Detection of specific sequences in nucleic acids

• US 5,242,794 A
• Filed: 06/05/1989
• Issued: 09/07/1993
• 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): Wax, Robert A.
• Assistant Examiner(s): Grimes, Eric
• Application Number:
  US07/361,407
• Time in Patent Office: 1,555 Days
• Field of Search: 435/6
• US Class Current: 435/6.16
• 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

The invention provides a method for diagnosis of genetic abnormalities or other genetic conditions which can be readily automated. The method is used to determine the presence or absence of a target sequence in a sample of denatured nucleic acid and entails hybridizing the sample with a probe complementary to a diagnostic portion of the target sequence (the diagnostic probe), and with a probe complementary to a nucleotide sequence contiguous with the diagnostic portion (the contiguous probe), under conditions wherein the diagnostic probe remains bound substantially only to the sample nucleic acid containing the target sequence. The diagnostic probe and contiguous probe are then covalently attached to yield a target probe which is complementary to the target sequence, and the probes which are not attached are removed. In the preferred mode, one of the probes is labeled so that the presence or absence of the target sequence can then be tested by melting the sample nucleic acid-target probe duplex, eluting the dissociated target probe, and testing for the label. In another embodiment, the testing is accomplished without first removing probes not covalently attached, by attaching a hook to the probe that is not labeled, so that the labeled target probe may be recovered by catching the hook. In both instances, the presence of both the diagnostic probe and the contiguous probe is required for the label to appear in the assay. The above method is then applied to the detection of genetic diseases.

Claims

1. A method of detecting the presence of a target sequence in duplex DNA, comprising:

(a) denaturing the duplex DNA;

(b) reacting the denatured DNA 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 said 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, wherein at least one of the first and second probes is labeled;

(c) ligating said first and second probes which are hybridized to the target sequence in step (b); and

(d) testing for the presence of ligated first and second probes formed in step (c).

2. The method of claim 1, wherein said ligating includes treating the first and second probes with DNA ligase a ligase enzyme.

3. The method of claim 1, for use in detecting a target sequence which contains a genetic mutation from a known normal sequence, which further includes the step of comparing the amount of ligated probe from step (c) with a calibrated standard of nucleic acid known to have such mutation.