Detection of specific sequences in nucleic acids

• US 5,521,065 A
• Filed: 06/08/1994
• Issued: 05/28/1996
• Est. Priority Date: 12/13/1984
• Status: Expired due to Term

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applied Biosystems, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Hunkapiller, Michael W., Glazer, Alexander N., Whiteley, Norman M.
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): MYERS, CARLA J
• Application Number:
  US08/257,219
• Time in Patent Office: 720 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 435/91.52, 935/16, 935/17, 935/77, 935/78, 536/24.3
• US Class Current: 435/6.18
• CPC Class Codes: C12Q 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6862 Ligase chain reaction [LCR]
• CPC Class Codes: C12Q 2521/501 Ligase
• CPC Class Codes: C12Q 2533/107 Probe or oligonucleotide ligation
• CPC Class Codes: C12Q 2535/131 Allele specific probes
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2561/125 Ligase Detection Reaction [LDR]
• CPC Class Codes: C12Q 2565/101 Interaction between at least two labels
• CPC Class Codes: Y10S 435/803 Physical recovery methods, e.g. chromatography, grinding
• CPC Class Codes: Y10S 435/81 Packaged device or kit

Abstract

A method of testing for the presence or absence of a target sequence in a mixture of single-stranded nucleic acid fragments is disclosed. The method involves reacting a mixture of single-stranded nucleic acid fragments with a first probe which is complementary to a first region of the target sequence, and with a second probe which is complementary to a second region of the target sequence, where the first and second target regions are contiguous with one another, under hybridization conditions in which the two probes become stably hybridized to their associated target regions. Following hybridization, any of the first and second probes hybridized to contiguous first and second target regions are ligated, and the sample is tested for the presence of expected probe ligation product. The presence of ligated product indicates that the target sequence is present in the sample.

Claims

1. A method of testing for the presence or absence of a target sequence in a mixture of single-stranded nucleic acid fragments, comprising:

reacting a mixture of single-stranded nucleic acid fragments with a first probe which is complementary to a first region of the target sequence, and with a second probe which is complementary to a second region of the target sequence, where the first and second target regions are contiguous with one another, under hybridization conditions in which the two probes become stably hybridized to their associated target regions,

ligating first and second probes which have hybridized to said contiguous first and second target regions, and

testing for the presence of ligated first and second probes, where the presence of the ligation product of said first and second probes indicates the presence of the target sequence in said mixture.

2. The method of claim 1, wherein one of the first and second probes is labeled.

3. The method of claim 2, wherein said testing includes separating labeled, ligated probes from labeled, non-ligated probes, and assaying for the presence of labeled, ligated probes.

4. The method of claim 1, wherein said testing further includes dissociating ligated probe from the target sequence prior to said testing.

5. The method of claim 1, wherein said ligating is carried out using a ligase enzyme.

6. The method of claim 1, wherein the target sequence tested for contains a genetic mutation.

7. The method of claim 1, wherein said nucleic acid mixture is tested for the presence of a normal target sequence and for the presence of a corresponding target sequence containing a genetic mutation.