Nucleic acid sequence detection employing amplification probes

Details Nucleic acid sequence detection employing amplification probes Nucleic acid sequence detection employing amplification probes

1997-12-05

2001-01-23

Horlick, Kenneth R. (Department: 1656)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S091100, C435S091200, C536S024300, C536S024310, C536S024330, C536S025320

Reexamination Certificate

active

06177243

ABSTRACT:

INTRODUCTION
1. Technical Field
The field of this invention is nucleic acid sequence detection.
2. Background
The amount of information concerning the genomes of a large variety of species is increasing exponentially. The availability of known sequences creates an enormous market for the detection of particular sequences present as DNA or RNA, whereby one can detect the presence of genes, their transcription or mutations, such as lesions, substitutions, deletions, translocations, and the like. By knowing sequences of interest, one can detect a wide variety of pathogens, particularly unicellular microorganisms and viral strains, and genetic diseases including the presence of genes imparting antibiotic resistance to the unicellular microorganisms, as illustrative of only a few of the available possibilities. In addition, there are needs within the extensive areas of genetic counseling, forensic medicine, research, and the like, for nucleic acid sequence detection technology.
In many instances, the target nucleic acid sequence is only a very small proportion of total nucleic acid in the sample. Furthermore, there may be many situations where the target nucleic acid of interest and other sequences present have substantial homology. It is therefore important to develop methods for the detection of the target nucleic acid sequence that are both sensitive and accurate.
Several enzymatic amplification methods have been developed, such as the polymerase chain reaction (PCR), the ligase chain reaction (LCR), NASBA, and self-sustained sequence replication (SSR). The first and most notable method that has received extensive use is PCR. Starting with specific primers, nucleoside triphosphate monomers, the target strand of DNA and a polymerase enzyme, one can greatly amplify the target DNA sequence of interest. This technology is extremely powerful and has been applied to a myriad of research applications, but it has a number of drawbacks which limit its use in a variety of areas. General availability is limited by the restrictive nature of licenses by the owners of the patent rights. In addition, the method requires an enzyme. While the availability of thermally stable enzymes has greatly enhanced the applicability of PCR, there is nevertheless the inconvenience that denaturation of the enzyme occurs during thermocycling. Also, the sample may include inhibitors of the enzyme requiring isolation of the nucleic acid sample free of inhibiting components. In addition, the methodology is sensitive to amplifying stray sequences, which then overwhelm the target sequence of interest, obscuring its presence. There is also the fact that the reagents are expensive and the amplified DNA usually requires verification. These comments apply equally to the other enzymatic amplified techniques noted above, such as LCR, NASBA, and SSR.
There is, therefore, substantial interest in identifying alternative techniques which allow for the detection of specific DNA sequences and avoid the deficiencies of the other systems. Also of interest is the development of devices for automatically carrying out these alternative nucleotide sequence detection techniques, where these automatic devices will reduce the opportunity of error introduction and provide for consistency of assay conditions.
RELEVANT LITERATURE
Barany, Proc. Natl. Acad. Sci. USA (1991) 88:189-193; Gautelli et al., Proc. Natl. Acad. Sci. USA (1990) 87:1874-1878. Segev Diagnostics, Inc. WO 90/01069. Imclone Systems, Inc. WO 94/29485. U.S. Pat. Nos. 5,185,243, 4,683,202 and 4,683,195.
SUMMARY OF THE INVENTION
Methods and compositions are provided for detecting nucleic acid sequences by using a pair of probes, in each of which at a different end there is a portion of the chain which serves as one half of a stem, which portion will be referred to as a side chain. The side chains comprise a cross linking system, which has a photoactivatable entity, normally coupled to a passive reactive entity. Upon orientation of the side chains in spacial proximity as a result of binding of the probes to a contiguous homologous target sequence and activation of the cross linking system associated with the side chains, the probes are joined together by a covalent linkage. The method employs adding the probes to the target nucleic acid under conditions of base pairing, activating the cross-linking system, so that primarily only those side chains in spacial proximity form a covalent bond, melting double-stranded nucleic acid and repeating the cycle. Where only one set of probes is used, the expansion is linear; where complementary sets of probes are used, in the re-annealing process the probes in addition to binding to target nucleic acid, will also bind to cross-linked probes. In this manner, one may obtain a linear or geometric increase in the number of cross-linked probes as the cycle of steps is repeated, wherein the process is initiated by the presence of target DNA.
In a preferred embodiment, the probes have non-cross-linking duplex forming side chains, where at least one side chain is in the form of a duplex prior to hybridization with the target DNA. The side chains are characterized that at least one of the side chains has a photoactivatable group and the other of the side chains has a recipient group which reacts with the photoactivatable group to form a covalent bond.
The methods comprise combining the probes whose sequences are homologous to adjacent sequences in the target DNA under conditions, which may be successive or simultaneous, which result in melting of the side chain duplexes and hybridization of the probes to the target DNA. After sufficient time for hybridization between the probes and the target DNA to occur, the hybridization medium is irradiated to photoactivate the photoactivatable groups, which will react with the recipient group to cross-link the probes bound to target DNA or dimerized probes.


REFERENCES:
patent: 4582789 (1986-04-01), Sheldon, III et al.
patent: 4617261 (1986-10-01), Sheldon, III et al.
patent: 4705886 (1987-11-01), Levenson et al.
patent: 4751313 (1988-06-01), Levenson et al.
patent: 4754065 (1988-06-01), Levenson et al.
patent: 4820630 (1989-04-01), Taub
patent: 4851330 (1989-07-01), Kohne
patent: 4883750 (1989-11-01), Whiteley et al.
patent: 4965188 (1990-10-01), Mullis et al.
patent: 5082934 (1992-01-01), Saba et al.
patent: 5118801 (1992-06-01), Lizardi et al.
patent: 5139940 (1992-08-01), Isaacs et al.
patent: 5185243 (1993-02-01), Ullman
patent: 5219734 (1993-06-01), Royer et al.
patent: 5230781 (1993-07-01), Middendorf et al.
patent: 5270183 (1993-12-01), Corbett
patent: 5302347 (1994-04-01), van den Berg et al.
patent: 5312728 (1994-05-01), Lizardi et al.
patent: 5333675 (1994-08-01), Mullis et al.
patent: 5366603 (1994-11-01), Middendorf et al.
patent: 5424413 (1995-06-01), Hogan et al.
patent: 5449602 (1995-09-01), Royer et al.
patent: 5451503 (1995-09-01), Hogan et al.
patent: 5475610 (1995-12-01), Atwood et al.
patent: 5602756 (1997-02-01), Atwood et al.
patent: 5616301 (1997-04-01), Moser et al.
patent: 5629149 (1997-05-01), Santamaria et al.
patent: 5843650 (1998-12-01), Segev
patent: 5846706 (1998-12-01), Segev
patent: 0 097 373 (1984-01-01), None
patent: 0 320 308 (1989-06-01), None
patent: 0 336 731 (1994-05-01), None
patent: 0 324 616 (1995-03-01), None
patent: 90/01069 (1989-07-01), None
patent: 94/29485 (1994-12-01), None
Guatelli et al., “Isothermal, In Vitro Amplification of Nucleic Acids by a Multienzyme Reaction Modeled After Retroviral Replication,”PNAS, 87:1874-1878 (1990).
Barany, “Genetic Disease Detection and DNA Amplification Using Cloned Thermostable Ligase,”PNAS, 88:189-193 (1991).
Nilsson et al., “Padlock Probes: Circularizing Oligonucleotides for Localized DNA Detection,”Science,265:2085-2088 (1994).

Affiliated with

Also associated with

Rating

Nucleic acid sequence detection employing amplification probes does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Nucleic acid sequence detection employing amplification probes, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Nucleic acid sequence detection employing amplification probes will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-2554814