Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-07-02
2004-02-03
Siew, Jeffrey (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C435S183000, C536S023100
Reexamination Certificate
active
06686157
ABSTRACT:
FIELD OF THE INVENTION
The disclosed invention is generally in the field of assays for detection of nucleic acids, and specifically in the field of nucleic acid amplification.
BACKGROUND OF THE INVENTION
A number of methods have been developed which permit the implementation of extremely sensitive diagnostic assays based on nucleic acid detection. Most of these methods employ exponential amplification of targets or probes. These include the polymerase chain reaction (PCR), ligase chain reaction (LCR), self-sustained sequence replication (3SR), nucleic acid sequence based amplification (NASBA), strand displacement amplification (SDA), and amplification with Q&bgr; replicase (Birkenmeyer and Mushahwar,
J. Virological Methods,
35:117-126 (1991); Landegren,
Trends Genetics,
9:199-202 (1993)).
While all of these methods offer good sensitivity, with a practical limit of detection of about 100 target molecules, all of them suffer from relatively low precision in quantitative measurements. This lack of precision manifests itself most dramatically when the diagnostic assay is implemented in multiplex format, that is, in a format designed for the simultaneous detection of several different target sequences.
Fluorescence in situ hybridization is a useful method of determining the physical position of sequences relative to each other in the genome. However, the ability to detect sequences decreases as the size of the target sequence decreases so that detection of targets that are less than 500 bases in length is very difficult or impossible.
Rolling Circle Amplification (RCA) driven by DNA polymerase can replicate circular oligonucleotide probes with either linear or geometric kinetics under isothermal conditions (Lizardi et al.,
Nature Genet.
19: 225-232 (1998); U.S. Pat. No. 5,854,033 to Lizardi; PCT Application No. WO 97/19193). If a single primer is used, RCA generates in a few minutes a linear chain of hundreds or thousands of tandemly-linked DNA copies of a target which is covalently linked to that target. Generation of a linear amplification product permits both spatial resolution and accurate quantitation of a target. DNA generated by RCA can be labeled with fluorescent oligonucleotide tags that hybridize at multiple sites in the tandem DNA sequences. RCA can be used with fluorophore combinations designed for multiparametric color coding (PCT Application No. WO 97/19193), thereby markedly increasing the number of targets that can be analyzed simultaneously. RCA technologies can be used in solution, in situ and in microarrays. In solid phase formats, detection and quantitation can be achieved at the level of single molecules (Lizardi et al., 1998).
Ligation-mediated Rolling Circle Amplification (LM-RCA) involves circularization of a probe molecule hybridized to a target sequence and subsequent rolling circle amplification of the circular probe (U.S. Pat. No. 5,854,033 to Lizardi; PCT Application No. WO 97/19193). During amplification, the probe can become separated from the target sequence as it rolls. This can diminish the quality of spatial information obtained about the target.
It is therefore an object of the present invention to provide a method and compositions for detecting nucleic acid sequences in situ with a combination of specificity and sensitivity.
It is another object of the present invention to provide a method and compositions for detecting the amount and location of nucleic acid sequences with a combination of specificity and sensitivity.
BRIEF SUMMARY OF THE INVENTION
Disclosed is a method and compositions for the sensitive detection of the amount and location of specific nucleic acid sequences. The method makes use of a branched oligomer, referred to as a lollipop oligomer, that has a tail portion, a right arm portion, and a left arm portion. These three components are joined at a common junction making a three-tailed structure. The two arms each end with sequences complementary to adjacent sequences in a target sequence. This allows the right and left arms to be ligated together when the oligomer is hybridized to the target sequence, thus topologically linking the oligomer to the target sequence. The tail portion can then be detected at the location of the target sequence. By using the tail of the oligomer to prime rolling circle replication of a DNA circle, a long tandem repeat DNA is associated with the target sequence. Rolling circle replication does not disturb association of the arms and the target sequence, thus maintaining close association of the tandem repeat DNA and the target sequence.
The topological locking of the probe to the target is important for detection systems in which the amplified product may float away from the target. This may happen if the target sequence is extremely small or if the assay is being preformed on a substrate such as a metaphase chromosomes or any array that does not trap the tandem repeat DNA. By using multiple different primer (tail) sequences and corresponding DNA circles the method can be multiplexed, with the amplification product of each different circle being separately detectable. Lollipop oligomers can be circularized by chemical or enzymatic ligation.
The disclosed method is useful for detecting any desired sequence. In particular, the disclosed method can be used to localize or amplify signal from any desired sequence. For example, the disclosed method can be used to probe transgenic cells, bacterial or yeast colonies, cellular material (for example, whole cells, DNA fibers, interphase nuclei, or metaphase chromosomes on slides, arrayed genomic DNA, RNA). The disclosed method is particularly useful for detecting sequence variants of a target sequence. For example, insertions, deletions, repeats, and single nucleotide polymorphisms (SNP), can be detected. Specificity of these detections is aided by sensitivity of ligation of the arm ends to mismatches.
The disclosed method is applicable to numerous areas including, but not limited to, disease detection, mutation detection, RNA expression profiling, gene discovery, gene mapping (molecular haplotyping), agricultural research, and virus detection. Preferred uses include SNP detection in situ in cells, on microarrays, on DNA fibers, and on genomic DNA arrays; detection of RNA in cells; RNA expression profiling; molecular haplotyping; mutation detection; abnormal RNA (for example, overexpression of an oncogene or absence of expression of a tumor suppressor gene); expression in cancer cells; detection of viral genome in cells; viral RNA expression; detection of inherited diseases such as cystic fibrosis, muscular dystrophy, diabetes, hemophilia, sickle cell anemia; assessment of predisposition for cancers such as prostate cancer, breast cancer, lung cancer, colon cancer, ovarian cancer, testicular cancer, pancreatic cancer.
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Bray-Ward Patricia
Kumar Gyanendra
Lane Michael J.
Ward David C.
Molecular Staging Inc.
Needle & Rosenberg P.C.
Siew Jeffrey
Tung Joyce
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