Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-12-30
2001-01-16
Riley, Jezia (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S091200, C436S022000, C436S023000, C436S024000, C436S024000, C436S024000, C436S024000, C436S025000
Reexamination Certificate
active
06174680
ABSTRACT:
FIELD OF THE INVENTION
The present invention is directed to methods for identifying and labeling glycosylase-recognizable sites on nucleic acids, and to novel compounds that bind to such glycosylase-recognizable sites on nucleic acids. In a preferred embodiment the method can be used to identify mutations and/or polymorphisms on a nucleic acid segment, or in an arbitrary mixture of nucleic acid segments or genes.
BACKGROUND OF THE INVENTION
The detection of mutations has been an area of great interest in recent years. For example, mutations in certain genes have been associated with a variety of disorders—ranging from blood disorders to cancers. Genetic tests are thus becoming an increasingly important facet of medical care. Consequently, there has been an emphasis on the ability to rapidly and efficiently detect mutations and polymorphisms.
Many electrophoretic techniques have been developed to rapidly screen DNAs for sequence differences by which such mutations can be detected. Denaturing Gradient Get Electrophoresis (DGGE) [Myers, R. M., Maniatis, T. and Lerman, L.,
Methods in Enzymology,
155, 501-527 (1987)], Constant Denaturant Gel Electrophoresis (CDGE) [Borresen, A. L., et al., Proc. Nat. Acad. Sci. USA, 88, 8405 (1991)], Single Strand Conformation Polymorphism (SSCP) [Orrita, M., et al.,
Proc. Nat. Acad. Sci. USA,
86, 2766-2770 (1989)], Heteroduplex Analysis (HA) [Nagamine, C. M., et al.,
Am. J. Hum, Genet.,
45,377-399 (19?9)] and Protein Truncation Test (PTT) [Roest, P. A. M., et al., Hum. Molec. Genet., 2,1719-1721 (1993)] are frequently used methods. Many labs use combinations of these methods to maximize mutation detection efficiency. All these methods require gel electrophoresis. Methods that do not require gel electrophoresis also exist. For example, selective hybridization on immobilized target sequences allows screening for rare known mutations [Zafiropoulos, A., et al.,
Biotechniques
223, 1104-1109 (1997)], while mass-spectrometry has been used to detect mutations by analyzing molecular weight of proteins [Lewis, J. K., et al.,
Biotechniques
24, 102-110 (1998)].
A fundamental problem with currently existing mutation and polymorphism detection methods is that they only screen for mutations on a single gene at a time (i.e. the method is directed to looking at a ‘gene of interest’, that is suspected of having a mutation). Given that the human genome has 50,000-100,000 genes, this is a severe limitation. It is likely that unknown mutations and polymorphisms in several other genes both known and unknown, exist simultaneously with mutations/polymorphisms in the ‘gene of interest’. However, mutations in those other genes would likely not be identified. Therefore a method that can perform ‘mutation/polymorphism scanning’ in for a wide array of genes simultaneously, without the initial need for identifying the gene one is screening would be useful. Gel-electrophoresis-based methods are essentially restricted to examining mutations in a single gene at a time. Attempts have been made to devise non-gel electrophoretic methods to identify mutations, that would not be restricted to a single gene [Cotton et al.,
Proc. Natl. Acad. Sci. USA vol.
85, pp 4397-4401, (1988)] [Nelson, S. F. et al.,
Nature Genetics,
4, 11-8, (May 1993)] [Modrich, P., et al., Methods for Mapping Genetic Mutations. U.S. Pat. No. 5,459,039, (1995)]. These methods, however, have had limited success [Nollau P and Wagener C.,
Clinical Chemistry
43: 1114-1128 (1997)] since they are complicated, typically requiring several enzymatic steps and they result in a large number of false positives, i.e. they frequently score mutations and polymorphisms in normal DNA. It would be desirable to have a method that allows highly sensitive and specific identification and rapid purification of sites that contain mutation/polymorphism over large spans of the genome.
Although DNA arrays and methodologies that can simultaneously scan a large set of DNA fragments for gene expression (e.g. the ‘repertoire’ and amount of genes expressed in normal vs. cancer cells) are known [Wodicka L,
Nature Biotechnology
15: 1359-1367 (1997); Lockhart, D J,
Nature Biotechnology
14: 1675-1680 (1996); Schena, M.,
Trends Biotecnnol
16: 301-306, (1998); Yang, T. T.,
Biotechniques
18: 498-503, (1995)], the ability to scan a large set of random DNA fragments for unknown mutations is a much more demanding process on which the technology is lagging [Ginot F.,
Human Mutation
10: 1-10 (1997)]. Thus far DNA array-based methods to scan for polymorphisms (SNPs) and mutations has been restricted to specific genes [Lipshutz, R. J.,
Biotechniques
19: 442-447 (1995); Wang, D. G.,
Science
280: 1077-1082 (1998)]. Whereas detection of unknown mutations over several genes requires a selectivity and sensitivity not currently achievable by present arrays [Ginot F.,
Human Mutation
10: 1-10 (1997)]. For example, when it comes to unknown mutation detection, even a single gene with a coding sequence of the size of APC (8.5 kb) is difficult to screen in a single experiment, especially when an excess normal alleles is simultaneously present [Sidransky D.,
Science
278: 1054-1058 (1997)]. A method that permits identification of mismatches over large spans of the genome would be desirable.
The process of mismatch repair of nucleic acids has also received considerable attention in recent years with the elucidation of systems in microorganisms such as
E. coli,
and more recently, mammals including humans. For example, continuous cellular damages occur to nucleic acids during the cell life cycle; for example damage resulting from exposure to radiation, or to alkylating and oxidative agents, spontaneous hydrolysis and errors during replication. Such damages must be repaired prior to cell division. There are a number of different cellular repair systems and a variety of components that participate in these systems. One component is represented by the class of DNA repair enzymes known as mismatch repair glycosylases. These enzymes convert mismatches in DNA to aldehyde-containing abasic sites. These abasic sites can also occur by other means. For example, they can occur spontaneously, or following deamination of cytosine to uracil and subsequent removal of uracil by uracil glycosylase [Lindahl and Myberg, 1972; Lindahl, 1982 & 1994; Demple and Harrison, 1994; von Sonntag 1987; Loeb and Preston, 1986]. It has been estimated that almost 10,000 abasic sites are generated per cell per day [Lindahl and Nyberg, 1972]. Finally abasic sites are generated by DNA damaging agents such as ionizing radiation [von Sonntag, 1987], reactive oxygen intermediates [Ljungman and Hanawalt, 1992; Lindahl, 1994], antibiotics [bleomycin-iron complexes, neocarzinostatin, Povirk and Houlgrave, 1988], or alkylation agents [methylmethanesulfonate, dimethylsulfate etc., Loeb and Preston—1986]. Unrepaired abasic sites can be lethal or promutagenic lesions since during DNA replication DNA polymerases insert primarily adenines opposite them [Kunkel et al.—1983; Loeb and Preston—1986]. Closely-spaced abasic sites generated within a few base pairs of each other by damaging agents may be a particularly significant set of lesions, as they may hinder repair [Chaudhry and Weinfeld, 1995a, 1997; Harrison et al., 1998], or they can be enzymatically converted to double strand breaks or other complex multiply-damaged sites [Dianov et al., 1991]. It has been postulated that such complex forms of DNA damage may be particularly difficult for cells to overcome [Ward 1985, 1988; Wallace, 1988; Goodhead, 1994; Chaudhry and Weinfeld, 1995a and b, and 1997; Rydberg, 1996; Hodgkins et al., 1996; Nikjoo et al., 1998; Harrison et al., 1998]. Quantification of the overall number of abasic sites directed to looking at abasic sites result
Dana-Farber Cancer Institute Inc.
Nixon & Peabody LLP
Riley Jezia
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