Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-02-28
2003-06-17
Siew, Jeffrey (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S007100, C435S091100, C435S091200, C435S287200, C536S022100, C536S023100, C536S024300, C536S024310, C536S024320, C536S024330
Reexamination Certificate
active
06579680
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to the field of molecular biology, and more particularly to the field of assays that involve nucleic acid hybridization probes. Hybridization assays are employed in the detection and identification of specific nucleic acid sequences in genes, or RNA molecules, for analysis of gene expression and genetic polymorphisms. Such assays are used for medical diagnostics, as well as in pharmacological, food technology, agricultural, and biological research.
BACKGROUND
Numerous methods have been used for detecting variation in nucleic acid sequence. These methods include, for example, electrophoretic mobility shifts due to conformational changes induced by single or multiple base variation, and direct mass spectrometric sequence determination. Yet, among these varied methods, hybridization techniques are particularly well suited to detect specific target sequences in a complex mixture where nucleic acid hybridization probes are used. These hybridization techniques can be broadly classified as being either homogeneous or heterogeneous assays.
Sometimes referred to as homogeneous assays, several solution-phase detection schemes have been developed. By “homogeneous” we mean assays that are performed without separating unhybridized targets from probe-target hybrids. These schemes often make use of the fact that the immediate chemical environment can affect the fluorescence of many fluorescent labels. Oligonucleotide probes that are complementary to contiguous regions of a target DNA strand are employed. One probe contains a fluorescent label on its 5′ end and the other probe contains a different fluorescent label on its 3′ end. Upon hybridization to contiguous regions on a target sequence, the 5′ and 3′ ends of the probes are brought in close proximity, such that one quenches the fluorescence of the other. When the sample is stimulated by light of an appropriate frequency, fluorescence resonance energy transfer (“FRET”) from one label to the other occurs. This energy transfer produces a measurable change in spectral response, indirectly signaling the presence of the target. The labels are sometimes referred to as FRET pairs. The altered spectral properties can be subtle, and the changes, if one is not careful, can be small relative to background signal. This characteristic can be seen as a potential shortcoming of this type of hybridization. Although this technique works well with a small number of samples, the relatively limited, low through-put of homogeneous hybridization, however, can be another potential shortcoming, since homogeneous assays are not as amenable to high volume genetic processing as an array.
Recently, so-called molecular beacons have been developed and used to detect the presence of unlabeled target sequences in homogeneous solution. Molecular beacons are oligonucleotides that possess a hairpin structure in which the loop portion of the molecule is a probe sequence that is complementary to a target sequence in the nucleic acid to be detected, and the stem is formed by the annealing of complementary arm sequences. (For instance, U.S. Pat. No. 5,925,517, entitled “Detectably Labeled Dual Conformation Oligonucleotide Probes, Assays and Kits,” issued to Tyagi et al., describes one form of molecular beacon, while PCT International Publication No. WO 99/21881 entitled “Methods, Kits, and Compositions Pertaining to Linear Beacons,” by Gildea et al. discloses another form. Pertinent content of these two patent documents is incorporated herein by reference.) A fluorophore is covalently attached to the end of one arm and a quencher dye is covalently attached to the end of the other arm. The stem keeps the fluorophore-quencher pair in close proximity to each other, thereby quenching the fluorescence of the fluorophore. Only upon hybridization to a perfectly matched, that is, completely complementary, target nucleic acid sequence is the hairpin-stem structure disrupted, resulting in a fluorescence signal. Due to the constrained nature of the hairpin structure, molecular beacons can recognize their perfect complementary sequence with enhanced specificity compared to their linear counterparts. Thus, molecular beacons have been used to easily discriminate between targets that differ from one another by even only a single nucleotide.
Although effective, the use of molecular beacons also has some drawbacks. Because hairpin formation and stability is critically dependent upon oligonucleotide sequence, molecular beacons can be difficult to design. As a consequence, several preliminary experiments need to be conducted to ensure the proper formation and stability of the hairpin structure. These additional activities add to both the time and expense of the assay. Until very recently, molecular beacons have been used only in homogeneous solutions. In addition, like other homogeneous assays, molecular beacon techniques also suffer from limited, low through-put hybridization and analysis. To be used in large-scale parallel analysis, molecular beacons must be attached to a solid surface. Attachment of these hairpin structures on a surface while maintaining proper fluorescence, quenching, and hybridization has, however, proven to be non-trivial, if not difficult, due to steric effects of the substrate on the hairpin.
For large-scale, parallel analysis of target DNA sequences, workers look to array technology. High through-put hybridization analysis has been achieved with the advent of DNA microarrays, also known as DNA “chips”, which permit the possibility of applying high probe densities on small substrates. A typical DNA chip experiment uses a conventional, heterogeneous, hybridization assay. Heterogeneous hybridization typically comprises a number of steps: immobilizing probe nucleic acid sequences on paper, beads, or plastic surfaces, adding an excess of labeled targets that are complementary to the sequence of the probe: hybridizing; removing excess labeled targets and unhybridized targets, and detecting the targets that remain bound to the immobilized probes. Unhybridized targets are removed by washing of the hybrids. In experiments such as these, careful control of both surface chemistry and washing conditions is required to minimize background signal from the nonspecific adsorption of labeled target sequences.
Recently a heterogeneous, nucleic acid hybridization assay that enhances discrimination of single nucleotide polymorphisms (SNPs) has been reported in the scientific literature—a paper entitled “Enhanced Discrimination of Single Nucleotide Polymorphisms by Artificial Mismatch Discrimination,”
Nature Biotechnology,
15, 331-335 (1997), the relevant sections of which are incorporated herein by reference. Developed by Z. Guo et al., this process is based on differences in thermodynamic stability of duplexes formed by an oligonucleotide containing an artificial mismatch site and two different labeled targets. From this work, U.S. Pat. No. 5,780,233, entitled “Artificial Mismatch Hybridization,” was issued to Guo et al, (the '233 Patent). The pertinent content of the '233 Patent is incorporated herein by reference. This patent describes a process for hybridizing labeled first and second target sequences to an immobilized oligonucleotide. The immobilized oligonucleotide has a nucleic acid sequence that is complementary in part to both the first and second targets, including at the position of sequence variation, but comprises at least one “artificial” mismatch relative to the first target, and an “artificial” mismatch and a “true” mismatch relative to the second target. The oligonucleotide forms a first duplex and a second duplex with the first and second targets, respectively, wherein the first duplex has a melting temperature that is higher than that of the second duplex.
Even though the process described by Guo et al. has potential advantages, it still may not possess the degree of sensitivity required for certain applications, or as workers in the field may wish to achieve. Primarily, the problem with the
Frutos Anthony Glenn
Lahiri Joydeep
Pal Santona
Quesada Mark A.
Corning Incorporated
Kung Vincent T.
Siew Jeffrey
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