Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-12-29
2002-10-08
Brusca, John S. (Department: 1631)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C536S023100
Reexamination Certificate
active
06461820
ABSTRACT:
3. FIELD OF THE INVENTION
The present invention relates to the detection and localization of base-pair mismatches and other perturbations in base-stacking within an oligonucleotide duplex.
4. DESCRIPTION OF RELATED ART
It is now well known that mutations in DNA can lead to severe consequences in metabolic functions (e.g., regulation of gene expression, modulation of protein production) which ultimately are expressed in a variety of diseases. For example, a significant number of human cancers are characterized by a single base mutation in one of the three ras genes (Bos, 1989). In order to unravel the genetic components of such diseases, it is of utmost importance to develop DNA sensors that are capable of detecting single-base mismatches rapidly and efficiently and to establish routine screening of disease-related genetic mutations based on such sensors (Skogerboe, 1993; Southern, 1996; Chee, 1996; Eng, 1997).
Various methods that have been developed for the detection of differences between DNA sequences rely on hybridization events to differentiate native versus mutated sequences and are limited by the small differences in base-pairing energies caused by point mutations within extended polynucleotides (Millan, 1993; Hashimoto, 1994; Xu, 1995; Wang, 1996; Lockhart, 1996; Alivisatos, 1996; Korriyoussoufi, 1997; Elghanian, 1997; Lin, 1997; Herne, 1997). Typically, a nucleic acid hybridization assay to determine the presence of a particular nucleotide sequence (i.e. the “target sequence”) in either RNA or DNA comprises a multitude of steps. First, an oligonucleotide probe having a nucleotide sequence complementary to at least a portion of the target sequence is labeled with a readily detectable atom or group. When the labeled probe is exposed to a test sample suspected of containing the target nucleotide sequence, under hybridizing conditions, the target will hybridize with the probe. The presence of the target sequence in the sample can be determined qualitatively or quantitatively in a variety of ways, usually by separating the hybridized and non-hybridized probe, and then determining the amount of labeled probe which is hybridized, either by determining the presence of label in probe hybrids or by determining the quantity of label in the non-hybridized probes. Suitable labels may provide signals detectable by luminescence, radioactivity, colorimetry, x-ray diffraction or absorption, magnetism or enzymatic activity, and may include, for example, fluorophores, chromophores, radioactive isotopes, enzymes, and ligands having specific binding partners. However, the specific labeling method chosen depends on a multitude of factors, such as ease of attachment of the label, its sensitivity and stability over time, rapid and easy detection and quantification, as well as cost and safety issues. Thus, despite the abundance of labeling techniques, the usefulness, versatility and diagnostic value of a particular system for detecting a material of interest is often limited.
Some of the currently used methods of mismatch detection include single-strand conformation polymorphism (SSCP) (Thigpen, 1992; Orita, 1989), denaturing gradient gel electrophoresis (DGGE) (Finke, 1996; Wartell, 1990; Sheffield, 1989), RNase protection assays (Peltonen and Pulkkinen, 1986; Osborne, 1991), allele-specific oligonucleotides (Wu, 1989), allele-specific PCR (Finke, 1996), and the use of proteins which recognize nucleotide mismatches, such as the
E. coli
mutS protein (Modrich, 1991).
In the first three methods, the appearance of a new electrophoretic band is observed by polyacrylamide gel electrophoresis. SSCP detects the differences in speed of migration of single-stranded DNA sequences in polacrylamide gel electrophoresis under different conditions such as changes in pH, temperature, etc. A variation in the nucleotide base sequence of single-stranded DNA segments (due to mutation or polymorphism) may lead to a difference in spatial arrangement and thus in mobility. DGGE exploits differences in the stability of DNA segments in the presence or absence of a mutation. Introduction of a mutation into double-stranded sequences creates a mismatch at the mutated site that destabilizes the DNA duplex. Using a gel with an increasing gradient of formamide (denaturation gradient gel), the mutant and wild-type DNA can be differentiated by their altered migration distances. The basis for the RNase protection assay is that the RNase A enzyme cleaves mRNA that is not fully hybridized with its complementary strand, whereas a completely hybridized duplex is protected from RNase A digestion. The presence of a mismatch results in incomplete hybridization and thus cleavage by RNase A at the mutation site. Formation of these smaller fragments upon cleavage can be detected by polyacrylamide gel electrophoresis. Techniques based on mismatch detection are generally being used to detect point mutations in a gene or its mRNA product. While these techniques are less sensitive than sequencing, they are simpler to perform on a large number of tumor samples. In addition to the RNase A protection assay, there are other DNA probes that can be used to detect mismatches, through enzymatic or chemical cleavage. See, e.g., Smooker and Cotton, 1993; Cotton, 1988; Shenk, 1975. Other enzymatic methods include for example the use of DNA ligase which covalently joins two adjacent oligonucleotides which are hybridized on a complementary target nucleic acid, see, for example Landegren (1988). The mismatch must occur at the site of ligation.
Alternatively, mismatches can also be detected by shifts in the electrophoretic mobility of mismatched duplexes relative to matched duplexes (Cariello, 1988). With either riboprobes or DNA probes, the cellular mRNA or DNA which may contain a mutation can be amplified using polymerase chain reaction (PCR) prior to hybridization. Changes in DNA of the gene itself can also be detected using Southern hybridization, especially if the changes are gross rearrangements, such as deletions and insertions.
DNA sequences of the specified gene which have been amplified by use of PCR may also be screened using allele-specific oligonucleotide probes. These probes are nucleic acid oligomers, each of which is complementary to a corresponding segment of the investigated gene and may or may not contain a known mutation. The assay is performed by detecting the presence or absence of a hybridization signal for the specific sequence. In the case of allele-specific PCR, the PCR technique uses unique primers which selectively hybridize at their 3′-ends to a particular mutated sequence. If the particular mutation is not present, no amplification product is observed.
In addition, restriction fragment length polymorphism (RFLP) probes for the gene or surrounding marker genes can be used to score alteration of an allele or an insertion in a polymorphic fragment. However, since the recognition site of restriction endonucleases ranges in general between 4 to 10 base pairs, only a small portion of the genome is monitored by any one enzyme.
Another means for identifying base substitution is direct sequencing of a nucleic acid fragment. The traditional methods are based on preparing a mixture of randomly-terminated, differentially labeled DNA fragments by degradation at specific nucleotides, or by dideoxy chain termination of replicating strands (Maxam & Gilbert, 1980; Sanger, 1977). Resulting DNA fragments in the range of 1 to 500 basepairs are then separated on a gel to produce a ladder of bands wherein the adjacent samples differ in length by one nucleotide. The other method for sequencing nucleic acids is sequencing by hybridization (SBH, Drmanac, 1993). Using mismatch discriminative hybridization of short n-nucleotide oligomers (n-mers), lists of constitutent n-mers may be determined for target DNA. The DNA sequence for the target DNA may be assembled by uniquely overlapping scored oligonucleotides. Yet another approach relies on hybridization to high-density arrays of oligonucleotides to determine genetic variation. Using a two-colo
Barton Jacqueline K.
Hill Michael G.
Kelley Shana O.
Brusca John S.
California Institute of Technology
Gray Cary Ware & Freidenrich LLP
Haile Lisa A.
Reynolds Kelly K.
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