to a repeat section and a flanking section of the target; and

performing the steps;

a) incubating the target with the probes to form a duplex,

b) incubating the duplex with a labelled oligonucleotide and/or at least one labelled nucleotide under chain extension conditions,

c) and monitoring chain extension as an indication of the length of the variable number repeat section of the target.

3. A method as claimed in claim 2, wherein a polymerase and/or ligase is used in step b).

4. A method as claimed in claim 2 or claim 3, wherein the polynucleotide target has a variable number tandem repeat section and two flanking sections, and wherein in step b) a labelled oligonucleotide is ligated to the probe strand of the duplex.

5. A method as claimed in claim 2 or claim 3, wherein the polynucleotide target has a variable number tandem repeat section and two flanking sections, and wherein in step b) the probe strand of the duplex is chain extended by addition of labelled nucleotides.